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https://openalex.org/W2156767781	https://europepmc.org/articles/pmc3055814?pdf=render	English	null	The creation of the health consumer: challenges on health sector regulation after managed care era	Globalization and health	2,011	cc-by	11,697	RESEARCH Open Access © 2011 Iriart et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: We utilized our previous studies analyzing the reforms affecting the health sector developed in the 1990s by financial groups to frame the strategies implemented by the pharmaceutical industry to regain market positions and to understand the challenges that regulatory agencies are confronting. Methods: We followed an analytical approach for analyzing the process generated by the disputes between the financial groups and the pharmaceutical corporations and the challenges created to governmental regulation. We analyzed primary and secondary sources using situational and discourse analyses. We introduced the concepts of biomedicalization and biopedagogy, which allowed us to analyze how medicalization was radicalized. Results: In the 1990s, structural adjustment policies facilitated health reforms that allowed the entrance of multinational financial capital into publicly-financed and employer-based insurance. This model operated in contraposition to the interests of the medical industrial complex, which since the middle of the 1990s had developed silent reforms to regain authority in defining the health-ill-care model. These silent reforms radicalized the medicalization. Some reforms took place through deregulatory processes, such as allowing direct-to-consumer advertisements of prescription drugs in the United States. In other countries different strategies were facilitated by the lack of regulation of other media such as the internet. The pharmaceutical industry also has had a role in changing disease definitions, rebranding others, creating new ones, and pressuring for approval of treatments to be paid by public, employer, and private plans. In recent years in Brazil there has been a substantial increase in the number of judicial claims demanding that public administrations pay for new treatments. Conclusions: We found that the dispute for the hegemony of the health sector between financial and pharmaceutical companies has deeply transformed the sector. Patients converted into consumers are exposed to the biomedicalization of their lives helped by the biopedagogies, which using subtle mechanisms present discourses as if they are objective and created to empower consumers. The analysis of judicialization of health policies in Brazil could help to understand the complexity of the problem and to develop democratic mechanisms to improve the regulation of the health sector. The creation of the health consumer: challenges on health sector regulation after managed care era Celia Iriart1, Tulio Franco2, Emerson E Merhy3 Correspondence: ciriart@salud.unm.edu 1Department of Family and Community Medicine and Robert Wood Johnson Foundation Center for Health Policy, University of New Mexico, MSC09 5060,1 University of New Mexico, Albuquerque, NM 87131-0001, USA Full list of author information is available at the end of the article Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Background These silent reforms radicalized the medicalization, now defined by some authors as biomedicalization [9]. Some reforms took place through deregulatory processes such as weak- ening the regulation regarding direct-to-consumer adver- tisements of prescription drugs in the United States. Others were developed more silently. This was the case of the hidden role, through paid experts, that the phar- maceutical industry has had in changing disease defini- tions, developing clinical guidelines for diagnosing new diseases or overdiagnosing others, and pressuring for approval of treatments to be paid by public systems and employer-based and private insurances. However, the most successful strategies were focused on converting patients into consumers. During the late 1980s and 1990s, pharmaceutical com- panies and health professionals were impacted by the managed care model of containing costs. The increased hegemony of financial capital in the health sector required changes in the way that business was con- ducted. The business model traditionally followed by health providers and the producers of drugs, devices, and equipment depends upon increasing consumption of health services and treatments. The pharmaceutical industry focused on health professionals, especially phy- sicians, to create or increase the demand for its pro- ducts. The financial groups administrating private, public, and employer-sponsored health plans have an opposite model. These companies realize more profits by cutting access to services and treatments, especially the more costly ones. For this reason, managed care organizations developed strategies to control costs using administrative procedures to limit physicians’ prescrip- tions and referrals. At the beginning of the 1990’s, financial groups oper- ating in the health sector introduced explicit and silent reforms following the U.S. model of managed care in several countries in Latin America, Asia, and Europe [2]. By silent reforms we mean changes in rules related to the health sector operation and/or conceptualization that most of the time avoids the legislative process and moreover, the public debate [3]. In Latin America and other developing countries, structural adjustment policies and neoliberal ideology created the context for health reforms that allowed the entrance of multinational financial companies into pub- licly financed programs and employer-based insurance [4,5]. Later, financial capital entered into the manage- ment of health care services: hospitals, home care, long- term care, nursing homes, etc. [7]. In other countries, different strategies are facilitated by the lack of regulation of other media instruments, such as the internet. Background new economic sectors, such as health. At the end of the 1980s and beginning of the 1990s the flows of financial capital into the health sector increased exponentially [1]. This process occurred first in the U.S. and after in many developed and developing countries, introducing new social actors, new rules, and new insurance models that have direct impact on the management and provision of health care services. Insurance companies had operated in the health sector before, but their operations were limited to selling life insurance policies and health insurance plans, mostly to individuals. The radical financialization of the world economy in the 1990s, supported by U.S. poli- cies that deregulated the financial markets, opened the As we demonstrated in our previous study the worldwide domain of financial capital, which has been increasing since the middle of the 1970s, defined the reforms of the health sector in the decades that followed [1-5]. Insurance companies and administrators of mutual and pension funds expanded their business opportunities by not only moving into different countries but by also entering into * Correspondence: ciriart@salud.unm.edu 1Department of Family and Community Medicine and Robert Wood Johnson Foundation Center for Health Policy, University of New Mexico, MSC09 5060,1 University of New Mexico, Albuquerque, NM 87131-0001, USA Full list of author information is available at the end of the article Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 2 of 12 masked most of the times as decisions based on scienti- fic evidence. doors for corporate groups to intensify their operations worldwide using new non-regulated financial tools [6]. From our previous study it was clear that the adminis- trative model implemented by the financial groups con- trolled expenditures limiting access to health services operating in contraposition to the interests of the medical industrial complex. The next step in our research agenda was to understand if the most powerful group of the medical industrial complex, the pharmaceutical industry, has remained quiet while the corporate groups managing financial capital were increasing their shares of the health market. We observed that as a consequence of the mas- sive adoption of managed care in the U.S. and other countries, the pharmaceutical arm of the medical indus- trial complex has developed health reforms, most of them without public debate, to regain market share and authority in defining the health-ill-care model [8]. Methods The study analyzed the ideological mechanisms and the practices developed by financial groups and other central actors in the health sector to change com- mon sense about the conceptualization of health-ill-care, including the idea that patients/users should be trans- formed into consumers/clients [4]. In the light of these processes, new questions arose: will the medical indus- trial complex, and particularly the pharmaceutical cor- porations, counter attack these reforms to regain market power and hegemony in defining the medical model? What kind of strategies will they implement if their tra- ditionally targeted groups (health care providers) were controlled by the strategies of the managed care groups? To respond to these questions we used theoretical concepts and research methods in the Foucaultian meaning of “toolboxes” [10]. This resulted in the utiliza- tion of theoretical concepts and research methods from different authors, and not unique approaches with the pretension to produce completed explanations about the analyzed situation. We looked for concepts with the potential to discover new interpretations and new dis- courses. In this direction, we utilized some concepts that proved their analytical power in other studies that we conducted, such as common sense and silent reforms. We found new ones, especially biomedicaliza- tion and biopedagogies, which we considered have the potential to bring new meanings to the analyzed situation. S l th d l i l h f l f We developed a non-systematic data collection and analysis following the ethnographic method of observing and analyzing data/information to widely describe the situation [12]. Following Clarke, we can define our study as a multisite/multiscape research, in the meaning that we examined multiple kinds of data from a particular situation of inquiry (p. 165) [11]. We utilize data from different sources, such as articles, news, or other data from list servers, materials researched for our teaching and other research activities, as well as, data and new insights of the situation gather by participating in national and international forums related to health sec- tor reforms. The initial materials gathered opened other pathways to obtain additional data, such as references to articles, names of companies, and individual and collec- tive social actors involved in the situation, and descrip- tion of regulations, among others. In addition, we cannot deny that our personal experiences receiving information as consumers from pharmaceuticals were also an important source of data and inquiry. Background Disease campaign awareness and funding of medical scientific and educational activities are also used to hide the promotion of drugs. In recent years we have observed in Brazil a substantial increase in the number of judicial claims demanding that private insur- ance and public administrations pay for non-approved treatments, including the experimental ones. This could be a strategy to regain market power in countries with health care systems led by public administrations, not driven by the market. These strategies could have ser- ious consequences for the regulatory process of the health sector. In the health sector the massive entrance of financial capital changed not only the modus operandi at the eco- nomic level but also the common sense regarding the ideas about health-ill-care. By common sense we refer to core ideas that underlie discourses on health in a spe- cific time and society. Common sense is shared meaning that provide direction for society, and act as social cement that fills gaps and artificially softens social con- tradictions. Common sense shapes the subjective assess- ment of a shared situation by people in different places in the social structure (p. 55) [3]. Cost containment, individual responsibility, cost-effectiveness, case manage- ment of patients, coordination of care, etc. are technical concepts that penetrated the sector and deeply trans- formed the conceptualization of health-ill-care. Profes- sional decisions were subordinated to administrative procedures which focused on maximizing profits The article will first present the strategies utilized by the pharmaceutical industry to regain market leadership in the United States. We consider it important to ana- lyze the changes operating in this country because the U.S. medical model influences the conceptualization and practice of medicine in the rest of the world, especially in developing countries. Second, we will analyze some contributions from the social sciences that allow a better comprehension of the phenomenon of the creation of Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 3 of 12 the health consumer as part of the biomedicalization led by the medical industrial complex. Finally, we will intro- duce the research that our team is conducting in Brazil analyzing if the judicial mandates obligating the public health system to financially cover new treatments are part of the strategies of the pharmaceutical industry to create the health consumer and the impact that the mandates have on the regulation of the health sector. Background answers, but to reinterpret some strategies by connect- ing different processes that appear disconnected in the discourses elaborated by the two most powerful groups operating in the health sector -the medical industrial complex and the financial groups. We understand power as multiple forces operating in a specific situation and time. Power is related to the concept of governabil- ity in the meaning of forces operating strategically to structure new subjectivities [12]. Methods The fundamental idea was to understand what the pharmaceutical industry was doing to counter attack the advances of the financial groups. To initiate the research process we followed Rabinow’s idea of utilizing a tradi- tional ethnographic method “to describe what is going on” (p. 236) [12]. As a first step, we defined the macro situation as the intercapitalistic disputes between the medical industrial complex and the financial groups operating in the health sector for increasing the share of the market and defining the medical model. At meso level we were interested in understanding if pharmaceu- tical groups were developing strategies to change regula- tions to impact the organization of the health sector. At micro level we wanted to learn if the pharmaceutical industry was also creating some mechanisms to recap- ture the supply side of the health care sector equation and/or developing mechanisms to capture the demand side (patients/users). The study followed an analytical approach that reinter- prets studies developed by other authors regarding the reforms that the pharmaceutical industry has developed during the past 20 years. The results of our study about the managed care reforms provided the knowledge that the financial groups developed strategies to control pro- viders of health care services in order to decrease expen- ditures. The study analyzed the ideological mechanisms and the practices developed by financial groups and other central actors in the health sector to change com- mon sense about the conceptualization of health-ill-care, including the idea that patients/users should be trans- formed into consumers/clients [4]. In the light of these processes, new questions arose: will the medical indus- trial complex, and particularly the pharmaceutical cor- porations, counter attack these reforms to regain market power and hegemony in defining the medical model? What kind of strategies will they implement if their tra- ditionally targeted groups (health care providers) were controlled by the strategies of the managed care groups? The study followed an analytical approach that reinter- prets studies developed by other authors regarding the reforms that the pharmaceutical industry has developed during the past 20 years. The results of our study about the managed care reforms provided the knowledge that the financial groups developed strategies to control pro- viders of health care services in order to decrease expen- ditures. Methods As researchers we are part of the situation as subjects that produce discourses about the situation, but also sub- jected to discourses that others produce [13]. To respond to these questions we used theoretical concepts and research methods in the Foucaultian meaning of “toolboxes” [10]. This resulted in the utiliza- tion of theoretical concepts and research methods from different authors, and not unique approaches with the pretension to produce completed explanations about the analyzed situation. We looked for concepts with the potential to discover new interpretations and new dis- courses. In this direction, we utilized some concepts that proved their analytical power in other studies that we conducted, such as common sense and silent reforms. We found new ones, especially biomedicaliza- tion and biopedagogies, which we considered have the potential to bring new meanings to the analyzed situation. Several methodological approaches were useful for creating a dialog between different sources of data. We will mention two as the most important to our study: a) situational analysis that provides elements to map differ- ent discourses and, b) discourse analysis which we used as a deconstructive reading and interpretation of power relationships to gain a comprehensive understanding of the effects of these dynamics in creating new subjectiv- ities [10,11]. We are not trying to provide unequivocal The extensive preliminary analysis allowed us to notice that pharmaceuticals were turning to their favor the strategy initiated by managed care organizations of transforming patients/users into clients/consumers. Page 4 of 12 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 From the preliminary analyses we also understood that the pharmaceutical industry was taking part in a silent process of health reforms to regain market power that impact different levels: policy, economic, ideological, and social. In addition, the team was interested in understanding how the process operates at macro, meso, and micro levels. The separation into these levels had only methodological purposes; the process is intri- cate and operates most of the times simultaneously at all levels. We decided that the line of analysis that will allow a wide and deep comprehension of the process will be through the utilization of analytical categories that capture the reforms that pharmaceutical companies were involved in and benefit from. g Changes in regulations Using these categories, we developed a systematic lit- erature and document review to find the data (primary and secondary) that describes changes in regulations and scientific norms, and in business models consistent with the creation of health consumers. With the docu- ments identified from different sources (academic arti- cles, books, brochures, websites of professional, patient and consumer associations, adds and news in television, magazines, and newspapers, among others), we initiated a process of mapping the information obtained in each category from different sources to describe the situation and the results of the changes in creating the health consumer and radicalizing the medicalization. We fol- lowed the procedures described by Clarke and we recommend her book to learn about the operational steps that we will not present here because an in-depth explanation of these complex methods merit a separate article [11]. As a first step in converting patients/users into clients/ consumers, the pharmaceutical industry required some changes at the regulatory level. Several laws and modifi- cations of existing regulations were approved in the U.S. that facilitated the transformation of the business model of pharmaceutical companies. For this article we will consider two important regulatory changes, one that facilitated the increase of the number of pharmaceutical products in the market, and another that enhanced the ability of the companies to offer the products to consu- mers. The first one is the act that introduced user fees to be paid by pharmaceutical companies to the Food and Drug Administration (FDA) to initiate the approval process for new drugs. The second one is related to the changes introduced on the regulation of the direct-to- consumer advertisements. Introducing user fees for drug approvals In 1992, the U.S. Congress approved the Prescription Drug User Fee Act (PDUFA) with relatively little public debate [14]. The Act allowed pharmaceutical companies to pay fees to speed the approval process and the FDA the ability to use these fees to finance the specific areas. After the Act was launched, new approvals were being issued in unprecedented numbers and review times were at his- toric lows. Important new drugs for cancer, AIDS, heart diseases, and stroke were approved using this process, benefiting many patients. But also numerous new drugs for chronic diseases, mental health and lifestyle condi- tions, some of them not very useful or really new and sometimes dangerous, were approved, opening a new dimension for the pharmaceutical business. Methods The broad categories that we defined were changes in regulations, changes in defining, branding and overdiagnosing diseases, and mechanisms utilized to capture the demand side. Com- plementing this process, part of our team started a lit- erature review to describe the increasing involvement of the judicial branch in the regulatory process in the health sector in Brazil. study of the hidden processes and social actors that could be behind the judicialization of the health sector in countries were health is considered a common good not a commodity. The medical industrial complex: strategies to regain market leadership To revitalize its role in the health sector the pharmaceu- tical industry needed to find new strategies [14]. The strategies were selected to create a new disease/ill/care model, make drugs for healthy people, and to exploit the idea developed by managed care organizations to convert users/patients into clients/consumers [15]. In order to accomplish these goals, the industry has been successfully lobbying for regulatory changes and in developing strategies to change the common sense related to the conceptualization of health-ill-care. We will analyze some of these changes and strategies in the following sections. g Changes in regulations According to Angell, in 2002 the FDA approved 78 drugs. From those only 17 contained new active ingredients and just The result of the process was the analytical descrip- tion of selected strategies implemented by the pharma- ceutical industry and their reinterpretation applying the theoretical concepts of biomedicalization and biopeda- gogies. This reinterpretation allows us to increase the understanding of the effects that the discourses cur- rently modeling the health sector have in creating new subjectivities and biosocialities, some of them as part of the hegemonic discourse and others questioning it. The study contributes to the field of health policy by highlighting how silent reforms introduced first by financial companies and after by the medical industrial complex, are challenging the regulation of the health sector. The described analytical approach allowed us to reinterpret the situation and show the need for further Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 5 of 12 limiting the power of the promotion by scaring the pub- lic. In 1997, the FDA rolled back this requirement and it opened the way for a new era in DTCA [16]. Compa- nies increased exponentially the amount of DTCA. In 2001, the big companies spent $1.8 billion in DTCA, but in 2003 they spent $3 billion [14]. The regulatory capacity of the agency is almost null to control this increased amount of advertisements, especially if we consider that in 2001 drug companies also substantially increased the ads in newspapers and magazines, exceed- ing largely the number of ads in medical journals for that year. Drug companies are required by law to have their DTCA reviewed by the FDA when they launch a new campaign. However in 2001, the agency had only 30 reviewers to control 34,000 DTCA. In addition, the agency cannot verify whether the companies submit all the ads [19]. seven were classified by the agency as improvements over existing drugs (p. 16-17) [16]. The speedy process established in the U.S. determined that many pharma- ceutical companies chose this country for the introduc- tion of new drugs into the market more than any other country in the world. The approval of this Act has also had consequences in other countries. g Changes in regulations The most important consequence was that in 1990s the World Bank (WB) and the Inter- American Development Bank (IADB) promoted, in sev- eral Latin American countries, the creation of regulatory agencies following the FDA model, especially the user fee provision. The reasoning of the WB and IADB was that the user fees will counter the fiscal deficit by gener- ating revenues to finance the new governmental agen- cies. Additionally, according to these international lending agencies, the efficiency of the bureaucratic pro- cess of drug approval will be improved. Argentina (1993) and Brazil (1999) created the agencies to regulate drugs, foods, and medical technologies following the FDA model of charging pharmaceutical companies fees to initiate an approval process [17,18]. Most of the DTCA are for chronic and lifestyle condi- tions, as well as behavioral/mental health problems. The ads will train consumers about signs and symptoms, as well as brand name medications, and direct them to consult with their physicians. Perhaps, it would be more appropriate to say, to convince their physicians about the diagnosis and treatment. These advertisements are now the biggest investment of pharmaceutical compa- nies in marketing and include ads in television, radio, magazines, internet, and mail advertisements, among others. But more than providing useful information to patients for specific health problems, the ads market the diseases/conditions (old, new, and rebranded) for which the pharmaceuticals have drugs to sell. The idea, pro- moted by the new pharmaceutical paradigm, is not just to sell pills to cure diseases, but to create a state of fear of becoming sick, aging, and dying [15,20]. Changing the rules for advertising drugs The new reg- ulation speeding the approval process increased the amount of pharmaceutical products in the market, but additional strategies were required to increase the demand of the products. The capture of the supply side (providers) of the health care equation developed by managed care organizations, mobilized pharmaceutical companies to develop strategies to capture the demand side. The industry envisioned the idea to convert patients into consumers with the right to be informed about pharmaceutical products as they have the right to receive information about other goods and services [14]. In other countries, pharmaceutical companies are pressuring regulators to allow DTCA for prescription drugs and to modify the classification of some drugs to be sold over-the-counter. g Changes in regulations While drug companies are waiting for regulation changes, they are using other stra- tegies to reach consumers, such as media disease aware- ness campaigns, promotion of drugs presented as news in television and other media, and developing health teaching materials for schools, among others. The cen- tral idea is to increase the capacity of consumers to self- diagnose and self-prescribe [21,22]. For years medical associations and also pharmaceutical executives opposed direct-to-consumer advertisements (DTCA) of prescription drugs, arguing that it will inter- fere in the physician-patient relationship. But during the 1980s the increasing power of consumer organizations created the possibility of advertisements as a way to empower patients [14]. This idea was captured by the managed care industry to promote the concept that an informed patient could make rational decisions about their health needs and not be manipulated by prescribers. Using this concept, innovators within the pharmaceutical industry convinced those who initially questioned that the DTCA of prescription drugs was a way to empower patients. Also, they promoted the idea that the DTCA will create more awareness about diseases and conditions not well known, but which were silently affecting huge number of people. Changing health definitions, branding diseases and overdiagnosing others Since the middle 1990s, pharmaceutical companies have had more influence within the expert committees reviewing disease/condition definitions and describing new diseases and health risks to be covered by private, employer-based, and public insurance plans [16]. The majority of these changes are defined by medical specia- lists in committees organized by official agencies, DTCA already was approved and regulated by the FDA, but the requirements were that the advertisements must present the entire list of side effects and cautions, Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 6 of 12 professional organizations, and international institutions, like the World Health Organization (WHO). Most of the time, the decisions of these bodies of experts, pre- sented as based on scientific research, are adopted worldwide. disorder to obsessive-compulsive disorder to pre- menstrual dysphoric disorder. And ‘lifestyle market- ing’ has now extended to the promotion of many blockbuster ‘maintenance drugs’ intended for daily, lifelong consumption, such as drugs for allergies, insomnia, and acid reflux disease” (p. e189) [25]. In middle 1990s, committees of experts redefined sev- eral diseases/conditions and health risks. These commit- tees decreased the parameters for high blood pressure and defined prehypertension; decreased the level of fast- ing glucose to define diabetes mellitus and of serum cholesterol for hypercholesterolemia; also experts decreased the Body Mass Index values for being over- weight and obese [16,23]. In the U.S., these changes resulted in the following increases in potential cases: 14% for diabetes; 35% for hypertension; 86% for hypercholesterolemia, and 42% for overweight. This opened the possibility for 140,630,000 additional indivi- duals to be diagnosed with some of these diseases/con- ditions and treated with drugs and other medical interventions [23]. Previously, acid reflux was known simply as heartburn and treated with a glass of milk or an over-the-counter antiacid. In the 1990s in the U.S., Glaxo began promot- ing one of its drugs to treat heartburn under a new name, GERD (gastroesophageal reflux disease), and described it as having serious health consequences if not treated (p. 86) [16]. Social phobia disorder, which is a very rare condition, was renamed social anxiety disorder by SmithKline just after September 11, 2001. The company launched an ambitious campaign promoting its antidepressant Paxil for this use. The commercial showed images of the World Trade Center towers collapsing (p. 88) [16]. Changing health definitions, branding diseases and overdiagnosing others Cor- porations are branding social, cultural, and political situations as diseases, defining them as conditions that could be medicated. The list is long and includes atten- tion deficit disorder, not only in children but now also in adults, depression, erectile dysfunction, female sexual dysfunction, bipolar disorder, restless legs, social anxiety, and panic attack, among many others. This is a dangerous approach that implies moving the bell curve of a population to the left in order to capture more people with low risk to prevent them from devel- oping the disease/condition. While this approach may be useful for preventing exposure to hazards, such as tobacco or contaminants, to use this approach for iden- tifying people at risk for conditions that require medica- tions creates a potentially dangerous situation exposing healthy people to adverse effects of drugs [24]. The idea of health promotion has been deeply medicalized because healthy behaviors are now related to the capa- city of individuals to control their risk of becoming sick. The social, economic, and political factors that create health risks and diseases are denied under the renewed logic of a positivist science. This logic reinstalled biolo- gical factors as the central causes for diseases, and medi- cal procedures and individual behavior changes as the only way to cure or alleviate them. In addition to their promotional efforts to create pub- lic awareness of new and old diseases/conditions, phar- maceutical companies create short questionnaires to test the risk for diseases/conditions. These questionnaires are extensively utilized in primary care settings by physi- cians, physician assistants, and nurse practitioners, espe- cially for diagnosing mental health problems for which they are not very well trained to diagnose. Using these tools, non-specialized professionals feel confident about their diagnoses and their capacity to medicate adults and children with psychiatric drugs. The strategies implemented by pharmaceutical companies successfully expanded the market through increasing the amount of professionals diagnosing and prescribing. This strategy, together with the increased investments in directed-to- consumer marketing generated the results for which they were developed. The top 25 drugs directly mar- keted to consumers in the U.S. rose by 34% from 1998 to 1999, while other prescriptions rose only 5.1% [9]. The described changes in definitions of diseases/con- ditions were not the only targets of pharmaceutical companies. These companies moved from promoting drugs to treating diseases, to promoting diseases/condi- tions to fit their drugs. Changing health definitions, branding diseases and overdiagnosing others During the 1980s and 1990s companies in the U.S. started promoting lifestyle drugs, including those for cosmetic and sexual enhancement. The crossover to curative medicine occurred with psy- chotropic drugs that have a wide range of active proper- ties, allowing pharmaceutical companies to expand the spectrum of diseases/conditions for which the drugs could be promoted. As Applbaum explains, Capturing the demand side The questionnaires contain a few general questions about common signs and symp- toms. Depending on the amount of positive answers, the respondent is recommended to consult with a doctor. Despite the fact that the test may conclude that a per- son is not at risk, the recommendation is that he/she should learn more about the disease/condition because the questionnaire may not have evaluated all risks [28]. g p The strategy proved to be successful for the pharma- ceuticals. Patient and consumer organizations were powerful allies for pressuring the Congress for regulatory changes needed by the pharmaceutical industry [14]. Patient associations have demonstrated to be good allies in pressuring for approvals of new drugs and for classify- ing new diseases and conditions to become recognized, and consequently, to be reimbursed by private and employer-based health plans and public programs. Patient testimonies have been very useful in convincing the FDA scientific reviewers to approve new drugs, despite the fact that in some cases serious concerns about drug safety existed. Also these groups have been very instrumental in pressuring reticent physicians to prescribe drugs for off-label uses. In the U.S., pharmaceu- tical companies cannot promote off-label use of their products but can inform physicians about them. Patients, well trained by the industry, are the best way to create the demand for these off-label treatments [16]. Patient advocacy organizations that offer lists of physicians to patients and also recommend treatments become power- ful allies of the pharmaceutical industry [14]. Some pharmaceutical companies fund cable television channels dedicated solely to provide health information. Special programs and the technology to watch them are offered free to hospitals and have been installed in patient areas. The programs combine specific informa- tion with ads and logos of the company that sponsor them [16]. Moreover, health news segments and health information on television are also new ways to inform consumers about research findings or to create awareness of diseases/conditions and the availability of treatments. Pharmaceutical companies produce this information that is then offered free of charge to the media [29]. Awareness campaigns and health fairs in school and community settings also are tools that the companies have utilized since the middle of 1990s. In 1994, Eli Lily sponsored a National Depression Awareness Day in a Maryland high school, reaching 1,300 students [14]. Other companies followed this initiative on college cam- puses. Capturing the demand side Another strategy that pharmaceutical companies uti- lized, initially in the U.S., to regain market positions was to support patients and consumer associations. The pharmaceutical groups learned from the women’s health movements and AIDS patient groups how valuable advocates could be for demanding increases for research funds, the approval of new drugs, and the coverage of “one class of antidepressants, the specific serotonin reuptake inhibitors, is marketed for eight distinct psychiatric conditions ranging from social anxiety Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 7 of 12 new treatments by public programs, and employer-based and private health plans [9]. have helped them to live successful lives [16,26]. Health fairs in community settings also offer a good opportu- nity to expand the number of potential consumers of pharmaceutical products. Companies offer free tests for cholesterol, diabetes, high blood pressure, etc. and infor- mation about these diseases/conditions. Another inter- esting way that pharmaceutical companies use to create awareness of diseases and treatments is to post commu- nication tools on their websites to facilitate the “dialo- gue between physicians and patients.” Some tools are for patients and others for physicians [27]. Pharmaceutical companies targeted patient and consu- mer organizations offering funds to help them to create awareness of diseases/conditions in the general public and among policymakers [14]. Education was the new strategy to a) pressure health plans to pay for costly drugs, especially new drugs or off-label indications of approved ones, most of the time with not much more benefit than the older ones but more expensive; b) dis- credit some treatments, such as psychotherapies, as costly and not as efficacious as new drugs for treating mental health disorders; and c) convince health plans about the improvement outcomes of some drugs beyond the specific indications, e.g. a drug like Fosomax was not only presented as a bone density builder but as a breakages preventer [15]. Pharmaceutical companies create new tools to facili- tate the identification of potential consumers of their drugs. Short questionnaires to test the risk for diseases/ conditions are available in magazines, as well as at web- sites of pharmaceutical companies, patient associations, and associations for specific diseases (e.g. American Dia- betes Association) [26,28]. Moreover, pharmaceutical companies also send questionnaires by mail to potential consumers with information about specific drugs. Addi- tionally, these questionnaires are available in primary care settings and schools. Capturing the demand side The awareness campaigns commonly go a step further by promoting the use of the drugs that the spon- sor makes. More importantly, they market the condition, teaching about very general signs and symptoms that mobilize people to request medical help [16]. For their awareness campaigns, as well as for television shows, ads, and other types of promotions, the companies also contract celebrities to talk to the public about their struggles against some diseases and how medications Other companies created subsidiaries to develop dis- ease management services for managed care organiza- tions and big employers that manage their own health insurance plans, such as Ford and General Motors [14]. The service consists in providing the health plan mem- bers with state-of-the-art information about disease/con- ditions and how to maintain medication compliance. The websites also offer tools to engage patients on healthy behaviors, such as exercise and nutrition. The sites provide people with health toolboxes that allow them to register doctor appointments, medication times, and medication renewal dates. The websites would then prompt patients to comply with their treatments and Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 8 of 12 transformation. Advances in biomedical technosciences, such as molecular biology, genomization, medical diag- nostic and treatment technologies, as well as computer and communicational developments created the possibi- lities for radicalizing the medicalization. Biomedicaliza- tion implies a “shift from enhanced control over external nature (i.e. the world around us) to the harnes- sing and transformation of the internal nature (i.e. biolo- gical processes of human and non-human life forms), often transforming life itself” (p. 164) [9]. Biotechnolo- gies, including drugs and other devices available to patients/consumers, create new biomedicalized subjec- tivities, identities, and biosocialities. New forms of social relationships are constructed around and through such identities (p. 165) [9]. Examples of these kind of new forms of social relationships are the social networks using websites, blogs, and other internet forums dedi- cated to health issues. It is important to remark that these new identities do not always imply the acceptance of the biomedicalized discourses and practices: some of these forum/groups are questioning the moral mandate and other forms of biomedicalization [33]. The process is not unidirectional and different discourses are created by a multiplicity of individuals and organizations. New organizational developments and regulatory measures could transform the current situation. Capturing the demand side check other biological and behavioral indicators (blood pressure, weight, nutrition, physical activities, among others), and to enter the data to monitor improvements. These databases provide manager companies a large quantity of information about demographic profiles of how certain patients are prescribed, use medications, and other health data. Companies are able to perform sophisticated data mining to understand patient and physician behavior in regards to a specific health pro- blem or condition and consequently develop more pre- cise promotion of their drugs. For Merck, a pioneering company in developing this type of information manage- ment capabilities, sales increased 23% in 2000. The sub- sidiary created by Merck, Medco, managed the pharmaceutical benefits of 51 million Americans in this year [14]. In the following years, free and paid websites of this kind flourished, especially for chronic diseases, lifestyle conditions, and mental health disorders. Subscriptions to these websites opened the doors for multiple compa- nies and organizations (not only pharmaceuticals) to send information about diseases/conditions, and to pro- mote drugs, diet products, medical procedures, etc. The worldwide capability of these tools allows companies to operate beyond the geographical boundaries and avoid regulations in place in specific countries. The transformation of medicalization into biomedicali- zation required the confluence of several processes in which the financialization of the health sector had an important role. This process increased exponentially the corporatization of the sector and the commodification of their products at different levels: provision, research, and education. These changes mobilized others stake- holders such as the pharmaceutical industry to develop new strategies. Some of these strategies, as we analyzed previously, included enhancing the direct relationship with potential clients of health products (diagnostic pro- cedures and treatments), as well as health promotion and prevention programs and services. Biomedicalizing life to regain the hegemony of the health sector In previous sections we analyzed the strategies utilized by the pharmaceutical industry to regain market posi- tions and leadership in defining the health-ill-care pro- cess in the U.S. From the U.S., multinational companies export, through their subsidiaries in other countries, to the rest of the world their new business models and strategies to regain market power. Models and strategies that the companies modify accordingly with the health sector organization and the regulatory environment in each country. Next we will consider some concepts from social sciences theories to better understanding the deep implications of these strategies for the lives of people. The technoscientific medicine and its subfields, such as public health, have developed illusory discourses in which death not only could be postponed, but also pre- vented [20]. The dream of being eternally young with plenty of energy penetrated all social, gender and age groups. In order to obtain this goal, the message is that people should exercise strict control and surveillance over the risks that could threaten their lives. Moreover, the messages about health-ill-care are presented as social/moral mandates, meaning that if individuals are not proactively controlling their health, the results of their behaviors are at a great cost to society. This goes beyond using specific medical interventions to recover health from illnesses or diseases; it supposes the biome- dicalization of health promotion and prevention, Medicalization is a concept developed in the 1970s to define aspects of life as medical problems previously outside of the jurisdiction of medicine [30-32]. Profound transformations in medicine, facilitated by advances in technosciences, started in the 1980s. To understand these transformations Clarke et al. developed the con- cept of biomedicalization, based on Foucault’s theory of biopower and later developments by Rainbow [9]. While medicalization focused on illnesses, rehabilitation, and care; biomedicalization focuses on health as an interna- lized moral mandate of self-control, surveillance, and Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 9 of 12 Individuals are offered a number of ways to under- stand and change their behaviors, as well as encouraged to take action to educate other members of their families and communities to have healthy lives. Most of the pedagogical tools are created to govern bodies and to provide the social meanings by which individuals come to know themselves and others but not the social- political environment. Biomedicalizing life to regain the hegemony of the health sector Moreover the scientific truths are recontextualized in different social and cultural sites to inform and persuade people on how they should under- stand their bodies and how to live their lives. In this light, health information is developed to facilitate the incorporation of the “outside” world (the social and eco- nomic wellbeing of others) into the “inside” (psyche and body) of the individual (p 49) [36]. requiring the internalization of the social mandate of being healthy, and of surveillance practices at the indivi- dual level. To be healthy in the context of biomedicalization implies that it is each individual’s responsibility to test for diseases/conditions, and to utilize drugs, devices, and other technomedical products and services to control the risk of developing diseases or aggravating a condi- tion. The services to accomplish the social/moral man- date include internet tools and other communicational mechanisms that introduce information through the intimacy of personal computer, home entertainment devices, as well as school and other small environments. Individuals are taught about diseases/conditions, how to test for them, and how to access services and products to preserve their health. In principle more access to medical and health knowl- edge and information could be considered democratic and it needs to be welcomed. However, in practice we should critically analyze how the data is created, by whom, and what are the interests behind this information market. In addition, the commodification of this process in capitalist societies implies that the access to informa- tion is stratified, non-democratic, and differentially affecting social groups and countries. People from the lower classes receive messages reinforcing the social/ moral mandate to control their health in order that they do not become a burden to society. For these populations the biopedagogies are implemented at schools, health fairs, community events, and media (especially televi- sion). Well intentioned professionals working for public health agencies, schools, and non-governmental organi- zations also reproduce these messages and biopedagogies without understanding how they operate and the conse- quences for the biomedicalization of life. People will use their limited income in testing for glucose, cholesterol, high blood pressure, and other conditions and accessing treatments (conventional or alternative) to control these risks. However, as we can observe in health statistics, dis- advantaged groups will fail to reach the healthy outcomes of the upper classes, instead they will be left with the guilt of not eating healthy, exercising, and having non- stressful lives. Biomedicalizing life to regain the hegemony of the health sector The messages hide that most of the health problems of these groups are not caused by their bad genes triggered by inadequate lifestyle habits, but by the unequal distribution of wealth. p In the context of biomedicalization health professionals, in their traditional role as leaders of the process of cure or alleviation of disease, are more and more dispensable for the new business model developed by the pharmaceutical industry. As we described previously the advances in com- puterization and data banking facilitate the capture, sto- rage, and analysis of enormous amounts of data from individuals. All of this information is utilized to generate messages to reach millions of people interested in health issues. The communication strategies can be developed considering differences in social classes, ages, genders, nationalities, cultures/ethnicities, diseases/conditions/risks, and so forth. There are numerous products and services that the pharmaceutical industry promotes under the con- cept of “educating people” to prevent the risks of becom- ing ill that do not require health provider mediation for their consumption. Also, we have described previously that pharmaceutical companies developed tools to teach patients how to direct their physicians and other health professionals to prescribe the desired product. Young peo- ple are especially vulnerable to these kinds of marketing messages that promote self-diagnosed and self-prescribed behaviors [21,22]. The concept of biopedagogy is also useful to comple- ment the understanding of the analyzed phenomena. This concept, drawing from Foucault’s biopower theory, is described by Wright as the normalizing and regulating practices traditionally reserved to schools, but currently appropriated by other learning and communicational spaces, and disseminated more widely through the web and other forms of media [34]. Biopedagogies place indi- viduals under constant surveillance and towards increased self-monitoring by elevating their knowledge around diseases/conditions, as well as learning how to be healthy. Using Berenstein, Wright states that we are now living in “totally pedagogized societies” where methods to evaluate, monitor and survey the body are encouraged across a range of cultural practices (p.8) [35]. Judicialization of health policies in Brazil: a new path on the creation of the health consumer? In addition, if the treat- ments the judicial system mandates the UHS to pay are predominantly those offered by private providers, the public system is subsidizing them, when public hospitals and clinics are confronting increasing budgetary restric- tions. In fact, the judicial claims appear to benefit more the middle and upper classes. A study analyzing 2,927 judicial claims demanding the state of São Paulo to pay for medications not covered by the UHS, shows that 73% of claimants reported that they live in high and middle income neighborhoods, while only 27% live in low income areas. Moreover, 74% of the cases were represented by private lawyers while only 26% were represented by public defenders [39]. Most of the health care services under the Brazilian Unified Health System (UHS) are offered through an extensive network of public primary care clinics and hospitals, as well as public health programs managed by municipalities. Brazilian citizens also have the right to receive health care services, paid with public funds, through private providers, if the public system does not offer the needed services. In part, because this private- public arrangement to guarantee the constitutional right to universal access to health, the public system is confronting increasing costs not matched by sufficient budget allocations. This obligates governmental adminis- trators to deny access to some health care services and put limitations on the kind of services that can be pro- vided. In particular, these limitations affect specialized and costly treatments, most of which are offered by the private sector. By appealing to the constitutional right, an increased number of individuals are interposing judi- cial claims demanding that the public system covers health care services that their physicians recommend to them. These are the nature of the problems that our team is starting to investigate in Brazil. Our empirical work will focus on understanding the implications for regulatory agencies of the judicialization of the health sector, as well as the role that the medical industrial complex plays in this process. We are also studying if the Brazi- lian pharmaceutical industry is adapting the strategies observed in the U.S. which turns users/patients into consumers by marketing new treatments, e.g., those approved but not covered by the public system, off-label indications, or not approved at all by the regulatory agencies in Brazil. Judicialization of health policies in Brazil: a new path on the creation of the health consumer? In this section, we will introduce the research that we are initiating in Brazil to investigate if the processes of capturing the demand side are operating through the judicialization of health policies and how they are affect- ing the capacity of the governmental administrations to regulate the health sector. Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 10 of 12 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 10 of 12 The importance of understanding if the judicialization of the health policies in Brazil is another strategy that the pharmaceutical industry is using to regain market power resides in two important facts: 1) in this country health is considered a common good not a private one; and 2) the governmental agencies led the regulatory process in a public-private environment of health care service provision. Brazil approved in 1988 a constitu- tional amendment declaring that health is a right that the state must guarantee and that each Brazilian has the right to universal and integral access to health care. The constitutional nature of the right to health opens the option for citizens to utilize the judicial branch to demand the fulfillment of their rights when they believe that the constitutional mandate is not followed. public system is in accordance with the constitutional rule to guarantee the health right, but it creates a conflict between the health and the judicial systems. Officials from health agencies consider that the judicial branch is assum- ing authority for decisions that need to be made by health specialists. They complain especially when the solicited medications, treatments, or diagnostic procedures are new, experimental, or off-label indications, implying that the efficacy and safety are not well established. The judicial branch is playing a more central role in defining the health policies by focusing only on the con- stitutional right. But this branch of the government may not be considering the powerful actors that could be using the justice system to mandate the inclusion of new treatments to be paid by the UHS. Moreover, increasing the number of services provided with limited funding allocation may threaten the constitutional right itself. Fewer people may be accessing basic needed ser- vices, favoring only small numbers of people that receive costly specialized treatments. Judicialization of health policies in Brazil: a new path on the creation of the health consumer? In situations like Brazil where health is a right guaranteed by the constitution, unneeded con- sumerism of health services promoted by the medical industrial complex may be seriously threatening the sta- bility and continuity of the public health care system, as well as increasing health inequities. We are only at the beginning of this research agenda that will provide important information to improve the regulation of the health sector and demonstrate the need for a close dia- logue among branches of the government. Data from the Superior Tribunal of Justice in Brazil indicate that in 2001 only two claims related to health issues were interposed. However, by 2004 the number of cases increased to 672 [37]. The Health Secretary of Rio de Janeiro started recording the numbers of judicial claims in 1991. Since then and until 1999, the number of cases slowly increased, especially in relation to some diseases. Starting in 2000, the number of cases requiring the Justice to obligate the state to pay for procedures to treat different health problems denied by public medical institutions increased exponentially. At the end of 2002, the Health Secretary counted 2,733 judicial actions against the State of Rio de Janeiro. In March 2006 the number of claims rose to 7,758 [38]. In the majority of cases, the judges ruled in favor of the citizens, obligating the public health system to pay for the demanded services [39]. To include more medications, diagnostic procedures, and treatments to be paid by the Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Iriart et al. Globalization and Health 2011, 7:2 http://www.globalizationandhealth.com/content/7/1/2 Page 11 of 12 Page 11 of 12 generated by the organization of societies. The analysis of judicialization of health policies, as we are observing in Brazil, could help to understand the extension and complexity of the problem and help to create a dialogue between professionals working in health and judicial sys- tems. Public institutions should generate and distribute scientific, non-biased health information and alert the population about the biased websites and other sources of data gathering and information spreading that are paid by private groups to pursue their commercial inter- ests. The regulation of this type of communication and the means to transmit the messages should be strength- ened, but it will require a profound and broad debate within each country. Regulating information is a delicate matter. Acknowledgements A fi i f hi Acknowledgements A first version of this article was presented in July 3, 2010 at the “Symposium on Health Systems, Health Economics and Globalization: Social Science Perspectives,” London U.K., organized by the Economic and Social Research Council; LSE Health; King’s College of London; and JNU. We are grateful for the comments of the discussant Professor Sudip Chaudhuri. CI was supported by the Robert Wood Johnson Foundation Center for Health Policy at the University of New Mexico, USA. The Writing Studio at the College of Education, University of New Mexico, provided invaluable support in editing the manuscript, our special recognition to Dr. Rebel Palm and Ms. Natasha Howard. We also want to thank the two anonymous reviewers for their suggestions and comments. Only the authors are responsible for the final product Acknowledgements A first version of this article was presented in July 3, 2010 at the “Symposium on Health Systems, Health Economics and Globalization: Social Science Perspectives,” London U.K., organized by the Economic and Social Research Council; LSE Health; King’s College of London; and JNU. We are grateful for the comments of the discussant Professor Sudip Chaudhuri. CI was supported by the Robert Wood Johnson Foundation Center for Health Policy at the University of New Mexico, USA. The Writing Studio at the College of Education, University of New Mexico, provided invaluable support in editing the manuscript, our special recognition to Dr. Rebel Palm and Ms. Natasha Howard. We also want to thank the two anonymous reviewers for their suggestions and comments. Only the authors are responsible for the final product A first version of this article was presented in July 3, 2010 at the “Symposium on Health Systems, Health Economics and Globalization: Social Science Perspectives,” London U.K., organized by the Economic and Social Research Council; LSE Health; King’s College of London; and JNU. We are grateful for the comments of the discussant Professor Sudip Chaudhuri. CI was supported by the Robert Wood Johnson Foundation Center for Health Policy at the University of New Mexico, USA. We recognize that the medical model dominated by the technosciences is not the only one operating around the world. Other models, more centered on the needs of the patients than on the consumption of technical pro- cedures, are also operating and disputing the conceptua- lization of the health-ill-care model. Judicialization of health policies in Brazil: a new path on the creation of the health consumer? Regulation could be a good instrument to pro- tect the people from spurious commercial interests, but also it could be used to decrease the voices that oppose the biomedicalization. Councils integrated with mem- bers from community groups, professional associations, health advocate organizations, among others, could be a tool to advise and control governmental regulatory agencies. Professional and consumer associations, pub- licly funded and supervised, could increase the amount of non-biased information. All efforts need to have the objective to increase the visibility of discourses defend- ing the individual and collective right to live healthy lives non-governed by self-surveillance and consumption of bioproducts, but more connected with the need to collectively improve working and living conditions. Author details 1 1Department of Family and Community Medicine and Robert Wood Johnson Foundation Center for Health Policy, University of New Mexico, MSC09 5060,1 University of New Mexico, Albuquerque, NM 87131-0001, USA. 2Departamento de Planejamento em Saúde, Instituto de Saúde da Comunidade, Universidade Federal University Fluminense, Av. Marquês de Paraná, 303-Anexo, 4o. Andar, Centro Niterói, RJ, CEP: 24030-210, Brasil. 3Instituto de Psiquiatria, Programa de Psiquiatria, Universidade Federal do Rio de Janeiro, Av. Vensceslau Brás 71, Fundos, Botafogo, 22290-040 - Rio de Janeiro, RJ - Brasil. Numerous researchers and activists are creating and spreading useful information for people making informed decisions about their health and to discrimi- nate between their individual problems and those Acknowledgements A fi i f hi It is especially true for countries such as Brazil, with a successful recent his- tory of creating a public health care system to guarantee the right to health of its population. However, we also observe that the interests linked to the biomedical model based on technosciencies are pressuring the regu- latory agencies to change the model so that these inter- ests can regain power. Efforts to improve the regulation, as well as new mechanisms, should be developed. We also want to thank the two anonymous reviewers for their suggestions and comments. Only the authors are responsible for the final product. Conclusions In this article we have analyzed how the leading force in the biomedicalization process –the pharmaceutical industry– has developed strategies to increase their share of the health market. The dispute for the hege- mony of the health sector between financial companies and the medical industrial complex has deeply trans- formed the sector. The big contenders (financial and industrial capital) obtained a part in the distribution of the market and economic surpluses. Public systems and employer-based insurances, as well as health workers (professionals and non-professionals), and moreover the people, are those affected by the transformations. The regulation of the sector had become largely complex. We are in the presence of an intense process of control and regulation of bodies “from inside out” as a type of biomedical governance (p.181) [9]. Patients converted into consumers are exposed to the biomedicalization of their lives helped by the biopedagogies that operate through subtle mechanisms which present information as objective and created to empower consumers. Regula- tory agencies in developed and developing countries lag behind in their capacity to regulate data gathering and communication tools that the multinational corpora- tions are creating to directly reach the population. However, it is important to remark that we consider biomedicalization as a contradictory process that arose as part of intercapitalistic disputes and has multiple allies and opponents. The powerful forces that create biomedicalized discourses operate in a situation where other discourses try to deterritorialize, desestratify, and create “lines of flight” opening the possibility for new situations to emerge [40]. Our intention in analyzing the process is to contribute to the multiplication of more democratic voices. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 19. Adams C: FDA inundated trying to assess drug ad pitches. Wall Street Journal; 2002, B1. 19. Adams C: FDA inundated trying to assess drug ad pitches. 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https://openalex.org/W4205353011	https://figshare.com/articles/journal_contribution/Intercity_innovation_collaboration_and_the_role_of_high-speed_rail_connections_evidence_from_Chinese_co-patent_data/18013830/1/files/32738469.pdf	English	null	Intercity innovation collaboration and the role of high-speed rail connections: evidence from Chinese co-patent data	Regional studies	2,022	cc-by	3,539	Online Appendix In this appendix, we report several robustness checks we have carried out. Section 1.1 reports the placebo test to check whether a counter-factual treatment would generate positive and signiﬁcant eﬀects. Together with the event study, it is to ensure our analysis satisﬁes the common trend assumption. Section 1.2 reports the robustness check to see our main results hold when we use the marketization index as an alternative measure of institutional distance. Section 1.3 conducts sub- sample analysis and explores the heterogenous HSR eﬀect among cities of diﬀerent sizes. Section 1.4 reports the instrument variable regression to test the potential endogeneity problem. 1.1 Placebo Test In conducting a placebo test, we aim to check whether the identiﬁed eﬀect could be contaminated by the pre-treatment trend. We create a placebo treatment of HSR connections using two years prior to the actual opening year and remove all observations with actual HSR connections. Figure A1 shows how the placebo variable is created. Figure A1: Visual Illustration of Placebo Variable Construction Note: This is a graphical illustration of how the placebo variable is constructed. The X-axis is the time horizon from 2005 to 2018, while the Y-axis is three typical city-pairs. City-pair 1 and 2 are HSR- connected during a year, while city-pair 3 is never connected. To constructed the placebo variable, ﬁrst, all post-connection period observations, as indicated by the green color, are deleted. Second, a placebo, as indicated by the red color, is created for the connected city-pairs for two periods before the true connection year. If the regression result shows the signiﬁcance of the placebo eﬀect, then the equal trend assumption is rejected. Note: This is a graphical illustration of how the placebo variable is constructed. The X-axis is the time horizon from 2005 to 2018, while the Y-axis is three typical city-pairs. City-pair 1 and 2 are HSR- connected during a year, while city-pair 3 is never connected. To constructed the placebo variable, ﬁrst, all post-connection period observations, as indicated by the green color, are deleted. Second, a placebo, as indicated by the red color, is created for the connected city-pairs for two periods before the true connection year. If the regression result shows the signiﬁcance of the placebo eﬀect, then the equal trend assumption is rejected. Table A1 reports the regression results using placebo variable designed. Column (1) shows that the coeﬃcient of Placebo is insigniﬁcant at 0.105, suggesting that right before the HSR connection there is no signiﬁcant trend between the connected and the unconnected. The interaction term of Placebo with Dist.S suggests between 0 to 250km, the placebo eﬀect is insigniﬁcant and with a low positive eﬀect. The interaction term of Dist.M suggests that city-pairs between 250 to 600km apart experienced a 27.4% reduction during two years prior to the HSR connection. The interaction term of Dist.L is insigniﬁcant. Table A1 reports the regression results using placebo variable designed. 1.1 Placebo Test Column (1) shows that the coeﬃcient of Placebo is insigniﬁcant at 0.105, suggesting that right before the HSR connection there is no signiﬁcant trend between the connected and the unconnected. The interaction term of Placebo with Dist.S suggests between 0 to 250km, the placebo eﬀect is insigniﬁcant and with a low positive eﬀect. The interaction term of Dist.M suggests that city-pairs between 250 to 600km apart experienced a 27.4% reduction during two years prior to the HSR connection. The interaction term of Dist.L is insigniﬁcant. 1 Table A1: The Placebo Test Table A1: The Placebo Test Dependent Variable: Co-patent Counts (1) (2) Plcebo 0.105 (0.069) 0.061 (0.087) Plcebo × Dist.S(0∼250km) 0.043 (0.103) Plcebo × Dist.M(250∼600km) −0.206∗∗(0.091) Plcebo × Dist.L(600∼1000km) −0.035 (0.096) Plcebo × Distance 0.00004 (0.0001) Plcebo × SameProv 0.012 (0.105) Control V ariables Yes Yes City, Y ear FE Yes Yes Observations 55,013 55,013 Akaike Inf. Crit. 81,965.050 82,073.530 Note: There are 23,924 city-pair-year observations were omitted from total 79,058 observations. All 55,013 are non-connected city-pair-year observations. Overall, the parallel trend required in the DID method is satisﬁed. For the middle ranged city-pairs, there is a noticeable fall before HSR connection. We argue that it does not change the results because even if the trend for the middle ranged city-pair is to decrease the co-patents, the DID estimate can be interpreted as the lowered bound. Moreover, the parallel assumption on the city-pairs within 250 km holds well and hence the main results are robust. Column (2) conﬁrms the parallel assumption between the connected and the unconnected and also between city-pairs within and across provinces. 1.2 Marketization Index as a Measure of Institution Proximity In this study, we follow Hong and Su (2013) and use a province-border measure of institutional friction. It implies that cities belonging to the same province are considered institutionally ap- proximate and incur no institutional friction because they are subject to the same political, legal, and regulatory inﬂuences. To test the robustness of our results, we use the marketization index as an alternative measure of institutional friction. Marketization index (MI) is also a frequently used measure of institutional quality in China (Chen et al., 2006; Li et al., 2006; Firth et al., 2009) since it indicates how Chinese provinces progress toward the realization of a fully-ﬂedged market economy under economic reform (Bin et al., 2020). The index is composed of ﬁve pillars that reﬂect the diﬀerent aspects of the marketization process, namely development of interme- diate organization and law, government-market relation, private economic development, product market development, and factor market development (Wang et al., 2017). A provinces score in the marketization index is the aggregate score of all sub-indices in ﬁve pillars. MI was developed by the National Economic Research Institute (NERI), an inﬂuential think tank in China, and the 2 ﬁrst MI report was published in 2000. Following Mao and Mao (2021), we calculate the intercity institutional friction (or institutional distance) using the following equation: Institutional.dist = 5 X k [(Ik,i −Ik,j)2/Vk]/5 (1) (1) where Ik, i is the marketization index k for city i, Vk is the variance of institutional score of kth pillar. As the marketization index is measured at the provincial level, we additionally deﬁne In- stitutional distance between cities of the same province is zero because cities in the same province tend to share the same regulations, policies, and laws. Table A2 presents the scores of marketi- zation index for the provinces and municipalities obtained from the report by Wang et al. (2017). At a glance, the marketization index is positively correlated with the development level in each region. 1.2 Marketization Index as a Measure of Institution Proximity Table A2: Marketization Index for Provinces and Municipalities 1 Shanghai 9.5 Fujian 8.0 Hubei 6.5 Hebei 6.0 Yunnan 4.9 2 Zhejiang 9.4 Shandong 7.6 Sichuan 6.5 Heilongjiang 5.6 Ningxia 4.7 3 Jiangsu 9.3 Chongqing 7.2 Hunan 6.3 Hainan 5.4 Guizhou 4.5 4 Guangdong 9.0 Liaoning 7.0 Jiangxi 6.2 Shaanxi 5.2 Gansu 4.0 5 Beijing 8.7 Anhui 6.7 Jilin 6.1 Inner Mongolia 5.2 Xinjiang 3.8 6 Tianjin 8.5 Henan 6.6 Guangxi 6 Shanxi 5.1 Qinghai 3.1 Note: The marketization index is the average from 2005 to 2016 for each province and municipality. Table A2: Marketization Index for Provinces and Municipalities As reported in Table A3, in the new regressions, we replace SamePro with Institutional.Dist. The regression results in Columns (2), (3), and (4) show that the coeﬃcients of institutional distance are negative and signiﬁcant, indicating that institutional distance has a negative eﬀect on intercity co-patent. In Column (4), the coeﬃcient of interaction between HSR connection and institutional distance is negative and signiﬁcant, suggesting that institutional distance constrains the HSR eﬀect. These results are consistent with one that uses province-border as a measure of institutional distance. They thus conﬁrm that our main thesis regarding the eﬀect of institutional distance remains robust even if we use the marketization index as an alternative measure of institutional distance. The caveat of using the marketization index is that it is a measurement at the provincial level. The political distance between cities of the same province is deﬁned as zeros. So the institutional distance here can be interpreted as a combination of provincial marketization index and province-border deﬁnition. It is worth noting that there’s diﬀerence between the two deﬁnitions. The province-border deﬁnition disregards the “quality” of institutions, i.e., it does not measure good or bad institutions; instead, it measures whether organizations in diﬀerent cities are subject to the same administration and hence similar political, economic, and legal environment. As Hong and Su (2013) noted, 3 Table A3: The Baseline Regression (Using Marketization Index as Institutional Distance) Table A3: The Baseline Regression (Using Marketization Index as Institutional Distance) Dependent Variable: CoPatent (1) (2) (3) (4) Connect 1.974∗∗∗ 0.006 −0.024 0.243∗∗∗ (0.059) (0.043) (0.056) (0.069) Distance −0.001∗∗∗ −0.001∗∗∗ −0.001∗∗∗ −0.001∗∗∗ (0.0001) (0.00003) (0.0001) (0.00004) Instutional.Dist 0.028 −1.238∗∗∗ −1.049∗∗∗ −1.191∗∗∗ (0.074) (0.038) (0.050) (0.045) Dist.S(0∼250km) 0.406∗∗∗ (0.096) Dist.M(250∼600km) 0.003 (0.078) Dist.L(600∼1000km) −0.011 (0.060) Connect × Dist.S(0∼250km) 0.261∗∗∗ (0.075) Connect × Dist.M(250∼600km) 0.146∗∗ (0.073) Connect × Dist.L(600∼1000km) −0.039 (0.070) Connect × Distance −0.00004 (0.0001) Connect × Instutional.Dist −0.165∗∗ (0.069) CityPair Controls No Yes Yes Yes City&Y ear FE No Yes Yes Yes Observations 79,058 79,058 79,058 79,058 Akaike Inf. Crit. 201,826.100 171,405.200 171,173.400 171,299.200 Note: ∗p<0.1; ∗∗p<0.05; ∗∗∗p<0.01. Robust standard errors are clustered at the city level. Note: ∗p<0.1; ∗∗p<0.05; ∗∗∗p<0.01. Robust standard errors are clustered at the city leve Chinese local governments prefer to match local ﬁrms with universities to form collaboration, so as to keep innovative resources within their territory. They often implement policies and regulations to encourage such local preferences. Under such incentives and expectations, innovative organizations under the administration of the same government are more likely to collaborate with each other. In contrast, the marketization index is a measurement of institutional “quality”. A higher score of marketization index is considered an indication of a better institutional setting for organizations to carry out economic activities Chinese local governments prefer to match local ﬁrms with universities to form collaboration, so as to keep innovative resources within their territory. They often implement policies and regulations to encourage such local preferences. Under such incentives and expectations, innovative organizations under the administration of the same government are more likely to collaborate with each other. In contrast, the marketization index is a measurement of institutional “quality”. Table A3: The Baseline Regression (Using Marketization Index as Institutional Distance) A higher score of marketization index is considered an indication of a better institutional setting for organizations to carry out economic activities 4 4 1.3 Heterogenous Eﬀect on Cities of Diﬀerent Sizes 1.3 Heterogenous Eﬀect on Cities of Diﬀerent Sizes Table A4: Heterogeneous Eﬀect of HSR Connection on Cities of Diﬀerent Sizes Dependent Variable: Co-patent Counts Major-Major Major-Small Small-Small (1) (2) (3) (4) (5) (6) Connect −0.076 −0.060 −0.031 −0.038 −0.035 −0.037 (0.065) (0.087) (0.055) (0.066) (0.165) (0.174) Connect × Dist.S(0∼250km) 0.208∗ 0.226∗∗ 0.248 (0.111) (0.094) (0.206) Connect × Dist.M(250∼600km) 0.094 0.139∗∗ 0.175 (0.083) (0.067) (0.200) Connect × Dist.L(600∼1000km) −0.038 −0.051 0.074 (0.078) (0.069) (0.227) Connect × Distance −0.00001 0.00002 0.0001 (0.0001) (0.0001) (0.0001) Connect × SameProv 0.294∗∗ 0.330∗∗∗ 0.266 (0.120) (0.089) (0.183) City Controls Yes Yes Yes Yes Yes Yes City, Y ear FE Yes Yes Yes Yes Yes Yes Observations 14,350 14,350 49,140 49,140 15,568 15,568 Akaike Inf. Crit. 54,521.920 54,544.550 98,083.250 98,155.330 15,699.140 15,700.630 Note: ∗p<0.1; ∗∗p<0.05; ∗∗∗p<0.01. Robust standard errors are reported. e A4: Heterogeneous Eﬀect of HSR Connection on Cities of Diﬀerent Sizes Note: ∗p<0.1; ∗∗p<0.05; ∗∗∗p<0.01. Robust standard errors are reported. To test the heterogeneity of treatment eﬀect among cities of diﬀerent sizes, we further conduct a subsample analysis on cities of diﬀerent sizes. We split Chinese cities into two types, major and small cities. Distinguishing between major and small cities can be challenging since there is no oﬃcial deﬁnition of major or small cities in China. Following previous research (e.g. Chen and Fang, 2018) we use the popular ﬁve-tier city classiﬁcation framework that was developed by Yicai Global, a leading media group in China. It ranks cities in accordance with ﬁve dimensions of commercial attractiveness, including the concentration of commercial resources, the extent to which a city serves as a business hub, vitality of urban residents, diversity of lifestyle, and future dynamism. It thus classiﬁes 19 cities as ﬁrst-tier cities, 30 as second-tier cities, and the rest as tier 3 to tier 5. Following this classiﬁcation, we deﬁne the ﬁrst and second-tier cities as major cities, and the rest as small cities. There are a total of 49 major cities, and 236 small cities. To disentangle the eﬀect of HSR, we separate the observations into three types: major-major, major- small, and small-small. We run sub-sample regressions of the three types of city-pairs. Table A3: The Baseline Regression (Using Marketization Index as Institutional Distance) Table A4 reports the HSR eﬀects on city-pairs of diﬀerent size combinations. The analysis reveals three additional intriguing results. First, the most signiﬁcant HSR eﬀect comes from between major- small city-pairs. HSR connection increases the number of co-patents by 22.6% within 250 km, 13.9% within 600 km, and 33% within the same province for the major-small city-pairs. Second, 5 for large-large city-pairs, the signiﬁcant HSR eﬀect (20.8 % increase) is only found within 250 km, and the eﬀect is more pronounced (29.4% increase) within the same province. Third, HSR connection has no eﬀect on innovation collaborations in small-small city-pairs. 1.4 Instrument Variable Regression Our results may be aﬀected by endogeneity that may occur as a result of missing variables or reverse causality. As we have included city and year ﬁxed eﬀect and a number of control variables in our regressions, endogeneity due to missing variables should not be a concern but reverse causality can be. This is because policymakers in China may have planned the HSR network to connect cities with the high potential of future economic connection. If that is the case, the endogeneity problem could create upward bias and the estimates could be spurious. One way to tease out the endogeneity is to use instrument variable, which correlates with the endogenous variable HSR connection but does not directly correlate with the outcome variable, intercity co-patents. Such variables are surely diﬃcult to ﬁnd. We follow the literature and use two instrument variables, historic rail connection, and city altitude (Baum-Snow et al., 2017; Dong et al., 2019). Historic rail connection is closely correlated to the modern-day HSR layout but is arguably not directly correlated with intercity innovation collaboration today, as Baum-Snow et al. (2017) argued the old rail system served very diﬀerent functions, such as shipping raw materials and manufactures between larger cities and to provincial capitals according to the dictates of national and provincial annual and ﬁve-year plans. We use the Chinese rail system in 1961 as the ﬁrst instrument variable of modern-day HSR network. The second instrument is geographical elevations of cities; due to the construction cost, cities with higher altitudess are less likely connected to the HSR network. Table A5: The First Stage Regression of 2SLS Dependent variable: connect 2018 Elevation.l −0.00003 (0.00002) Elevation.s −0.0002∗∗∗(0.00001) Historic.l 0.183∗∗∗(0.018) Histroic.s 0.128∗∗∗(0.012) Observations 5,647 R2 0.158 Note: This is the ﬁrst stage regression of 2SLS. The endogenous variable “connect2018” is regressed on all the instrument variable and exogenous variable. In this table, we only show the coeﬃcients of the four instrument variables. Table A5: The First Stage Regression of 2SLS Dependent variable: connect 2018 Elevation.l −0.00003 (0.00002) Elevation.s −0.0002∗∗∗(0.00001) Historic.l 0.183∗∗∗(0.018) Histroic.s 0.128∗∗∗(0.012) Observations 5,647 R2 0.158 Note: This is the ﬁrst stage regression of 2SLS. The endogenous variable “connect2018” is regressed on all the instrument variable and exogenous variable. In this table, we only show the coeﬃcients of the four instrument variables. 1.4 Instrument Variable Regression Table A5: The First Stage Regression of 2SLS Dependent variable: connect 2018 Elevation.l −0.00003 (0.00002) Elevation.s −0.0002∗∗∗(0.00001) Historic.l 0.183∗∗∗(0.018) Histroic.s 0.128∗∗∗(0.012) Observations 5,647 R2 0.158 Note: This is the ﬁrst stage regression of 2SLS. The endogenous variable “connect2018” is regressed on all the instrument variable and exogenous variable. In this table, we only show the coeﬃcients of the four instrument variables. Due to that the IVs are time-invariant—that is, the instruments can only predict whether s are time-invariant—that is, the instruments can only predict whether Due to that the IVs are time-invariant—that is, the instruments can only 6 cities are connected by 2018 but not when—we use a long-diﬀerence model to examine the eﬀects of HSR-connection on intercity innovation collaboration. The long-diﬀerence model converts the panel data into a cross-sectional one by taking the logarithmic diﬀerence of co-patent count between 2005 and 20181. The ﬁrst stage is to regress the endogenous variable, Connectij, on the instrument variable and exogenous variable as shown in Table A5. Due to the dyadic nature of our observation, each observation involves two cities. Each two cities are arranged in their GDP size and deﬁned as the larger or smaller city, as indicated by the suﬃx “.L” and “.S”. The ﬁrst stage result shows that for the larger city, elevation does not predict the HSR connection in 2018 while the elevation of smaller cities is negatively correlated with the prospect of HSR connection. That means the policymaker has planed the HSR network to connect the larger cities regardless of the geographical condition, while a harsh geographical environment could impede the HSR connection for the smaller cities. Historical rail connections positively predict the modern-day HSR connection for both smaller and larger cities. Geographical elevation and historic connection are strong instruments as indicated by the signiﬁcant coeﬃcients. Table A6: The Long Diﬀerence OLS and IV regression Results Dependent variable: Copatent count (2005-2018) Collaboration partners (2005-2018) OLS IV OLS IV (1) (2) (3) (4) Connect 0.151∗∗∗(0.049) −0.040 (0.191) 0.092∗∗∗(0.036) −0.130 (0.139) Connect × Dist.S(0∼250km) 0.310∗∗∗(0.104) 0.655∗∗(0.300) 0.207∗∗∗(0.076) 0.433∗∗(0.218) Connect × Dist.M(250∼600km) −0.126 (0.078) 0.173 (0.229) −0.092 (0.057) 0.083 (0.166) Connect × Dist.L(600∼1000km) −0.088 (0.079) 0.077 (0.219) −0.010 (0.057) 0.207 (0.159) Observations 5,647 5,647 5,647 5,647 R2 0.246 0.243 0.276 0.271 Note: The table compares the OLS and IV regression results. It only presents the endogenous variables. 1To implement a Two-Stage Least Square approach means we cannot have Negative Binomial regression. So instead we use OLS and take the logarithmic of the outcome variable. Following Dong et al. (2019), because there are many zero we use log(x + 1) method. 1.4 Instrument Variable Regression Table A6: The Long Diﬀerence OLS and IV regression Results Table A6 reports the 2SLS long-diﬀerecnce regerssion results. Columns (1) and (2) show the eﬀects on intercity co-patent count and Columns (3) and (4) are on collaborative partnerships. Column (1) presents the OLS results and it shows that co-patents in city-pairs that are HSR- connected tend to grow by 31% for cities within 250 km. For city-pairs that are beyond that range, they expect to grow by 15.1% in co-patents. This ﬁnding closely aligns with our previous results using Negative Binomial panel regression. Column (2) shows the IV 2SLS results, where the standard errors are inﬂated as expected. The coeﬃcient of HSR connection within 250km remains 7 signiﬁcant and changes to 0.655, suggesting that the IV result generates an unbiased estimate higher than the OLS result. 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https://openalex.org/W2944189233	https://www.frontiersin.org/articles/10.3389/fphar.2019.00562/pdf	English	null	A Model for Apoptotic-Cell-Mediated Adaptive Immune Evasion via CD80–CTLA-4 Signaling	Frontiers in pharmacology	2,019	cc-by	11,639	A Model for Apoptotic-Cell-Mediated Adaptive Immune Evasion via CD80–CTLA-4 Signaling Abraam M. Yakoub1* and Stefan Schülke 2 1Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, CA, United States, 2 Vice President’s Research Group: Molecular Allergology, Paul-Ehrlich-Institut, Langen, Germany Abraam M. Yakoub1* and Stefan Schülke 2 1Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, CA, United States, 2 Vice President’s Research Group: Molecular Allergology, Paul-Ehrlich-Institut, Langen, Germany Apoptotic cells carry a plethora of self-antigens but they suppress eliciting of innate and adaptive immune responses to them. How apoptotic cells evade and subvert adaptive immune responses has been elusive. Here, we propose a novel model to understand how apoptotic cells regulate T cell activation in different contexts, leading mostly to tolerogenic responses, mainly via taking control of the CD80–CTLA-4 coinhibitory signal delivered to T cells. This model may facilitate understanding of the molecular mechanisms of autoimmune diseases associated with dysregulation of apoptosis or apoptotic cell clearance, and it highlights potential therapeutic targets or strategies for treatment of multiple immunological disorders. Edited by: Maria Gerosa, University of Milan, Italy Keywords: apoptosis, immunotolerance, autoimmunity, costimulatory pathway, CD80, CTLA-4, coinhibitory pathway, immune evasion Reviewed by: Satish Ramalingam, SRM Institute of Science and Technology, India Roberta Gualtierotti, University of Milan, Italy Reviewed by: Satish Ramalingam, SRM Institute of Science and Technology, India Roberta Gualtierotti, University of Milan, Italy APOPTOSIS Apoptosis, or programmed cell death (PCD), is the physiological form of cell death that plays an important role in tissue homeostasis and regeneration, as well as maintenance of robust organ functions. While cell death by necrosis may have immunostimulatory and inflammatory effects (Sauter et al., 2000), apoptosis shows no immunostimulatory capacities, and may serve beneficial functions to the host (Sauter et al., 2000; Mahajan et al., 2016). *Correspondence: Abraam M. Yakoub abraamyakoub@gmail.com i Our understanding of many aspects of apoptosis has constantly increased over the past ~30 years, due to tremendous effort and a myriad of studies using various model systems. For example, mouse models, due to the power of mouse genetics and with the availability of gene-targeting approaches, have been a mainstay tool to understand both the various functions of apoptosis-related genes in development and the association between gene functions or apoptosis states and disease phenotypes in mammals (see, for instance, Reyes et al., 2010; Gómez-Sintes et al., 2011; Yamaguchi et al., 2011; Wu et al., 2015). Given that the apoptosis machinery is evolutionarily conserved, from worms to mammals, other eukaryotic models have also been used such as drosophila, especially considering the ease of genetic screens and availability of a large number of fly lines (see, for instance, Richardson and Kumar, 2002; Gullaud et al., 2003; Denton et al., 2008; Xu et al., 2009). Even yeast, which, while it lacks main regulators of mammalian apoptosis such as caspases and the B cell lymphoma 2 (Bcl-2) family members, was used to study apoptosis via heterologous expression of many such genes, given the advantages of the yeast model (e.g., easy manipulation via molecular biology or genetics, low cost, and the availability of powerful tools such as the yeast two-hybrid system) (Fleury et al., 2002; Kazemzadeh et al., 2012). In addition to the in vivo models, Specialty section: This article was submitted to Inflammation Pharmacology, a section of the journal Frontiers in Pharmacology Received: 29 August 2018 Accepted: 06 May 2019 Published: 31 May 2019 PERSPECTIVE published: 31 May 2019 doi: 10.3389/fphar.2019.00562 PERSPECTIVE Citation: In the intrinsic pathway, activation of apoptosis is triggered by either developmental signals or genotoxic substances resulting in the release of many proteins including cytochrome C from the mitochondria by pro-apoptotic members of the Bcl-2 family (Nagata and Tanaka, 2017). The released cytochrome C subsequently mediates the formation of apoptosomes in the respective cell’s cytosol, which are multiprotein complexes consisting of cytochrome C, pro-caspase 9, and apoptotic protease-activating factor 1 (APAF1) that process pro-caspase 9 to its mature form (Liu et al., 1996; Zou et al., 1997, 1999). Mature caspase 9 finally mediates the maturation of inactive pro-caspase 3 to its active form caspase 3 (Nagata and Tanaka, 2017). In the extrinsic pathway of apoptosis, binding of FasL (Fas Ligand, expressed on the surface of the apoptosis-inducing cell) to Fas (CD95, tumor necrosis factor receptor superfamily member 6) on the cell destined to undergo apoptosis results in a conformational change in the Fas trimer allowing for the formation of the death-inducing signaling complex (DISC) (Nagata and Tanaka, 2017). DISC is a multiprotein complex containing the Fas-associated death domain protein (FADD) and pro-caspase 8 (Chinnaiyan et al., 1995; Kischkel et al., 1995; Muzio et al., 1996). DISC activation results in the production of mature caspase 3 by DISC-matured caspase 8 (Nagata and Tanaka, 2017). Finally, caspase 3 activated by both apoptosis pathways triggers the apoptosis program via the cleavage of >500 cellular substrates (Nagata and Tanaka, 2017). While FasL expression is restricted to cytotoxic T lymphocytes, T helper type-2 (Th2) cells, and Natural Killer (NK) cells (Kägi et al., 1994; Lowin et al., 1994), Fas is expressed by most cell types (Nagata and Tanaka, 2017). Therefore, FasL-Fas interaction-induced apoptosis is very important for tissue homeostasis. Besides FasL, other ligands such as tumor necrosis factor-alpha (TNF-α), lymphotoxin-alpha (LT-α), TNF-like protein-1A (TL1A), and Apo2L/TNF-related apoptosis- inducing ligand (TRAIL) can also trigger Fas-dependent apoptosis via the extrinsic pathway (Yamada et al., 2017). Besides IL-10, ACs were shown to induce the production of many anti-inflammatory cytokines such as transforming growth factor beta (TGF-β), platelet activating factor (PAF), and prostaglandin E2 (PGE2) (Voll et al., 1997b; Cvetanovic and Ucker, 2004). In addition, macrophage exposure to ACs caused a reduction in the macrophages’ expression of the pro-inflammatory and immunostimulatory cytokines tumor necrosis factor (TNF)-α, IL-12, IL-1β, IL-18, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Fadok et al., 1998; Kim et al., 2005). Citation: Yakoub AM and Schülke S (2019) A Model for Apoptotic-Cell-Mediated Adaptive Immune Evasion via CD80–CTLA-4 Signaling. Front. Pharmacol. 10:562. doi: 10.3389/fphar.2019.00562 May 2019 \| Volume 10 \| Article 562 1 Frontiers in Pharmacology \| www.frontiersin.org A Model for Apoptotic Immune Evasion Yakoub and Schülke Early, in 1997, a pioneering study (Voll et al., 1997a) showed that peripheral blood-derived macrophages exposed to ACs exhibited enhanced production of the immunosuppressive cytokine interleukin (IL)-10, which is an important immune regulatory molecule that prevents inflammatory immune responses, tissue damage, and the development of autoimmunity. Recently, ACs were shown to induce upregulation of the transcription factor aryl hydrocarbon receptor (AhR) in a Toll-like receptor (TLR) 9-dependent manner, which enhanced production of IL-10 to mediate AC-dependent immunosuppression (Shinde et al., 2018). Consequently, AhR knockout induced autoimmune responses and systemic lupus erythematosus (SLE) disease in a mouse model (Shinde et al., 2018). However, it is important to note that, while IL-10 is mainly considered to have anti-inflammatory effects on a wide range of target cells, recent findings suggest a more complex modulatory function of this important cytokine. Because of its role as an important B cell growth and differentiation factor (that promotes B cell proliferation and IgG production), IL-10 was suggested to contribute to the pathology of SLE via activation of autoreactive B cells (reviewed in Geginat et al., 2016). IL-10 levels were shown to increase in SLE patients and polymorphisms in the IL-10 promoter were strongly associated with SLE development (Peng et al., 2013). In line with these findings, neutralization of IL-10 blocked autoantibody production in SLE patients (Llorente et al., 1995). However, both the source of the pathogenic IL-10 production in SLE patients and its possible contribution to other autoimmune diseases remain to be further characterized (Geginat et al., 2016). in vitro models have also been frequently used to understand the signaling pathways or molecular interactions that regulate apoptosis at the cellular level, in physiological or disease conditions (Calissano et al., 2009; Spencer and Sorger, 2011). Importantly, with the advent of stem cell technologies and in vitro differentiation methods, many human (stem cell-derived) cell types, including neurons, were used to understand apoptosis-related molecular disease mechanisms in the human genetic background (Csöbönyeiová et al., 2016; Fang et al., 2018). Induction of cell death by apoptosis in mammals is initiated by two major signaling cascades: the “extrinsic” and “intrinsic” pathways of apoptosis (Nagata and Tanaka, 2017). Citation: Therefore, ACs are able to modulate the activation state of, and cytokine secretion from, antigen-presenting cells (APCs) which influences both innate and subsequent adaptive immune responses to the ACs. This immune modulation also has consequences for T cell activation upon encountering ACs. For example, suppression of macrophage-derived IL-12 production may prevent the differentiation of self-reactive T helper type-1 (Th1) CD4+ cells and autoimmunity (Trembleau et al., 1995), while AC-induced IL-10 represses the expression of MHC-II and costimulatory molecules required for antigen presentation and subsequent T cell activation (Couper et al., 2008). APOPTOTIC CELLS AND ADAPTIVE IMMUNITY It was initially thought that apoptotic cells (ACs) might be immunologically null, however a plethora of evidence has since then indicated that ACs are immunologically active, exerting, in most cases, anti-inflammatory and immunosuppressive effects. The Route to T Cell Activation An Integrative Model of Apoptotic-Cell- Mediated Immune Evasion and Tolerance It was suggested that CTLA-4 and PD-1 may play redundant or complementary functions that differentially target different stages of tolerance (priming, activation, or reactivation of T cells, respectively) (Linsley and Ledbetter, 1993; June et al., 1994; Dahl et al., 2000).h gi Although CD80 can bind to and activate CD28, significant evidence suggests that it also contributes a strong coinhibitory function. In fact, the binding of CD80 to the coinhibitory receptor CTLA-4 occurs with higher affinity than its binding to the costimulatory receptor CD28 (KD = 0.2 and 4 μM, respectively) (van der Merwe and Davis, 2003; Collins et al., 2005; Butte et al., 2008). Furthermore, the crystal structure of the CD80-CTLA-4 complex showed that CD80 homodimers bind bivalent CTLA-4 homodimers in an unusually stable, high-avidity complex (Ikemizu et al., 2000; Stamper et al., 2001). CTLA-4-mediated coinhibitory signaling is critical for negative regulation of T cell activation and proliferation, as evidenced by the severe lymphoproliferation and multi-organ inflammatory lymphocytic infiltrates observed in mice lacking CTLA-4 signaling (Tivol et al., 1995) or in cancer patients receiving anti-CTLA-4 antibodies (Pardoll, 2012). The role of the costimulatory/coinhibitory molecules in immune responses to ACs in vivo (in mice) has also been suggested by the finding that antigen-coupled ACs (derived from splenocytes) induced T cell tolerance via enhanced IL-10 and PD-L1 expression on AC-ingesting macrophages. These ACs also enhanced Treg activation that maintained immunotolerance in that model (Kushwah et al., 2010). PD-L1 upregulation was dependent on IL-10, as IL-10 neutralization with antibodies reduced the PD-L1 response to ACs. Even when the TCR and CD28 are ligand-activated, CTLA-4 activation can inhibit cell cycle progression and cause proliferative arrest of T cells by suppressing IL-2 production (Krummel and Allison, 1996; Walunas et al., 1996). It thus seems that CTLA-4 has a superdominant, overarching role in the regulation of T cell activation. CTLA-4 selectively reverses CD28-mediated costimulation (Walunas et al., 1996; Tai et al., 2007). Moreover, since CTLA-4 is a higher affinity CD80/86 binding partner, CTLA-4 can compete out CD28 for CD80/CD86 binding, also leading to suppression of T cell activation. The Route to T Cell Activation Upon T cell receptor (TCR) activation by ligand binding, such as by specific-antigen-bound major histocompatibility complex May 2019 \| Volume 10 \| Article 562 Frontiers in Pharmacology \| www.frontiersin.org 2 A Model for Apoptotic Immune Evasion Yakoub and Schülke (MHC) on the surface of APCs, the TCR-associated CD3 chains become tyrosine-phosphorylated leading to recruitment of kinases and scaffold proteins and formation of a supramolecular complex that triggers signaling pathways and transcriptional cascades responsible for T cell differentiation and clonal expansion, as well as effector cell generation (Rathmell et al., 2003; Smith-Garvin et al., 2009; Marko et al., 2010; Schultze et al., 2012). Among those signaling and transcriptional events are the upregulation of the glucose receptor Glut 1 (Frauwirth et al., 2002) and the glutamine receptors Snat1 and Snat2 (Carr et al., 2010), and the activation of the MAPK and PI3K/ Akt pathways (Kannan et al., 2012). Collectively, these events are required to fulfill the metabolic needs of the proliferating T cells and to support cell cycle progression and cytokine production (Appleman et al., 2002).fi ligands were later discovered and reported to modulate APC-T cell interaction (for review, see Kow and Mak, 2013; Attanasio and Wherry, 2016; Schildberg et al., 2016). ligands were later discovered and reported to modulate APC-T cell interaction (for review, see Kow and Mak, 2013; Attanasio and Wherry, 2016; Schildberg et al., 2016). An Integrative Model of Apoptotic-Cell- Mediated Immune Evasion and Tolerance Strong evidence indicates an essential role for the coinhibitory pathway in suppressing adaptive immune responses. First, the role of the coinhibitory signaling in regulating self-tolerance or autoimmunity is supported by the finding that various coinhibitory ligands are expressed, besides APCs, on non-hematopoietic cells which was suggested to play a role in maintaining tissue tolerance by suppressing self-reactive T cells in the periphery (Anderson et al., 2016; Schildberg et al., 2016; Ward-Kavanagh et al., 2016; Janakiram et al., 2017). Notably, some tumors and infectious pathogens evade immune recognition by exploiting such natural tolerance mechanisms (Odorizzi and Wherry, 2012; Pardoll, 2012; Wang et al., 2013; Attanasio and Wherry, 2016; Baumeister et al., 2016). However, TCR ligation alone is insufficient to trigger or maintain robust T cell activation, as this process is tightly regulated by a complex array of costimulatory and coinhibitory ligands and receptors (Esensten et al., 2016). The prototypical costimulatory receptor is CD28, while the prototypical coinhibitory receptors are cytotoxic T lymphocyte antigen-4 (CTLA-4) and PD-1 (Buchbinder and Desai, 2016). Shared ligands between both types of receptors include CD80 (B7-1) and CD86 (B7-2). Stimulation of CD28 potentiates and sustains IL-2 production from T cells and prevents peripheral immunotolerance development (Bour-Jordan et al., 2011; Kow and Mak, 2013). These T cells activated by CD28-B7 signaling then mature and differentiate, subsequently inducing B cell proliferation and differentiation into plasma cells producing antigen-specific antibodies (Kow and Mak, 2013).i y Moreover, CTLA-4-mediated coinhibition was shown to be essential for terminating T cell activation as CTLA-4−/− mice develop massive lymphoproliferation and early death (Tivol et al., 1995; Waterhouse et al., 1995). CTLA-4 was also suggested as a master switch for peripheral tolerance (Bluestone, 1997). Importantly, the essential role of CTLA-4 in autoimmune regulation was further highlighted by the fact that blockade of CTLA-4 signaling in multiple animal models resulted in aggravation of autoimmune diseases (Karandikar et al., 1996; Luhder et al., 1998; Chitnis et al., 2001). Moreover, CTLA-4 gene single-nucleotide polymorphisms (SNPs) in humans were associated with autoimmune disorders. For example, a SNP in the 6.1-kb (kilobase) 3′ region of the CTLA-4 gene was associated with higher risk of Grave’s disease, autoimmune hypothyroidism, and type 1 diabetes mellitus (Ueda et al., 2003). Blockade of the CTLA-4- or PD-1-mediated coinhibitory signaling accelerated cardiac allograft rejection in C57BL/6 mice receiving BALB/c hearts (Ito et al., 2005). Frontiers in Pharmacology \| www.frontiersin.org An Integrative Model of Apoptotic-Cell- Mediated Immune Evasion and Tolerance Besides CD28 and CTLA-4, other costimulatory and coinhibitory receptors and Since ACs were reported to induce IL-10, and given that IL-10 is mostly an anti-inflammatory cytokine as discussed above, which is important for the induction of tolerance and suppression of dendritic cell maturation (Faulkner et al., 2000; Corinti et al., 2001), it seemed plausible to suggest that adaptive immune responses to ACs could be mediated by IL-10 effects. However, that hypothesis has been difficult to May 2019 \| Volume 10 \| Article 562 3 A Model for Apoptotic Immune Evasion Yakoub and Schülke there were also instances where ACs were reported to have immunostimulatory effects (Hoffmann et al., 2000; Ignatius et al., 2000; Feng et al., 2002; Buttiglieri et al., 2003; Goldszmid et al., 2003; Ishii et al., 2003; Casares et al., 2005). It is thus possible to propose a model (Figure 1) whereby exposure to ACs in a non-inflammatory/non-immunostimulatory context (that does not involve immunogenic stimuli that trigger T cell activation) mounts a tolerogenic response via T cell inhibition through the coinhibitory pathway, whereas exposure to activated ACs in an immunostimulatory context mounts an immunogenic response via T cell activation through the costimulatory pathway. In support of this model is our finding that under non-immunostimulatory conditions, the AC-induced CD80 upregulation on macrophages was coupled with CD28 downregulation on T cells (Yakoub et al., 2018), which may possibly enhance the coinhibitory functions of CD80 as it binds the coinhibitory receptor CTLA-4 with much higher affinity and stability than it does the costimulatory receptor CD28 as described above. fully establish due to different conclusions from various studies. For example, ACs were shown to induce IL-10 production by monocytes (Voll et al., 1997a), but not macrophages (Fadok et al., 1998). But antigen-coupled ACs enhanced IL-10 and PD-L1 expression in AC-ingesting macrophages and induced T cell tolerance in mice (Getts et al., 2011). Importantly, however, while statistically significant, the IL-10-induced PD-L1 upregulation in that study was subtle when compared to the IL-10-neutralized control (only ~20% increase in PD-L1 mean fluorescence intensity (MFI) over the control). Conversely, using macrophage cell lines and mouse primary macrophages, we found that the upregulation of another costimulatory/ coinhibitory molecule, CD80, was more pronounced (on average > 4-fold upregulation, relative to the unstimulated control) (Yakoub et al., 2018). An Integrative Model of Apoptotic-Cell- Mediated Immune Evasion and Tolerance In another study, dendritic cells exposed to ACs exhibited reduced T cell proliferation and activation and reduced lipopolysaccharide (LPS)-triggered upregulation of the costimulatory molecule CD86 (Stuart et al., 2002). In that study, ACs did not significantly affect IL-10 levels secreted by dendritic cells, and even IL-10 neutralization by soluble IL-10R did not affect expression of the costimulatory molecules on the dendritic cells (Stuart et al., 2002). Even further, ACs could inhibit LPS-induced activation of bone marrow-derived dendritic cells derived from IL-10-deficient mice; and neutralizing another anti-inflammatory cytokine, TGF-β1, could not suppress the inhibition of dendritic cell maturation by ACs (Stuart et al., 2002), contrary to what was suggested (Chen et al., 2001). In total, these reports suggest that the effect of ACs on adaptive immune responses cannot, or cannot completely, be attributed to secondary effects of the cytokine secretion modulated by ACs, and that ACs may have direct effects on the machinery regulating adaptive immune responses. CD80 binding to CTLA-4 conveys an inhibitory signal to T cell activation that overrides costimulatory signals, counteracting the initiation of T cell activation and proliferation and indeed inducing their apoptosis (Walunas et al., 1994). Moreover, other mechanisms could also contribute to AC-mediated suppression of adaptive immune responses. For example, ACs (derived from dendritic cells) engulfed by dendritic cells induced TGF-β1 secretion and differentiation of naïve T cells into Foxp3+ Tregs (Kushwah et al., 2010). Naïve T cells can differentiate upon antigen recognition into effector T cell subsets such as Th1, Th2, and Th17, or into immunosuppressive Tregs. AC-ingesting dendritic cells suppressed the development of the effector Th17 cells, but enhanced the development of Tregs where dendritic cell-T cell interaction and the costimulatory/coinhibitory signaling were suggested to play a role in Treg induction (Yamazaki et al., 2003; Torchinsky et al., 2009). Similar to the results in macrophages showing upregulation of CD80 by ACs (Yakoub et al., 2018), ACs were reported to induce CD80 and CD86 expression on in vitro differentiated human dendritic cells, which involved both soluble factors secreted and cell-cell contact to achieve the full effects of the ACs (Johansson et al., 2007; Pathak et al., 2012). Notably, however, these effects of ACs on CD80/86 expression on dendritic cells required “pre-activation” of the ACs with anti-CD3 and anti-CD28 antibodies, whereas non-pre-activated ACs showed no effect on CD80/86 expression on dendritic cells (Johansson et al., 2007; Pathak et al., 2012). Frontiers in Pharmacology \| www.frontiersin.org An Integrative Model of Apoptotic-Cell- Mediated Immune Evasion and Tolerance These results suggest a differential AC response between dendritic cells and macrophages, as such AC activation was not required to modulate CD80 levels on macrophages (Yakoub et al., 2018). In a similar concept, “heat-stressed,” but not unstressed, ACs induced the costimulatory molecules CD40, CD80, and CD86 on dendritic cells and secretion of the immunostimulatory IL-12, resulting in enhanced T cell responses (Feng et al., 2002).f y CD80 expressed on T cells was also shown to bind PD-L1 on APCs with an affinity greater than that of CD80-CD28 binding (Butte et al., 2007; Rollins and Gibbons Johnson, 2017), which may negatively regulate T cell activation. Similarly, CD80 on APCs, which ACs upregulate (Yakoub et al., 2018), was suggested to bind PD-L1 on T cells (Schildberg et al., 2016), which may downregulate T cell activation, if not directly, indirectly by competing out CD28 and thus reducing the costimulatory signal that is essential for T cell activation and sustenance of the adaptive immune response. Notably, PD-L1 expression on parenchymal tissues including pancreatic islets mediated tolerance and inhibited self-reactive CD4 T cells (Keir et al., 2006); and interference with CD80-PD-L1 binding enhanced activation of CD4 and CD8 T cells in vivo and accelerated development of autoimmune diabetes in NOD mice (Paterson et al., 2011). Interestingly, it was also proposed that CD80/86 binding to CTLA-4 and PD-L1 on T cells enhances T cell motility, reducing T cell-APC contacts and the strength of the immune synapse, while enhancing contacts with, and activation of, Tregs (Dilek et al., 2013). Moreover, binding of PD-L1 on ACs to PD-1 on T cells is also possible (Kushwah et al., 2010), which may also strengthen the coinhibitory signal While the immunosuppressive or tolerogenic effects of ACs are established by a plethora of evidence (Kabelitz and Janssen, 1997; Steinman et al., 2000; Fadok et al., 2001; Ferguson et al., 2002; Liu et al., 2002; Stuart et al., 2002; Morelli et al., 2003; Rovere-Querini et al., 2004; Gray et al., 2007), May 2019 \| Volume 10 \| Article 562 Frontiers in Pharmacology \| www.frontiersin.org 4 A Model for Apoptotic Immune Evasion Yakoub and Schülke lke A Model for Apoptotic Immune Evasion A B C A model for the regulation of T cell-mediated adaptive immune responses to apoptotic cells. (A) At the resting state, no activation of the TCR latory pathway takes place. An Integrative Model of Apoptotic-Cell- Mediated Immune Evasion and Tolerance (B) In immunostimulatory contexts (such as AC ingestion by APCs in the presence of LPS, TLR ligands, or antigens), TCR binding to AC antigens presented in the context of MHC-I/II and the costimulatory signaling mediated by binding of CD28 by or CD86 and CD80) take place. Thus, activation of the costimulatory pathway ensures effector T cell (Teff) activation and proliferation, leading an immune response to AC antigens. (C) In non-immunostimulatory contexts, such as AC ingestion by APCs in the absence of additional iating stimuli (e.g., TLR ligands or mycobacterial antigens), the costimulatory signaling mediated by CD28 is downregulated. Rather, CD80 is which binds mainly to CTLA-4 (CD80 binds CTLA-4 with much higher affinity than CD28), initiating the coinhibitory signaling that leads to Teff nd apoptosis. Even if some costimulatory signal is conveyed (by binding of some CD80 molecules to CD28), the CTLA-4-mediated gnal predominates and usually overrides costimulation. Other signals are possible (e.g., binding of CD80 to PD-L1 on T cells or binding of Cs to PD-1 on T cells) and might have some contribution to the overall coinhibitory signaling delivered to T cells, although their significance ntribution have yet to be established. Overall, the coinhibitory signaling to T cells triggers the inhibition of Teff functions; and in this context, of Tregs may also be enhanced A B A A A B B B B B C C FIGURE 1 \| A model for the regulation of T cell-mediated adaptive immune responses to apoptotic cells. (A) At the resting state, no activation of the TCR or the costimulatory pathway takes place. (B) In immunostimulatory contexts (such as AC ingestion by APCs in the presence of LPS, TLR ligands, or mycobacterial antigens), TCR binding to AC antigens presented in the context of MHC-I/II and the costimulatory signaling mediated by binding of CD28 by mainly CD86 (or CD86 and CD80) take place. Thus, activation of the costimulatory pathway ensures effector T cell (Teff) activation and proliferation, leading to mounting of an immune response to AC antigens. (C) In non-immunostimulatory contexts, such as AC ingestion by APCs in the absence of additional immunopotentiating stimuli (e.g., TLR ligands or mycobacterial antigens), the costimulatory signaling mediated by CD28 is downregulated. Rather, CD80 is upregulated, which binds mainly to CTLA-4 (CD80 binds CTLA-4 with much higher affinity than CD28), initiating the coinhibitory signaling that leads to Teff suppression and apoptosis. Sjögren’s Syndrome Sjögren’s Syndrome (SS) is an autoimmune disease targeting the salivary and lacrimal glands resulting in chronic dryness of mouth and eyes (Ainola et al., 2018). In SS, apoptotic particles blebbing from apoptotic epithelial cells, possibly caused by defects in the production of sex hormones, that contain typical SS autoantigens such as hY1RNA were shown to contribute to disease pathology (Ainola et al., 2018). In line with this increase in apoptosis rates, enhanced levels of both Fas and FasL were reported in salivary gland tissues, but not in lacrimal gland tissues or peripheral blood Rheumatoid Arthritis Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disorder characterized by progressive inflammatory bone and joint destruction. Immunologically, the autoimmune responses are mediated by either circulating autoantibodies directed against citrullinated peptides and rheumatoid factor or complement protein C3 (Abdolmaleki et al., 2018). The destructive autoimmune responses in RA are maintained by IL-6 and TNF-α secreted by tissue macrophages triggering the MMP and RANK-ligand- supported activation of osteoclasts (An et al., 2016). In RA patients, the sustained joint inflammation is maintained by an abnormal state of aberrant cell survival caused by perpetual T cell activation resulting in stimulation and proliferation of fibroblasts which was termed “apoptosis resistance” (Malemud, 2018). In line with these observations, anti-apoptotic proteins were shown to be upregulated in RA synovial fluids (reviewed in Williams et al., 2018). Therefore, induction of apoptosis has the potential to reduce joint damage and to further modulate autoimmune responses in RA by modulating the coinhibitory signaling to T cells as previously described. Experimental apoptosis induction has therefore been considered as potential therapeutic avenue in RA, e.g., by targeting intracellular apoptotic inhibitory molecules, but thus far has not reached clinical trials (Williams et al., 2018). p While the distinction between APC types (dendritic cells or macrophages) in terms of the type of immune response (immunogenic vs. tolerogenic) to ACs cannot be completely settled given the varying reports thus far in this regard, there is still some preliminary evidence to propose that macrophages might mainly mediate tolerogenic responses to ACs while dendritic cells might mainly mediate the immunogenic responses. Dendritic cells and macrophages exhibit distinct locales and may thus mediate differential, locale-specific, AC responses (T cell activation or inhibition). For example, in the intestinal lamina propria, both APC types present commensal microbe and dietary antigens to T cells, with dendritic cells inducing effector Th17 T cells, and macrophages inducing Tregs (Denning et al., 2007). Tregs induced by AC-presenting macrophages showed induced anti-inflammatory cytokine production and reduced immunostimulatory cytokines, and displayed an anergic phenotype after restimulation with the antigen (Denning et al., 2007). Because macrophages are more abundant than dendritic cells in the lamina propria, T cell tolerance thus becomes the predominant response in that context. An Integrative Model of Apoptotic-Cell- Mediated Immune Evasion and Tolerance Even if some costimulatory signal is conveyed (by binding of some CD80 molecules to CD28), the CTLA-4-mediated coinhibitory signal predominates and usually overrides costimulation. Other signals are possible (e.g., binding of CD80 to PD-L1 on T cells or binding of PD-L1 on APCs to PD-1 on T cells) and might have some contribution to the overall coinhibitory signaling delivered to T cells, although their significance and relative contribution have yet to be established. Overall, the coinhibitory signaling to T cells triggers the inhibition of Teff functions; and in this context, differentiation of Tregs may also be enhanced. May 2019 \| Volume 10 \| Article 562 Frontiers in Pharmacology \| www.frontiersin.org 5 A Model for Apoptotic Immune Evasion Yakoub and Schülke to T cells, although its significance needs to be established as previously discussed. the immunosuppressive properties of ACs may be exploited for therapeutic purposes. Whether ACs would induce a tolerogenic or immunogenic response may depend on presence of immunostimulatory conditions or cues, including: (1) the type of cell death induced in the ACs, e.g., caspase-dependent or independent (Larmonier et al., 2002); (2) type of the apoptosis-inducing agent or drug used in the experimental model (Casares et al., 2005; Obeid et al., 2007); (3) the stage of apoptosis (early vs. late) in the ACs (Weyd et al., 2013); (4) type of the apoptotic corpse (ACs or apoptotic blebs) ingested by the APC (Fransen et al., 2009); (5) cell-type of the ACs ingested by the APCs (Kushwah et al., 2010); (6) secretion of T cell immuostimulatory cytokines, such as TNF-α, or TLR ligands (Clayton et al., 2003); (7) type and ratio of the APCs (dendritic cells or macrophages) present in a particular tissue site (Denning et al., 2007); or (8) presence of potentially immunogenic or immunogenizing infectious pathogen (e.g., mycobacterial) antigens in the tissue microenvironment (Espinosa-Cueto et al., 2017). Systemic Lupus Erythematosus y p y SLE is a chronic systemic autoimmune disorder characterized by the presence of circulating nuclear antigens, including DNA and nucleosomes, and of autoantibodies against these nuclear antigens (Poon et al., 2014). SLE affects the skin, lungs, kidneys, and central nervous system. Impaired engulfment of ACs by phagocytes leads to accumulation of ACs in the lymph nodes and blood and in the skin after UV exposure. Impaired AC clearance eventually leads to secondary necrosis, which allows intracellular (self-) antigens, normally hidden within the AC to be exposed extracellularly, and thus recognizing these self-antigens by the immune system, causing the production of autoantibodies and autoimmunity. While mostly suggested to be caused by defective AC engulfment, super-stimulatory APCs that lead to hyperactive T cells were also suggested to recapitulate SLE in a mouse model (Zhu et al., 2005). Thus, targeting APC activity which is normally modulated by ACs in normal physiological conditions might be a successful strategy for therapy development for SLE. Rheumatoid Arthritis Importantly, macrophages were shown to be essential for clearing tumor ACs introduced into mice and eliciting tolerogenic responses to the ACs (Asano et al., 2011); as ablation of the spleen marginal zone macrophages in these mice diminished the immunosuppressive potential of the ACs and enabled triggering of an immune response to ACs (McGaha et al., 2011). It thus seems possible that the immunosuppressive or tolerogenic response to ACs is mainly mediated by macrophages as the APCs. Cancer Anti-tumor chemotherapeutic agents lead to production of massive cytotoxicity and generation of ACs. Given the adaptive immunosuppressive and tolerogenic effects of ACs, it is plausible to hypothesize that the apoptosis induced, and the ACs produced, by cancer treatments contribute to tumor survival indirectly by dampening immune responses to cancer cell antigens carried on these ACs. Thus, functionally blocking ACs in cancer may help reduce these undesirable effects of antineoplastic agents on anti-tumor immunity. In fact, some therapeutic strategies to target the coinhibitory molecules that mediate the adaptive immune responses to ACs have been investigated for their possible beneficial effects on anti-tumor immunity (Chen, 2004; Hirano et al., 2005; Curran et al., 2010). Diabetes Mellitus Type 1 diabetes mellitus can be caused by autoimmune responses to pancreatic beta-cell antigens resulting in insulin deficiency and hyperglycemia. While it was suggested that inefficient clearing of apoptotic pancreatic cells resulting in the release of damage-associated molecular patterns (DAMPs) in combination with autoantigens may contribute to the pathology of type 1 diabetes (Heimberg et al., 2001; O’Brien et al., 2006), ACs were also suggested as a tool to induce tolerance to beta- cell self-antigens. Indeed, ACs (apoptotic beta-cell infusion) could suppress beta-cell antigen-specific CD4+ T cell proliferation and delay the onset of diabetes in the diabetes-susceptible, autoimmune (NOD) mice (Xia et al., 2007; Marin-Gallen et al., 2010). Therefore, ACs show a promising potential for the treatment of type 1 diabetes. Although the costimulatory/coinhibitory pathway has been a tempting target for disease therapy, some challenges remain and are yet to be overcome in the future to enable full harnessing of the therapeutic potential of this pathway. For example, some therapies targeting the costimulatory/coinhibitory pathway have even been discontinued in medical practice due to limited effectiveness (Smith et al., 2013), or have failed clinical trials at early stages (Khoury and Sayegh, 2004). Some proposed therapies can also pose significant risks (Frebel and Oxenius, 2013). This all reflects the fact that we have not yet reached complete and thorough understanding of the costimulation/ coinhibition pathways and their intricate interactions with diseases. In conclusion, this perspective proposes a model to understand how ACs regulate the costimulatory/coinhibitory signaling pathways of regulating T cell activation in order to suppress adaptive immune responses, which may facilitate harnessing these molecular mechanisms in therapy development for various immunopathological conditions. Autoimmune Lymphoproliferative Syndrome y Autoimmune Lymphoproliferative Syndrome (ALPS) is an inherited autoimmune disorder characterized by spleno- and hepatomegaly, lymphadenopathy, autoimmune lesions in multiple organs, as well as autoimmune hemolytic anemia, thrombocytopenia, or leukocytopenia, caused by cell-type specific autoantibody production (Turbyville and Rao, 2010; Price et al., 2014; Yamada et al., 2017). In ALPS patients, these symptoms are caused by spontaneous mutations in the Fas, FasL, or caspase 10 genes, resulting in a defective apoptosis of antigen-activated T- and B cells in the periphery, and the impaired limitation of immune responses (Yamada et al., 2017). Since the pathology of ALPS is caused by a disruption of lymphocyte apoptosis, it is plausible to speculate that a decrease in production of ACs (which exert immunoinhibitory/anti-inflammatory effects as discussed) may contribute to the autoimmune pathology in ALPS patients. The role of ACs in ALPS pathogenesis and its possible exploitation for therapy is thus an interesting area for future research. CONCLUDING REMARKS While ACs have been investigated as tolerogenic or immunosuppressive “vaccines,” understanding the molecular mechanisms of the ACs’ immunomodulating effects, especially their interaction with the costimulatory/coinhibitory pathway, may encourage attempts at targeting specific molecules that ACs exploit in mediating their effects. In general, the costimulatory/coinhibitory pathway has been explored as target for therapeutic purposes. For example, targeting of either CD28, PD-1, PD-L1, or CTLA-4 has been investigated and some therapies targeting this machinery are already in clinical use (e.g., the anti-CTLA-4 antibody Ipilimumab for the treatment of advanced metastatic melanoma, and the anti-PD-1 antibodies Pembrolizumab and Nivolumab for the treatment of advanced melanoma, advanced non-small cell lung cancer, and metastatic renal cell carcinoma) (Dilek et al., 2013). ROLE OF APOPTOTIC-CELL-MEDIATED IMMUNOSUPPRESSION IN DISEASES An abundance of ACs (~70 billion) is produced daily in humans and defective clearance of these ACs has been associated with diseases, primarily autoimmune conditions. Therefore, we will briefly discuss some disease conditions, where we propose that May 2019 \| Volume 10 \| Article 562 Frontiers in Pharmacology \| www.frontiersin.org 6 A Model for Apoptotic Immune Evasion Yakoub and Schülke Donor’s ACs given to transplant patients may tolerize or repress the recipient’s immune responses to the transplant, prevent graft- versus-host reactions, and enhance graft survival (Kleinclauss et al., 2006; Wang et al., 2006, 2009; Bittencourt et al., 2011). lymphocytes of patients with SS, implicating Fas-mediated apoptosis in the destruction of salivary gland tissue (Ishimaru et al., 2001; Bolstad et al., 2003). The presence of both autoantigens and adjuvanting nucleic acids in these apoptotic particles was shown to stimulate plasmacytoid dendritic cells in salivary glands via TLR7 and TLR9, resulting in the activation of autoreactive T and B cells (Ainola et al., 2018). 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PD-1/PDL1 and CD28/CD80 pathways modulate natural killer T cell function to inhibit hepatitis B virus replication. J. Viral Hepat. 20, 27–39. doi: 10.1111/ jvh.12061 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Wang, Z., Shufesky, W. J., Montecalvo, A., Divito, S. J., Larregina, A. T., and Morelli, A. E. (2009). In situ-targeting of dendritic cells with donor-derived apoptotic cells restrains indirect allorecognition and ameliorates allograft vasculopathy. PLoS One 4:e4940. doi: 10.1371/journal.pone.0008442h Copyright © 2019 Yakoub and Schülke. 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https://openalex.org/W2476243991	https://www.mdpi.com/1996-1944/9/7/607/pdf?version=1469177835	English	null	Influence of Network Structure on Glass Transition Temperature of Elastomers	Materials	2,016	cc-by	15,015	q g Received: 23 June 2016; Accepted: 15 July 2016; Published: 22 July 2016 q g Received: 23 June 2016; Accepted: 15 July 2016; Published: 22 July 2016 Abstract: It is generally believed that only intermolecular, elastically-effective crosslinks inﬂuence elastomer properties. The role of the intramolecular modiﬁcations of the polymer chains is marginalized. The aim of our study was the characterization of the structural parameters of cured elastomers, and determination of their inﬂuence on the behavior of the polymer network. For this purpose, styrene-butadiene rubbers (SBR), cured with various curatives, such as DCP, TMTD, TBzTD, Vulcuren®, DPG/S8, CBS/S8, MBTS/S8 and ZDT/S8, were investigated. In every series of samples a broad range of crosslink density was obtained, in addition to diverse crosslink structures, as determined by equilibrium swelling and thiol-amine analysis. Differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) were used to study the glass transition process, and positron annihilation lifetime spectroscopy (PALS) to investigate the size of the free volumes. For all samples, the values of the glass transition temperature (Tg) increased with a rise in crosslink density. At the same time, the free volume size proportionally decreased. The changes in Tg and free volume size show signiﬁcant differences between the series crosslinked with various curatives. These variations are explained on the basis of the curatives’ structure effect. Furthermore, basic structure-property relationships are provided. They enable the prediction of the effect of curatives on the structural parameters of the network, and some of the resulting properties. It is proved that the applied techniques—DSC, DMA, and PALS—can serve to provide information about the modiﬁcations to the polymer chains. Moreover, on the basis of the obtained results and considering the diversiﬁed curatives available nowadays, the usability of “part per hundred rubber” (phr) unit is questioned. Keywords: elastomer; rubber; curatives; crosslinks; crosslink density; crosslink structures; glass transition; free volumes materials materials materials Katarzyna Bandzierz 1,, Louis Reuvekamp 2, Jerzy Dryzek 3, Wilma Dierkes 2, Anke Blume 2 and Dariusz Bielinski 1 Katarzyna Bandzierz 1,, Louis Reuvekamp 2, Jerzy Dryzek 3, Wilma Dierkes 2, Anke Blume 2 and Dariusz Bielinski 1 1 Institute of Polymer and Dye Technology, Lodz University of Technology, Lodz 90-924, Poland; dariusz.bielinski@p.lodz.pl 1 Institute of Polymer and Dye Technology, Lodz University of Technology, Lodz 90-924, Poland; dariusz.bielinski@p.lodz.pl 2 Elastomer Technology & Engineering, University of Twente, Enschede 7500 AE, The Netherlands; l.a.e.m.reuvekamp@utwente.nl (L.R.); w.k.dierkes@utwente.nl (W.D.); a.blume@utwente.nl (A.B.) 3 Institute of Nuclear Physics, Polish Academy of Sciences, Krakow 31-342, Poland; jerzy.dryzek@ifj.edu.pl * Correspondence: katarzyna.bandzierz@gmail.com; Tel.: +48-42-631-3214 2 Elastomer Technology & Engineering, University of Twente, Enschede 7500 AE, The Netherlands; l.a.e.m.reuvekamp@utwente.nl (L.R.); w.k.dierkes@utwente.nl (W.D.); a.blume@utwente.nl (A.B.) 3 Institute of Nuclear Physics, Polish Academy of Sciences, Krakow 31-342, Poland; jerzy.dryzek@ifj.edu.pl * Correspondence: katarzyna.bandzierz@gmail.com; Tel.: +48-42-631-3214 Inﬂuence of Network Structure on Glass Transition Temperature of Elastomers Katarzyna Bandzierz 1,, Louis Reuvekamp 2, Jerzy Dryzek 3, Wilma Dierkes 2, Anke Blume 2 and Dariusz Bielinski 1 1. Introduction Among them are tensile strength, mechanical modulus, hardness, swelling in solvents, glass transition temperature, and thermal stability. Crosslink density is usually analyzed by the equilibrium swelling of the elastomer in a thermodynamically-suitable solvent, and calculated on the basis of the Flory-Rehner equation [3]. Other methods of crosslink density determination refer to the Mooney-Rivlin model [4,5], or to nuclear magnetic resonance [6]. and start the chemical reactions for crosslinking. The curing process is, therefore, carried out at elevated temperatures (130–190 °C). The types of curatives are usually: (i) classical curing systems based on rhombic sulfur combined with an accelerator; (ii) sulfur donors; (iii) organic peroxides; or (iv) metal oxides. The curatives nowadays are abundant and characterize themselves with diverse structures, which increasingly often are very complex. Therefore, the curatives exhibit various and complicated reactivities, especially at elevated temperatures. This is particularly significant in case of sulfur compounds, rhombic sulfur and their mixtures. Hence, the transformation products and resulting crosslinks can be very diverse. The formed intermolecular elastically effective crosslinks, are usually classified as carbon-carbon (C–C) crosslinks and sulfidic crosslinks. The C–C crosslinks are created directly between the polymer chains. In contrast, the sulfidic crosslinks are made of a variable number of To introduce crosslinks into the elastomer, a curing process has to be carried out. It is conventionally performed with the use of curatives, which need high temperatures to be activated and start the chemical reactions for crosslinking. The curing process is, therefore, carried out at elevated temperatures (130–190 ˝C). The types of curatives are usually: (i) classical curing systems based on rhombic sulfur combined with an accelerator; (ii) sulfur donors; (iii) organic peroxides; or (iv) metal oxides. The curatives nowadays are abundant and characterize themselves with diverse structures, which increasingly often are very complex. Therefore, the curatives exhibit various and complicated reactivities, especially at elevated temperatures. This is particularly signiﬁcant in case of sulfur compounds, rhombic sulfur and their mixtures. Hence, the transformation products and resulting crosslinks can be very diverse. sulfur atoms. Depending on this number, sulfidic crosslinks are further subdivided into (i) monosulfidic (C–S–C); (ii) disulfidic (C–S2–C); and (iii) polysulfidic (C–Sx–C, x ≥ 3) crosslinks. All of these crosslinks show varying structure and length, resulting in various characteristics. As a result, they provide different properties to the material. 1. Introduction Elastomers are a unique class of materials. They can withstand high elastic deformations, and recover to their original size and shape immediately after removal of the force. The majority of elastomers are rubbers. To exhibit elasticity, their chains have to be connected to each other by chemical bonds, called crosslinks. The crosslinks prevent untangling of the chains and sliding over each other under load. When crosslinked, intrinsically-viscoelastic elastomers behave in a “hyperelastic” way, and plastic deformations do not occur (Figure 1). Materials 2016, 9, 607; doi:10.3390/ma9070607 www.mdpi.com/journal/materials 2 of 17 f 18 Materials 2016, 9, 607 Materials 2016, 9, (a) (b) Figure 1. Performance of an elastomer under loading force and after its removal: (a) plastic deformation of an uncrosslinked elastomer; and (b) elastic recovery of a crosslinked elastomer. l k d b l h h h h d l Figure 1. Performance of an elastomer under loading force and after its removal: (a) plastic deformation of an uncrosslinked elastomer; and (b) elastic recovery of a crosslinked elastomer. (b) (a) Figure 1. Performance of an elastomer under loading force and after its removal: (a) plastic deformation of an uncrosslinked elastomer; and (b) elastic recovery of a crosslinked elastomer. Figure 1. Performance of an elastomer under loading force and after its removal: (a) plastic deformation of an uncrosslinked elastomer; and (b) elastic recovery of a crosslinked elastomer. y p elastomer networks. It is defined as a number of crosslinks per unit volume in a polymer network [1]. Crosslink density influences, to a very high extent, various properties of elastomers [2]. Among them are tensile strength, mechanical modulus, hardness, swelling in solvents, glass transition temperature, and thermal stability. Crosslink density is usually analyzed by the equilibrium swelling of the elastomer in a thermodynamically-suitable solvent, and calculated on the basis of the Flory-Rehner equation [3]. Other methods of crosslink density determination refer to the Mooney-Rivlin model [4,5], or to nuclear magnetic resonance [6]. To introduce crosslinks into the elastomer, a curing process has to be carried out. It is conventionally performed with the use of curatives, which need high temperatures to be activated Hence, crosslink density is a basic structural parameter which characterizes three-dimensional elastomer networks. It is deﬁned as a number of crosslinks per unit volume in a polymer network [1]. Crosslink density inﬂuences, to a very high extent, various properties of elastomers [2]. 1. Introduction Therefore, it is generally assumed that after the crosslink density, the crosslink structure is the second most important parameter influencing the elastomer properties [7,8]. To characterize the various types of crosslinks, chemical analyses are used. The most commonly used is thiol-amine analysis [9], based on the selective cleavage of particular types of sulfidic crosslinks. This analysis was originally developed for natural rubber. Nowadays it is also often used for other types of elastomers [10,11]. Since the curing process is carried out at elevated temperatures, uncontrolled side reactions can also take place. Reactions of the curatives do not always lead to the formation of crosslinks to connect two polymer chains. Actually, part of the transformation products of the curatives can chemically attach to only one polymer chain. In this way, a non-elastic, intramolecular modification is formed as schematically presented in Figure 2 Depending on the curatives these modifications The formed intermolecular elastically effective crosslinks, are usually classiﬁed as carbon-carbon (C–C) crosslinks and sulﬁdic crosslinks. The C–C crosslinks are created directly between the polymer chains. In contrast, the sulﬁdic crosslinks are made of a variable number of sulfur atoms. Depending on this number, sulﬁdic crosslinks are further subdivided into (i) monosulﬁdic (C–S–C); (ii) disulﬁdic (C–S2–C); and (iii) polysulﬁdic (C–Sx–C, x ě 3) crosslinks. All of these crosslinks show varying structure and length, resulting in various characteristics. As a result, they provide different properties to the material. Therefore, it is generally assumed that after the crosslink density, the crosslink structure is the second most important parameter inﬂuencing the elastomer properties [7,8]. To characterize the various types of crosslinks, chemical analyses are used. The most commonly used is thiol-amine analysis [9], based on the selective cleavage of particular types of sulﬁdic crosslinks. This analysis was originally developed for natural rubber. Nowadays it is also often used for other types of elastomers [10,11]. is formed, as schematically presented in Figure 2. Depending on the curatives, these modifications exhibit various structures. They can be in a configuration of pendant groups or cyclic structures, containing sulfur. Furthermore, since the curatives are introduced in amounts of several parts per hundred rubber (phr), those modifications can be abundant. Since the curing process is carried out at elevated temperatures, uncontrolled side reactions can also take place. Reactions of the curatives do not always lead to the formation of crosslinks to connect two polymer chains. 1. Introduction It shows their influence on the network parameters and resulting properties Due to the increasing number of According to the literature [8,12], the elastomer properties are almost entirely dependent on the intermolecular crosslinks and their structure. At the same time, the presence of modiﬁcations to the polymer chains is barely mentioned. If these modiﬁcations are discussed, it is stated that they do not inﬂuence the material properties, or only to a limited extent. As a consequence, much attention is paid to the analysis of solely the crosslink density and their structures. In fact, there are no reliable direct methods to identify and quantify the modiﬁcations of the chains formed by the curatives. This causes difﬁculties in investigating these structures. The reactivity of the curatives can be very complex; therefore, it is a far-reaching simpliﬁcation to consider the curing process only as formation of intermolecular crosslinks. influence on the network parameters and resulting properties. Due to the increasing number of curatives available nowadays, there arises the need for comprehensive research on their role in the curing process. For this purpose, elastomers cured with various types of curatives were prepared. The structural parameters, such as crosslink density and crosslink structure were characterized, using chemical methods—equilibrium swelling and thiol-amine analysis. The behavior of the elastomers was investigated via monitoring the glass transition process. Two microscale techniques were used for this: differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The influence of the various curatives on the size of the free volumes and molecular structure was characterized by positron annihilation lifetime spectroscopy (PALS). The different results between the samples are discussed. They are explained on the basis of the curatives’ structures and modifications of the chains formed by them. 2. Experimental The present research discusses the unrecognized role of the elastomer curatives. It shows their inﬂuence on the network parameters and resulting properties. Due to the increasing number of curatives available nowadays, there arises the need for comprehensive research on their role in the curing process. For this purpose, elastomers cured with various types of curatives were prepared. The structural parameters, such as crosslink density and crosslink structure were characterized, using chemical methods—equilibrium swelling and thiol-amine analysis. The behavior of the elastomers was investigated via monitoring the glass transition process. Two microscale techniques were used for this: differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). 1. Introduction Actually, part of the transformation products of the curatives can chemically attach to only one polymer chain. In this way, a non-elastic, intramolecular modiﬁcation is formed, as schematically presented in Figure 2. Depending on the curatives, these modiﬁcations exhibit various structures. They can be in a conﬁguration of pendant groups or cyclic structures, containing sulfur. Furthermore, since the curatives are introduced in amounts of several parts per hundred rubber (phr), those modiﬁcations can be abundant. terials 2016, 9, 607 3 of Materials 2016, 9, 607 3 of 18 S S S S S2 Sy Sn Sz Sz S Acc Sy Sy C C (a) (c) (d) (b) (c) Figure 2. Structures formed as a result of curing intermolecular crosslinks: (a) carbon-carbon crosslinks; (b) sulfidic crosslinks, intramolecular modifications of the polymer chains: (c) cyclic sulfur structures; and (d) pendant groups. Figure 2. Structures formed as a result of curing intermolecular crosslinks: (a) carbon-carbon crosslinks; (b) sulﬁdic crosslinks, intramolecular modiﬁcations of the polymer chains: (c) cyclic sulfur structures; and (d) pendant groups. 3 of 17 18 Materials 2016, 9, 607 M i l 1 9 Figure 2. Structures formed as a result of curing intermolecular crosslinks: (a) carbon-carbon crosslinks; (b) sulfidic crosslinks, intramolecular modifications of the polymer chains: (c) cyclic sulfur structures; and (d) pendant groups. Figure 2. Structures formed as a result of curing intermolecular crosslinks: (a) carbon-carbon crosslinks; (b) sulﬁdic crosslinks, intramolecular modiﬁcations of the polymer chains: (c) cyclic sulfur structures; and (d) pendant groups. According to the literature [8,12], the elastomer properties are almost entirely dependent on the intermolecular crosslinks and their structure. At the same time, the presence of modifications to the polymer chains is barely mentioned. If these modifications are discussed, it is stated that they do not influence the material properties, or only to a limited extent. As a consequence, much attention is paid to the analysis of solely the crosslink density and their structures. In fact, there are no reliable direct methods to identify and quantify the modifications of the chains formed by the curatives. This causes difficulties in investigating these structures. The reactivity of the curatives can be very complex; therefore, it is a far-reaching simplification to consider the curing process only as formation of intermolecular crosslinks. The present research discusses the unrecognized role of the elastomer curatives. 1. Introduction The inﬂuence of the various curatives on the size of the free volumes and molecular structure was characterized by positron annihilation lifetime spectroscopy (PALS). The different results between the samples are discussed. They are explained on the basis of the curatives’ structures and modiﬁcations of the chains formed by them. 2 2 S l P 2.2. Sample Pre 2.2.1. Mixing 2.2. Sample Preparation 2.2.1. Mixing The samples were mixed in two steps. The first step was performed in an internal micromixer Brabender Plasti-Corder (Duisburg, Germany), with a mixing chamber of 75 mL. The mixing procedure employed was first plasticizing the E-SBR for one minute. After that, the 5 phr of ZnO and 1 phr of stearic acid were added. The total mixing time was 5 min, and the mixing temperature did not exceed 50 °C. The first mixing step was immediately followed by a second step, carried out on a David Bridge (Rochdale, UK) two-roll open mixing mill. After one minute of mixing on the two-roll mill, the curatives were added. In case of accelerated sulfur curing (series: DPG/S8, CBS/S8, MBTS/S8, ZDT/S8), the accelerators were firstly incorporated. After another 3 min, the sulfur was 2.2.1. Mixing The samples were mixed in two steps. The first step was performed in an internal micromixer Brabender Plasti-Corder (Duisburg, Germany), with a mixing chamber of 75 mL. The mixing procedure employed was first plasticizing the E-SBR for one minute. After that, the 5 phr of ZnO and 1 phr of stearic acid were added. The total mixing time was 5 min, and the mixing temperature did not exceed 50 °C. The first mixing step was immediately followed by a second step, carried out on a David Bridge (Rochdale, UK) two-roll open mixing mill. After one minute of mixing on the two-roll mill, the curatives were added. In case of accelerated sulfur curing (series: DPG/S8, CBS/S8, MBTS/S8, ZDT/S8), the accelerators were firstly incorporated. After another 3 min, the sulfur was added. The compounds were homogenized for 10 min, while the temperature did not exceed 40 °C. The compositions of the mixtures are given in Table 2. The samples were mixed in two steps. The ﬁrst step was performed in an internal micromixer Brabender Plasti-Corder (Duisburg, Germany), with a mixing chamber of 75 mL. The mixing procedure employed was ﬁrst plasticizing the E-SBR for one minute. After that, the 5 phr of ZnO and 1 phr of stearic acid were added. The total mixing time was 5 min, and the mixing temperature did not exceed 50 ˝C. The ﬁrst mixing step was immediately followed by a second step, carried out on a David Bridge (Rochdale, UK) two-roll open mixing mill. After one minute of mixing on the two-roll mill, the curatives were added. Arlanxeo, Maastrich Netherlands) e e a ) Siarkopol Tarnob Tarnobrzeg, Pol 2.2. Sample Preparation Arlanxeo, Maastrich Netherlands) ) Sia kopo a ob Tarnobrzeg, Pol 2.2. Sample Preparation 1500, with butadiene 2.1. Materials 1,2-vinyl, 10% 1,4-cis, and 75% 1,4-trans. The molecular weight of the SBR was Mn = 151,000 g/mol, Mw = 425,000 g/mol, and dispersity 2.8, as reported by Synthos. The Mooney viscosity was ML (1+4)100 °C = 50. Zinc oxide and stearic acid were obtained from Arlanxeo (Maastricht, The Netherlands). The curatives used are listed and characterized in Table 1. Acetone used for extraction, and toluene used for crosslink density analysis were of analytical grade, obtained from Assink Chemie (Enschede, The Netherlands). The chemicals for the thiol-amine probes: piperidine (99%), 2-propanethiol (≥98%), and 1-dodecanethiol (≥98%) were provided by Sigma-Aldrich (St. Louis, MO, USA). The polymer used in this study was a cold emulsion styrene-butadiene rubber (E-SBR) KER 1500, with 23.5% of bound styrene, produced by Synthos (Oswiecim, Poland). The ratio of styrene to butadiene units is therefore one to six or seven. The chemical structure of butadiene consists of 15% 1,2-vinyl, 10% 1,4-cis, and 75% 1,4-trans. The molecular weight of the SBR was Mn = 151,000 g/mol, Mw = 425,000 g/mol, and dispersity 2.8, as reported by Synthos. The Mooney viscosity was ML (1+4)100 ˝C = 50. Zinc oxide and stearic acid were obtained from Arlanxeo (Maastricht, The Netherlands). The curatives used are listed and characterized in Table 1. Acetone used for extraction, and toluene used for crosslink density analysis were of analytical grade, obtained from Assink Chemie (Enschede, The Netherlands). The chemicals for the thiol-amine probes: piperidine (99%), 2-propanethiol (ě98%), and 1-dodecanethiol (ě98%) were provided by Sigma-Aldrich (St. Louis, MO, USA). 4 of 17 Materials 2016, 9, 607 Name (Purity MB 2 2’ dibenzothi 2,2 -dibenzothia (94%; Arlanxeo (94%; Arlanxeo The Nethe The Net Table 1. Characterization of the curatives used in the experiments. Molec ( N S S N zyl disulfide Maastricht, C1 S S S S , aa i , rlands) NH G NH PG uanidine (97%; Table 1. Characterization of the curatives used in the experiments. 1500, with butadiene 2.1. Materials Molec ( N S S N zyl disulfide Maastricht, C1 S S S S , rlands) NH G NH PG uanidine (97%; Name (Purity; Producer) Structural Formula Molecular Formula, Molecular Weight (g/mol) MBTS 2,2’-dibenzothiazyl disulﬁde (94%; Arlanxeo, Maastricht, The Netherlands) S N S S S N N H N H NH S N S N H N S S N S S S S N S N S S N S S S S N S O P S Zn S P O O O S S MBTS 2,2’-dibenzothiazyl disulfide (94%; Arlanxeo, Maastricht, The Netherlands) C14H8N2S4 332.49 DPG 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) C13H13N3 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) C13H16N2S2 264.41 TMTD Tetramethylthiuram disulfide (96%; Arlanxeo, Maastricht, The Netherlands) C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulfide (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N′-dibenzylthio-carb amoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio phosphate [13] (50%; C32H68O4P2S4Zn C14H8N2S4 332.49 DPG 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) S N S S S N N H N H NH S N S N H N S S N S S S S N S N S S N S S S S N S O P S Zn S P O O O S S MBTS 2,2’-dibenzothiazyl disulfide (94%; Arlanxeo, Maastricht, The Netherlands) C14H8N2S4 332.49 DPG 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) C13H13N3 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) C13H16N2S2 264.41 TMTD Tetramethylthiuram disulfide (96%; Arlanxeo, Maastricht, The Netherlands) C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulfide (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N′-dibenzylthio-carb amoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The h l d C32H68O4P2S4Zn 772.47 C13H13N3 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) S S N H N H NH S N S N H N S S N S S S S N S N S S N S S S S N S O P S Zn S P O O O S S The Netherlands) DPG 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) C13H13N3 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) C13H16N2S2 264.41 TMTD Tetramethylthiuram disulfide (96%; Arlanxeo, Maastricht, The Netherlands) C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulfide (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N′-dibenzylthio-carb amoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The Netherlands) C32H68O4P2S4Zn 772.47 C13H16N2S2 264.41 TMTD Tetramethylthiuram disulﬁde (96%; Arlanxeo, Maastricht, The Netherlands) N H N H NH S N S N H N S S N S S S S N S N S S N S S S S N S O P S Zn S P O O O S S DPG 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) C13H13N3 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) C13H16N2S2 264.41 TMTD Tetramethylthiuram disulfide (96%; Arlanxeo, Maastricht, The Netherlands) C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulfide (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N′-dibenzylthio-carb amoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The Netherlands) C32H68O4P2S4Zn 772.47 C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulﬁde (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) N H N H NH S N S N H N S S N S S S S N S N S S N S S S S N S O P S Zn S P O O O S S DPG 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) C13H13N3 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) C13H16N2S2 264.41 TMTD Tetramethylthiuram disulfide (96%; Arlanxeo, Maastricht, The Netherlands) C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulfide (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N′-dibenzylthio-carb amoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The Netherlands) C32H68O4P2S4Zn 772.47 C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N1-dibenzylthio- carbamoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) N H N H NH S N S N H N S S N S S S S N S N S S N S S S S N S O P S Zn S P O O O S S 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) C13H13N3 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) C13H16N2S2 264.41 TMTD Tetramethylthiuram disulfide (96%; Arlanxeo, Maastricht, The Netherlands) C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulfide (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N′-dibenzylthio-carb amoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The Netherlands) C32H68O4P2S4Zn 772.47 C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The Netherlands) N H N H S N S N H N S S N S S S S N S N S S N S S S S N S O P S Zn S P O O O S S Arlanxeo, Maastricht, The Netherlands) 211.26 CBS N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) C13H16N2S2 264.41 TMTD Tetramethylthiuram disulfide (96%; Arlanxeo, Maastricht, The Netherlands) C6H12N2S4 240.43 TBzTD Tetrabenzylthiuram disulfide (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) C30H28N2S4 544.82 Vulcuren® 1,6-bis(N,N′-dibenzylthio-carb amoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) C36H40N2S6 693.11 ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The Netherlands) C32H68O4P2S4Zn 772.47 C32H68O4P2S4Zn 772.47 DCP Dicumyl peroxide (98%; Merck Schuchardt, Hohenbrunn, Germany) Materials 2016, 9, 607 5 of 18 O O DCP Dicumyl peroxide (98%; Merck Schuchardt, Hohenbrunn, Germany) C18H22O2 270.37 S8 Rhombic sulfur (99.9%; S S S S S S8 C18H22O2 270.37 S8 Rhombic sulfur (99.9%; Siarkopol Tarnobrzeg, Tarnobrzeg, Poland) Materials 2016, 9, 607 5 of 18 O O DCP Dicumyl peroxide (98%; Merck Schuchardt, Hohenbrunn, Germany) C18H22O2 270.37 S8 Rhombic sulfur (99.9%; Siarkopol Tarnobrzeg, Tarnobrzeg, Poland) S S S S S S S S S8 256.52 S8 256.52 Name (Purity; Producer) MBTS 2,2’-dibenzothiazyl disulf (94% A la eo Maa t i ) DPG 1,3-diphenylguanidine (97 A la eo Maa t i ht Th Arlanxeo, Maastricht, Th N th l d ) Netherlands) Name (Purity; Producer) MBTS 2,2’-dibenzothiazyl disulf (94%; Arlanxeo Maastric ) DPG 1,3-diphenylguanidine (9 Arlanxeo Maastricht Th Arlanxeo, Maastricht, Th Netherlands) Netherlands) MBTS 2,2’-dibenzothiazyl disulﬁde (94%; Arlanxeo, Maastricht, The Netherlands) 2,2’-dibenzothiazyl disulfid (94%; Arlanxeo, Maastricht The Netherlands) DPG 1,3-diphenylguanidine (97% Arlanxeo, Maastricht, The DPG 1,3-diphenylguanidine (97%; Arlanxeo, Maastricht, The Netherlands) (94%; Arlanxeo, Maastricht The Netherlands) DPG 1,3-diphenylguanidine (97% Arlanxeo, Maastricht, The Netherlands) CBS DPG 1,3-diphenylguanidine (97% Arlanxeo, Maastricht, The Netherlands) CBS N-cyclohexyl-1-benzothiazy 1,3-diphenylguanidine (97% Arlanxeo, Maastricht, The Netherlands) CBS N-cyclohexyl-1-benzothiazy sulfenamide (95%; Arlanxe Arlanxeo, Maastricht, The Netherlands) CBS N-cyclohexyl-1-benzothiazy sulfenamide (95%; Arlanxeo Maastricht, The Netherland Netherlands) CBS N-cyclohexyl-1-benzothiazy sulfenamide (95%; Arlanxeo Maastricht, The Netherland CBS N-cyclohexyl-1-benzothia sulfenamide (95%; Arlanx Maastricht, The Netherlan TMTD Netherlands) CBS N-cyclohexyl-1-benzothiaz N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) N cyclohexyl 1 benzothiaz sulfenamide (95%; Arlanxe Maastricht, The Netherland Maastricht, The Netherland TMTD TMTD Tetramethylthiuram disulfi TMTD Tetramethylthiuram disulfi (96%; Arlanxeo Maastrich Tetramethylthiuram disul (96%; Arlanxeo, Maastric The Netherlands) Netherlands) CBS N-cyclohexyl-1-benzothiaz N-cyclohexyl-1-benzothiazyl sulfenamide (95%; Arlanxeo, Maastricht, The Netherlands) N cyclohexyl 1 benzothiaz sulfenamide (95%; Arlanxe Maastricht, The Netherland Maastricht, The Netherland TMTD TMTD Tetramethylthiuram disulfi TMTD Tetramethylthiuram disulfi (96%; Arlanxeo Maastrich Tetramethylthiuram disul (96%; Arlanxeo, Maastric The Netherlands) CBS N-cyclohexyl-1-benzothiazy sulfenamide (95%; Arlanxeo Maastricht The Netherland N cyclohexyl 1 benzothiazy sulfenamide (95%; Arlanxe Maastricht, The Netherland TMTD Tetramethylthiuram disulfid (96% A l M t i ht TMTD Tetramethylthiuram disulﬁde (96%; Arlanxeo, Maastricht, The Netherlands) TMTD Tetramethylthiuram disulfi (96%; Arlanxeo, Maastrich The Netherlands) Tetramethylthiuram disulfid (96%; Arlanxeo, Maastricht The Netherlands) (96%; Arlanxeo, Maastricht The Netherlands) TBzTD The Netherlands) TBzTD Tetrabenzylthiuram disulf TMTD Tetramethylthiuram disulfid (96%; Arlanxeo, Maastricht, TMTD Tetramethylthiuram disulfid (96%; Arlanxeo, Maastricht, The Netherlands) The Netherlands) TBzTD Tetrabenzylthiuram disulfid TBzTD Tetrabenzylthiuram disulfid (70%; Shandong Yanggu H t i Ch i l Y TBzTD Tetrabenzylthiuram disulﬁde (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) Tetrabenzylthiuram disulfid (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) y (70%; Shandong Yanggu Huatai Chemical, Yanggu, China) ( g gg Huatai Chemical, Yanggu China) l TMTD thylthiura e Netherl TBzTD TBzTD zylthiura y Shandong ai Chemic TMTD Tetramethylthiuram disulfid Tetramethylthiuram disulfid (96%; Arlanxeo, Maastricht Th N th l d ) TBzTD TBzTD Tetrabenzylthiuram disulfid (70% Sh d Y Tetrabenzylthiuram disulﬁde (70%; Shandong Yanggu Huatai y (70%; Shandong Yanggu Huatai Chemical Yanggu ( g gg Huatai Chemical, Yanggu, China) China) C6H12N2S4 240.43 C30H28N2S4 C30H28N2S4 544.82 C30H28N2S4 544.82 544.82 C6H12N2S4 240.43 C30H28N2S4 C30H28N2S4 544.82 C30H28N2S4 544.82 544.82 TBzTD Tetrabenzylthiuram disulfid (70%; Shandong Yanggu H i Ch i l Y TBzTD Tetrabenzylthiuram disulfi (70%; Shandong Yanggu Huatai Chemical, Yanggu China) Huatai Chemical, Yanggu China) Vulcuren® China) Vulcuren® 1,6-bis(N,N′-dibenzylthio-c amoyl-dithio)-hexane (87% Vulcuren® 1,6-bis(N,N′-dibenzylthio-ca amoyl-dithio)-hexane (87% Arlanxeo, Maastricht, The Vulcuren® 1,6-bis(N,N1-dibenzylthio- carbamoyl-dithio)-hexane (87%; Arlanxeo, Maastricht, The Netherlands) Vulcuren® 1,6-bis(N,N′-dibenzylthio-ca amoyl-dithio)-hexane (87% Arlanxeo, Maastricht, The Netherlands) 1,6-bis(N,N -dibenzylthio- amoyl-dithio)-hexane (87 Arlanxeo, Maastricht, T Netherlands) Huatai Chemical, Yanggu China) Vulcuren® 1,6-bis(N,N′-dibenzylthio-ca Vulcuren® 1,6-bis(N,N′-dibenzylthio-ca amoyl-dithio)-hexane (87% A la eo Maa t i ht The 1,6-bis(N,N -dibenzylthio-ca amoyl-dithio)-hexane (87% Arlanxeo, Maastricht, The Netherlands) Arlanxeo, Maastricht, Th Netherlands) ZDT 2 ethylhexyl zinc Netherlands) ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50% ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50% Arlanxeo, Maastricht, The ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50%; Arlanxeo, Maastricht, The Netherlands) ZDT 2-ethylhexyl zinc dithio-phosphate [13] (50 Arlanxeo, Maastricht, T Netherlands) Materials 2016, 9, 607 DCP aterials 2016, 9, 607 DCP Dicumyl peroxide (98%; Me Schuchardt Hohenbrunn C36H40N2S6 693.11 C32H68O4P2S4Zn C32H68O4P2S4Zn 772.47 C32H68O4P2S4Zn 772.47 5 of 18 693.11 2H68O4P2S4Zn 772.47 270.37 C18H22O2 270.37 2.2.2. Curing After addition of the curing agents, the compounds curing properties were determined with an RPA2000 rubber process analyzer from Alpha Technologies (Bellingham, WA, USA). The increase in torque at 160 ˝C, 0.833 Hz and 2.79% strain was measured over a time period of 60 min. The optimal cure time (τ95) of the rubber compounds was determined. Rubber sheets with a thickness of 2 mm were cured in an electrically-heated Wickert laboratory press WLP 1600/54/3 (Landau, Germany) under 100 bar for the optimal cure time. The curing temperature for the S8 series was 180 ˝C, whereas for all of the other series it was 160 ˝C. The uncrosslinked reference (Ref.) was prepared by compression molding at a temperature of 160 ˝C for 10 min. 2.3. Extraction with Acetone To remove low molecular, non-polymer substances, the cured samples were extracted with acetone in a Soxhlet apparatus for 72 h. This was followed by drying them to a constant weight at 55 ˝C in a Heraeus (Hanau, Germany) oven with an air circulation system. The acetone-extracted samples were used for crosslink density analysis and DSC glass transition studies. 2 2 S l P 2.2. Sample Pre 2.2.1. Mixing In case of accelerated sulfur curing (series: DPG/S8, CBS/S8, MBTS/S8, ZDT/S8), the accelerators were ﬁrstly incorporated. After another 3 min, the sulfur was added. The compounds were homogenized for 10 min, while the temperature did not exceed 40 ˝C. The compositions of the mixtures are given in Table 2. 5 of 17 Materials 2016, 9, 607 Table 2. Composition of studied samples, given for 100 phr of E-SBR, 5 phr of zinc oxide, and 1 phr of stearic acid. Sample Name Amount of Curatives Added (phr) Ref. 0.0 DCP 0.35 0.60 0.75 1.20 2.20 - TMTD 1.5 3.0 4.5 5.5 7.0 10.0 TBzTD 2.5 5.0 8.0 11.0 15.0 22.0 Vulcuren® 3.0 5.0 7.0 9.0 11.0 18.0 S8 1.0 2.0 3.5 4.2 5.0 6.5 DPG/S8 1.5/1.5 2.0/2.0 2.5/2.5 3.0/3.0 3.6/3.6 4.1/4.1 CBS/S8 0.7/0.7 1.2/1.2 1.8/1.8 2.4/2.4 3.0/3.0 3.4/3.4 MBTS/S8 0.8/0.8 1.5/1.5 2.0/2.0 3.0/3.0 4.0/4.0 4.4/4.4 ZDT/S8 1.5/1.5 2.2/2.2 3.0/3.0 5.0/5.0 6.5/6.5 - .2.2. Curing 2.4. Crosslink Density Analysis To analyze the crosslink density, samples of approx. 0.05 g were swollen to equilibrium in toluene for four days at room temperature (23 ˘ 1 ˝C). This was followed by drying the samples to a constant weight for four days at 60 ˝C in a Heraeus (Hanau, Germany) oven with an air circulation system. The crosslink density was calculated on basis of the Flory-Rehner equation [3]: V “ ´ln p1 ´ Vr q ` Vr ` χV2 r V0 ´ Vr 1 3 ´ 2Vr f ¯ (1) (1) where: ν—crosslink density per unit volume (mol/cm3); Vr—volume fraction of rubber in a swollen sample (-); V0—solvent molar volume (for toluene: V0 = 106.9 cm3/mol); V0—solvent molar volume (for toluene: V0 = 106.9 cm3/mol); —functionality of crosslinks (f = 4, assuming the formation of tetra-functional crosslinks); f—functionality of crosslinks (f = 4, assuming the formation of tetra-functional crosslinks); χ—Flory-Huggins rubber-solvent interaction parameter (for investigated SBR-toluene system: χ = 0.378 [14]). χ—Flory-Huggins rubber-solvent interaction parameter (for investigated SBR-toluene system: χ = 0.378 [14]). The given crosslink density values are the average of four specimens of every sample. The relative standard deviation is approx. 3.0% on average. The given crosslink density values are the average of four specimens of every sample. The relative standard deviation is approx. 3.0% on average. Materials 2016, 9, 607 6 of 17 2.5. Crosslink Structure Analysis The structure of the crosslinks was determined by thiol-amine analysis [9]. This is based on selective cleavage of particular types of sulﬁdic crosslinks by two sets of thiol-amine chemical probes. Polysulﬁdic crosslinks were cleaved by the “soft” probe, composed of 2-propanethiol (0.4 M) and piperidine (0.4 M) in toluene. This analysis was run for 2 h under nitrogen gas atmosphere in a closed glass vial, using rubber samples pre-swollen in toluene for 12 h. Polysulﬁdic and disulﬁdic crosslinks were cleaved by the “hard” probe, which composition was 1-dodecanethiol (1.0 M) in piperidine. This analysis was run for 24 h under nitrogen gas atmosphere in a closed glass vial. After the thiol-amine analyses, the samples were removed from the probes immediately. The probe residues were removed from the samples by washing them at least ﬁve times with toluene for 30 min at room temperature. After this, the remaining crosslink density was analyzed by equilibrium swelling in toluene and calculated on the basis of the Flory-Rehner equation (according to the procedure described in Section 2.4.). 2.6. Buoyancy Method To determine the density of the uncrosslinked reference and the cured samples, a buoyancy method was used. The measurements were carried out in ethanol at a temperature of 23 ˝C. Analytical balance Radwag (Radom, Poland), equipped with a standard kit for density determination, was used. The given density is an average of six measurements. The relative standard deviation of the experiment was approx. 2%. 2.8. Dynamic Mechanical Analysis (DMA) Dynamic mechanical measurements were performed with use of a dynamic-mechanical analyzer DMA Viscoanalyseur VA2000 from Metravib (Limonest, France), in tension mode. The test frequency was ﬁxed at 10 Hz, and the strain at 0.1%, which was within the linear viscoelastic region of the studied samples. The dimensions of the test bar specimens were 40 ˆ 5 ˆ 2 mm3. Acquisition of the data was done using Dynatest 6.83 software. The measurements were carried out with a temperature sweep in the range of ´80 ˝C to 80 ˝C. In the temperature range of the E” and tan delta changes, the temperature interval was 1 ˝C. This allowed establishing an accurate determination of the Tg values. In the rest of the measurement, the interval was 5 ˝C. The initial stabilization time at ´80 ˝C was set to 15 min, whereas for other measurement points it was 3 min. 2.7. Differential Scanning Calorimetry (DSC) Differential scanning calorimetry (DSC) measurements were performed with use of a DSC Polyma 214 calorimeter from Netzsch-Geratebau (Selb, Germany). The samples of approx. 10 mg were placed in standard aluminum pans with pierced lids. The measurements were carried out in nitrogen atmosphere as follows: (i) cooled at a rate of 10 ˝C/min from 30 to ´110 ˝C; (ii)stabilized at ´110 ˝C for 3 min; and (iii) heated at a rate of 10 ˝C/min from ´110 to 30 ˝C. Prior extraction of the samples with acetone served to obtain smoother thermograms, without inﬂuencing the Tg values. A thermal history effect was checked by recording three measurement cycles for a representative set of samples. The same Tg values were obtained, which showed that the measurements were not sensitive to thermal history. The data shown here were, therefore, collected during the ﬁrst heating scan. The values of glass transition temperature were taken as the midpoints of the heat capacity change during the heating step. 3.1. Crosslink Density and Crosslink Structures In all studied series of samples, an increasing amount of the added curatives resulted in the formation of a higher crosslink density. This is due to more chemical species, which were involved in the formation of chemical bonds between the polymer chains. In Figure 3, the crosslink density and participation of the various crosslinks are presented. Curing with various curatives resulted in the formation of diversiﬁed crosslink structures. Use of organic peroxide, i.e., DCP, led to the formation of solely carbon-carbon crosslinks. During the curing process with pure TMTD, mainly C–C and monosulﬁdic crosslinks were formed. TBzTD acted upon curing in a similar way to TMTD and produced short crosslinks, mainly monosulﬁdic. TBzTD also belongs to the class of thiurams and has benzyl groups instead of the methyl groups present in a TMTD molecule. Use of Vulcuren® resulted in the formation of longer crosslinks, with major participation of the disulﬁdic ones. The structure of Vulcuren® is comparable with TBzTD and, additionally, has a six-carbon spacer between two disulﬁde groups. However, the crosslink structure of TBzTD- and Vulcuren®-cured samples differs noticeably. To produce a sufﬁcient crosslink density, the amount of incorporated TBzTD and Vulcuren® had to be very high. This is due to the high molecular weight of these molecules. Use of pure sulfur S8 led to the formation of predominantly polysulﬁdic and disulﬁdic crosslinks. This is caused by the fact that sulfur, alone, reacts slowly and mainly develops long crosslinks. In case of accelerated sulfur curing systems, DPG/S8, CBS/S8, MBTS/S8 and ZDT/S8, the ratio in phr between the accelerator and sulfur in all series was 1. This classiﬁes them as semi-efﬁcient curing systems. Accelerators, such as MBTS, DPG, and CBS, combined with rhombic sulfur S8, led to the formation of mainly polysulﬁdic and disulﬁdic crosslinks. The share of short crosslinks was very low for these samples. Curing with ZDT/S8 resulted in the formation of approximately equal participation of short C–C and monosulﬁdic crosslinks, medium-length disulﬁdic, and longer polysulﬁdic crosslinks. Materials 2016, 9, 607 8 of 18 CBS, combined with rhombic sulfur S8, led to the formation of mainly polysulfidic and disulfidic crosslinks. The share of short crosslinks was very low for these samples. Curing with ZDT/S8 resulted in the formation of approximately equal participation of short C–C and monosulfidic crosslinks, medium-length disulfidic, and longer polysulfidic crosslinks. Figure 3. 2.9. Positron Annihilation Lifetime Spectroscopy (PALS) The measurements of the positron lifetime spectra of the samples were performed using a fast–fast positron lifetime spectrometer with BaF2 scintillators. The time resolution (FWHM) of the lifetime spectrometer was approx. 250 ps. The positron source, 22Na enveloped in 7 µm Kapton foil, was 7 of 17 Materials 2016, 9, 607 located between the surfaces of two identical samples. This sandwich was positioned in front of the scintillation detectors of the spectrometer. The positron lifetime spectrum was measured for 24 h to obtain more than 2 ˆ 106 counts in the spectrum. Deconvolution of each spectrum was performed assuming three lifetime components. This was sufﬁcient to obtain a satisfactory χ2 value close to unity. The source component was also taken into account during the deconvolution procedure. The error bar of the free volume radius is expressed as the standard deviation of the measurement. 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetr 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry During the glass transition process, one of the properties that exhibit abrupt change is the heat capacity. The DSC technique serves to investigate this, and determine the glass transition temperature (Tg). The Tg measured in this way is sometimes referred to as “calorimetric”. Due to the fact that the sample is not exposed to external deformations, it is also called “static” Tg. Figure 4 shows changes in Tg as a function of crosslink density, for the uncrosslinked reference and the series of samples cured with various curatives. During the glass transition process, one of the properties that exhibit abrupt change is the heat capacity. The DSC technique serves to investigate this, and determine the glass transition temperature (Tg). The Tg measured in this way is sometimes referred to as “calorimetric”. Due to the fact that the sample is not exposed to external deformations, it is also called “static” Tg. Figure 4 shows changes in Tg as a function of crosslink density, for the uncrosslinked reference and the series of samples cured with various curatives. Figure 4. “Static” Tg as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 4. “Static” Tg as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 4. “Static” Tg as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 4. “Static” Tg as a function of crosslink density for the uncrosslinked reference and all cured series of samples. For the uncrosslinked reference (Ref.), the “static” Tg is approx. ´53 ˝C. It has no chemical crosslinks and the chains interact with each other via weak van der Waals forces. Hence, the value of Tg comes from the microstructure of SBR—its building blocks, the relative participation of styrene and butadiene, and molecular weight. Compared to the reference, for all cured samples the Tg values linearly increase with a rise in crosslink density. As a result of the crosslinking, polymer chains were united with short, strong covalent bonds. This made the cured material more compact and stiffer. Crosslinks are topological constraints, which hinder segmental mobility of the chains. The denser the network is, the shorter the molecular segments between crosslinks are. 3.1. Crosslink Density and Crosslink Structures Crosslink density and crosslink structures formed during the curing process with use of various curatives. The columns in each series correspond to the samples with an increasing amount of curatives, as listed in Table 2. Figure 3. Crosslink density and crosslink structures formed during the curing process with use of various curatives. The columns in each series correspond to the samples with an increasing amount of curatives, as listed in Table 2. Figure 3. Crosslink density and crosslink structures formed during the curing process with use of various curatives. The columns in each series correspond to the samples with an increasing amount of curatives, as listed in Table 2. Figure 3. Crosslink density and crosslink structures formed during the curing process with use of various curatives. The columns in each series correspond to the samples with an increasing amount of curatives, as listed in Table 2. Materials 2016, 9, 607 Figure 3. C various cur 8 of 17 3.2. Effect of Curing on the Glass Transition Temperature 3.2. Effect of Curing on the Glass Transition Temperature 3.2. Effect of Curing on the Glass Transition Temperature 3.2. Effect of Curing on the Glass Transition Temperature 3.2. Effect of Curing on the Glass Transition Temperature 3.2. Effect of Curing on the Glass Transition Temperature 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry The increase in the crosslink density is, therefore, followed by a rise in Tg. This is caused by higher thermal energy required to enable the molecular mobility of the polymer segments. p y g Actually, it is a well-known phenomenon [15,16] that the presence of crosslinks causes an increase in Tg. Fox and Loshaek [17] derived equations, predicting linear dependency of Tg on the crosslink density. This prediction concerned polymers with a ﬁxed composition and having a sufﬁciently low crosslink density. It assumed failure of linearity at higher crosslink densities. In the present research, good linearity is evident in the whole range of crosslink density for all series cured with various curatives. However, trends for particular series of samples show signiﬁcant differences. They come from other structural factors than the total crosslink density. Figure 4 shows that the increase in Tg is the lowest for the series cured with single-component curatives, such as DCP, TMTD, TBzTD, and Vulcuren®. The increase in Tg is higher in case of CBS/S8, DPG/S8 and pure sulfur S8 series. The largest change is for MTBS/S8 and ZDT/S8. To explain differences between the series, their structural features have to be discussed in detail. As a result of curing with an organic peroxide DCP, C-C crosslinks were formed. They are short and rigid and, hence, provide good elasticity and high stiffness to the material. It would be logical to assume that due to these characteristics they will cause a large increase in Tg. Actually, the situation 9 of 17 Materials 2016, 9, 607 is the opposite: an increase in the crosslink density caused little changes in Tg. Additionally, the DCP series is very much in line with the dependency for the TMTD series. Curing with TMTD also produced C–C and monosulﬁdic crosslinks. On the basis of these identical trends, a few remarks can be made. Firstly, DCP and TMTD led to formation of very short and undiversiﬁed crosslinks. Secondly, DCP and TMTD are curatives of low molecular weights and uncomplicated structures. Therefore, complex rearrangement reactions of these curatives were not possible. This made their reactivity at elevated temperature uncomplicated. Due to the low molecular weight, a part of the decomposition products possibly volatized and liberated from the system. Further, in addition to the formation of intermolecular crosslinks, the DCP and TMTD curatives modiﬁed the polymer chains by grafting on them. 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry However, due to their small size, the DCP and TMTD did not stiffen the polymer chains in this way. As a result, the chains remained ﬂexible and mobile. Due to all these factors, an increase in crosslink density caused very low rise in Tg. g For TBzTD and Vulcuren® series, the change of Tg with an increase in the crosslink density is also small. This might be caused by the presence of benzyl groups in the structure of those curatives. These groups are also present in the SBR structure as styrene units, which makes the TBzTD and Vulcuren® molecules alike to the polymer matrix. In spite of their large amount and bulkiness, they led to a minor change in heat capacity. Therefore, the increase in Tg was small. Additionally, despite the difference in the crosslink structure of TBzTD and Vulcuren® samples, their Tg changes are similar. This suggest that the crosslink structure is of minor importance. Curing with rhombic sulfur S8 led to a large increase in Tg as a function of the crosslink density. This increase is evidently higher than for the above discussed single-component curatives. Curing with pure sulfur is inefﬁcient—it has a slow curing rate. Moreover, it produces a large number of polysulﬁdic and intramolecular cyclic structures [18,19]. These numerous modiﬁcations restricted the free rotation possibilities of a single polymer chain. In this way, they hindered the mobility of the chains and considerably stiffened them. As a result, the Tg signiﬁcantly increased. g In case of accelerated sulfur curing, the increase in Tg as a function of crosslink density is strongly dependent on the type of accelerator used. The increase for the DPG/S8 and CBS/S8 series is lower, when compared to the sulfur cured samples (S8). This can arise from two facts. Firstly, when the accelerator is combined with sulfur, the formation of intermolecular crosslinks is more efﬁcient. It leads to lower quantity of intramolecular modiﬁcations of the polymer chains. Secondly, the DPG and CBS molecules have a low molecular weight (211.26 g/mol and 264.41 g/mol, respectively) and are small in size. Hence, the pendant groups of the accelerator fragments are not bulky. Therefore, they do not hinder the free rotation of the macromolecular segments to such a high degree like the cyclic sulfur structures do. These two effects can contribute to the observed trend lines as shown in Figure 4. 1 The number of the accelerators’ moles were calculated for the content of the pure active chemical substance in the commercially-available curatives, as listed in Table 1. 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry Additionally, DPG/S8 and CBS/S8 series exhibit very alike dependencies, because the accelerators are similar in molecular weight and size. As mentioned before, the ratio of accelerator to sulfur, expressed in “phr” unit, was 1. Considering the molecular weight of the curatives, and calculating their amount in moles, this ratio is slightly different. For DPG/S8 it equals 1.18, whereas for CBS/S8 it is 0.92 (Table 3). Table 3. Dependence between number of moles of accelerator 1 and number of moles of sulfur (Ratio = naccelerator/nsulfur). Sample Name Ratio (-) DPG/S8 1.18 CBS/S8 0.92 MBTS/S8 0.73 ZDT/S8 0.17 1 The number of the accelerators’ moles were calculated for the content of the pure active chemical substance in the commercially-available curatives, as listed in Table 1. Table 3. Dependence between number of moles of accelerator 1 and number of moles of sulfur (Ratio = naccelerator/nsulfur). 1 The number of the accelerators’ moles were calculated for the content of the pure active chemical substance in the commercially-available curatives, as listed in Table 1. Materials 2016, 9, 607 10 of 17 For the MBTS/S8 series, the increase in Tg is higher than for the series cured with pure sulfur. This is due to the structure of the MBTS molecule. It has a high molecular weight (332.49 g/mol) and aromatic heterocyclic moieties, which exhibit high stiffness. Fragments of the accelerator, together with sulfur, grafted onto the polymer chains during the curing process. Due to the bulkiness and inﬂexibility of these structures, this modiﬁcation caused considerable stiffening of the polymer chains. Consequently, it was followed by a large increase in Tg. The ratio between the number of moles of MBTS and S8 equals 0.73. It is lower than for the DPG/S8 and CBS/S8 series, in which the accelerator molecules are smaller. Furthermore, the MBTS/S8 samples have a similar crosslink structure compared to the DPG/S8 and CBS/S8 samples. However, the Tg values differ between those series. This proves that not the crosslink structure, but the accelerator type inﬂuences the behavior of the polymer chains. In case of the samples cured with ZDT/S8, the increase in Tg with rise in crosslink density is very large. It is much higher than for the S8 series, and the greatest from all the studied curing systems. This effect is due to the ZDT molecular weight of 772.47 g/mol, which is the largest of all used accelerators. 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry 3.2.1. Glass Transition Temperature Determined by Differential Scanning Calorimetry The ZDT structure is complex and has the bulky alkyl moieties. Similarly to the above discussed systems, modiﬁcations of the chains were formed by the curatives during the curing process. Due to their bulkiness, the mobility of the macromolecular segments was limited to a high extent. This caused a signiﬁcant increase in the Tg. Furthermore, ZDT is a polar molecule, which also possibly contributed to this rise. The ZDT fragments bound to the chains could interact with other similar fragments via intermolecular speciﬁc interactions. This additionally restricted the mobility of the polymer chains. As a result of curing, the crosslink structure developed in ZDT/S8 and sulfur S8 samples was similar. However, the Tg values of these two series differ signiﬁcantly. The comparable crosslink structure does not result in the same behavior of the polymer chains. It is the various types of intramolecular modiﬁcations, which explain the differences. In case of ZDT/S8, the bulky fragments of accelerator, together with sulfur, grafted of the polymer chains. In case of S8 series, the modiﬁcations were in a form of cyclic structures and pendant groups composed of sulfur. The combined ZDT/S8 curatives stiffened the polymer chains to a greater extent than solely sulfur S8. As shown in Table 3, one-to-one ratio between ZDT and S8 is as low as 0.17, when their number of moles is considered. This means that the number of ZDT molecules was in a very small amount in relation to the molecules of rhombic sulfur. In fact, “phr” is a simpliﬁed unit, used to easily calculate the amount of various additives in a rubber mix formulation on a weight basis. Due to its universality, it was widely accepted by rubber technologists and research scientists. However, as the above described examples show, the “phr” unit used to calculate the amount of curatives can be misleading. Curatives can exhibit diversified structure and molecular weight and this unit does not consider these important variables. Therefore, in case of substances, such as curatives, it is non-informational and indirect. To overcome this problem, the amount of curatives should be expressed in “mol” unit. It considers the molecular weight, so that the number of reacting molecules is clearly expressed. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis The “dynamic” Tg of the sulfur cured samples (S8), show a further increase in Tg. In case of MBTS/S8 cured samples, a higher increase in Tg is observed. This is due to the inflexible and bulky moieties of MBTS, combined with sulfur, causing a significant stiffening of the polymer chains. Finally, the most evident increase in Tg was observed for ZDT/S8. In this series the accelerator molecules are the largest and cause the highest stiffening of the polymer chains. Figure 5. Tan delta as a function of temperature for the uncrosslinked reference and the DPG/S8 cured series of samples. Figure 5. Tan delta as a function of temperature for the uncrosslinked reference and the DPG/S8 cured series of samples. a u y oie ie o , o i e i u u , au i g a ig i i a i e i g o e po y e chains. Finally, the most evident increase in Tg was observed for ZDT/S8. In this series the accelerator molecules are the largest and cause the highest stiffening of the polymer chains. Figure 6 depictures the “dynamic” Tg values as a function of the crosslink density, for all of the investigated samples. The “dynamic“ Tg shifts to higher values with increasing crosslink density when compared to the uncrosslinked reference. These changes of Tg are related to the chains’ relaxation. The “dynamic” Tg values show significant differences between the series cured with various curatives, as already observed in case of the “static” Tg. For the uncrosslinked reference, the “dynamic” Tg is approx. −42 °C. It is significantly larger than the “static” Tg (−53 °C). Such a difference is caused by frequency of 10 Hz in DMA measurement. Due to this, the “dynamic” Tg shifted to a higher temperature. In case of the cured samples, the lowest increase in the “dynamic” Tg is for the DCP and TMTD series. This corresponds to the dependencies in the “static” Tg. However, for the TBzTD and Vulcuren® samples, the increase in “dynamic” Tg is significantly larger than in case of the “static” Tg. This effect arises from differences in the measurement techniques of DSC and DMA. Both in DMA and DSC measurements, molecular motions of the polymer segments begin at a temperature associated with the glass transition. The polymer segments are restricted by the crosslinks and modifications. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis In DSC, the sample is static, whereas in DMA it is deformed. Thus, in DMA, the mobility of the chains is a response to the deformations of a particular frequency. As a result, Figure 6. “Dynamic” Tg, determined by a maximum of the tan delta peak, as a function of the crosslink density for the uncrosslinked reference and all cured series of samples. Figure 6. “Dynamic” Tg, determined by a maximum of the tan delta peak, as a function of the crosslink density for the uncrosslinked reference and all cured series of samples. associated with the glass transition. The polymer segments are restricted by the crosslinks and modifications. In DSC, the sample is static, whereas in DMA it is deformed. Thus, in DMA, the mobility of the chains is a response to the deformations of a particular frequency. As a result, Figure 6. “Dynamic” Tg, determined by a maximum of the tan delta peak, as a function of the crosslink density for the uncrosslinked reference and all cured series of samples. Figure 6. “Dynamic” Tg, determined by a maximum of the tan delta peak, as a function of the crosslink density for the uncrosslinked reference and all cured series of samples. g g g , possible on an experimental time scale. Curing with TBzTD and Vulcuren® led to the formation of The dynamic glass transition process is sometimes also ascribed to the maximum of the loss modulus (E’’) peak. Figure 7 presents the Tg values determined from E’’. The Tg for the uncrosslinked reference is approx. −50 °C. All cured series of samples show a tendency corresponding to the tan delta “dynamic” Tg. Differences between the maxima of the tan delta and For the uncrosslinked reference, the “dynamic” Tg is approx. ´42 ˝C. It is signiﬁcantly larger than the “static” Tg (´53 ˝C). Such a difference is caused by frequency of 10 Hz in DMA measurement. Due to this, the “dynamic” Tg shifted to a higher temperature. g g g possible on an experimental time scale. Curing with TBzTD and Vulcuren® led to the formation of The dynamic glass transition process is sometimes also ascribed to the maximum of the loss modulus (E’’) peak. Figure 7 presents the Tg values determined from E’’. The Tg for the uncrosslinked reference is approx. −50 °C. All cured series of samples show a tendency corresponding to the tan delta “dynamic” Tg. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis DMA tests were performed to study microscale changes in molecular mobility, as a response to externally applied mechanical deformations on the samples. In this way the “dynamic” Tg values were determined by the maximum of the tan delta peak. Figure 5 presents exemplary data from the DMA measurements, represented by the DPG/S8 series. They show the tan delta plotted versus the temperature for different crosslink densities. Figure 6 depictures the “dynamic” Tg values as a function of the crosslink density, for all of the investigated samples. The “dynamic“ Tg shifts to higher values with increasing crosslink density when compared to the uncrosslinked reference. These changes of Tg are related to the chains’ relaxation. The “dynamic” Tg values show signiﬁcant differences between the series cured with various curatives, as already observed in case of the “static” Tg. 11 of 17 the Materials 2016, 9, 607 DMA measur te e atu e f Figure 5. Tan delta as a function of temperature for the uncrosslinked reference and the DPG/S8 cured series of samples. Figure 5. Tan delta as a function of temperature for the uncrosslinked reference and the DPG/S8 cured series of samples. Materials 2016, 9, 607 12 of 18 stiff and bulky structures. They require high thermal energy to enable molecular mobility under dynamic conditions. The trend lines for the TBzTD and Vulcuren® series are similar to those of DPG/S8 and CBS/S8. The “dynamic” Tg of the sulfur cured samples (S8), show a further increase in Tg. In case of MBTS/S8 cured samples, a higher increase in Tg is observed. This is due to the inflexible and bulky moieties of MBTS, combined with sulfur, causing a significant stiffening of the polymer chains. Finally, the most evident increase in Tg was observed for ZDT/S8. In this series the accelerator molecules are the largest and cause the highest stiffening of the polymer chains. Figure 5. Tan delta as a function of temperature for the uncrosslinked reference and the DPG/S8 cured series of samples Figure 5. Tan delta as a function of temperature for the uncrosslinked reference and the DPG/S8 cured series of samples. Materials 2016, 9, 607 12 of 18 stiff and bulky structures. They require high thermal energy to enable molecular mobility under dynamic conditions. The trend lines for the TBzTD and Vulcuren® series are similar to those of DPG/S8 and CBS/S8. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis crosslink d The “dynamic” glass transition process is sometimes also ascribed to the maximum of the loss modulus (E”) peak. Figure 7 presents the Tg values determined from E”. The Tg for the uncrosslinked reference is approx. ´50 ˝C. All cured series of samples show a tendency corresponding to the tan delta “dynamic” Tg. Differences between the maxima of the tan delta and E” peaks are within the range of 8–11 ˝C. The values of E” “dynamic” Tg are close to values of “static” Tg. The “dynamic” glass transition process is sometimes also ascribed to the maximum of the loss modulus (E’’) peak. Figure 7 presents the Tg values determined from E’’. The Tg for the uncrosslinked reference is approx. −50 °C. All cured series of samples show a tendency corresponding to the tan delta “dynamic” Tg. Differences between the maxima of the tan delta and E’’ peaks are within the range of 8–11 °C. The values of E’’ “dynamic” Tg are close to values of “static” Tg. Figure 7. “Dynamic” Tg, determined by a maximum of E’’, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 7. “Dynamic” Tg, determined by a maximum of E”, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 7. “Dynamic” Tg, determined by a maximum of E’’, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 7. “Dynamic” Tg, determined by a maximum of E”, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. To evaluate the elastic properties of the studied samples, the height of the tan delta peaks was correlated with the crosslink density (Figure 8). The largest value was observed for the uncrosslinked reference, due to its high plasticity. In case of the cured samples, the increase in the crosslink density was followed by a decrease in the tan delta height. The elastic properties were, therefore, improved. This effect is caused by the higher number of crosslinks connecting the polymer To evaluate the elastic properties of the studied samples, the height of the tan delta peaks was correlated with the crosslink density (Figure 8). The largest value was observed for the uncrosslinked reference, due to its high plasticity. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis Differences between the maxima of the tan delta and For the uncrosslinked reference, the “dynamic” Tg is approx. ´42 ˝C. It is signiﬁcantly larger than the “static” Tg (´53 ˝C). Such a difference is caused by frequency of 10 Hz in DMA measurement. Due to this, the “dynamic” Tg shifted to a higher temperature. E’’ peaks are within the range of 8–11 °C. The values of E’’ “dynamic” Tg are close to values of “static” Tg. Figure 7. “Dynamic” Tg, determined by a maximum of E’’, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. g In case of the cured samples, the lowest increase in the “dynamic” Tg is for the DCP and TMTD series. This corresponds to the dependencies in the “static” Tg. However, for the TBzTD and Vulcuren® samples, the increase in “dynamic” Tg is signiﬁcantly larger than in case of the “static” Tg. This effect arises from differences in the measurement techniques of DSC and DMA. Both in DMA and DSC measurements, molecular motions of the polymer segments begin at a temperature associated with the glass transition. The polymer segments are restricted by the crosslinks and modiﬁcations. In DSC, the sample is static, whereas in DMA it is deformed. Thus, in DMA, the mobility of the chains is a response to the deformations of a particular frequency. As a result, segments of the chains undergo conformational rearrangements. It minimizes localized stress, as it is possible on an experimental time scale. Curing with TBzTD and Vulcuren® led to the formation of stiff and bulky structures. They require high thermal energy to enable molecular mobility under dynamic conditions. The trend lines for the TBzTD and Vulcuren® series are similar to those of DPG/S8 and CBS/S8. The “dynamic” Tg of the sulfur cured samples (S8), show a further increase in Tg. In case of MBTS/S8 cured samples, a higher increase in Tg is observed. This is due to the inﬂexible and bulky moieties of MBTS, combined with sulfur, causing a signiﬁcant stiffening of the polymer chains. Finally, the most evident increase in Tg was observed for ZDT/S8. In this series the accelerator molecules are the largest and cause the highest stiffening of the polymer chains. 12 of 17 Materials 2016, 9, 607 Figure 6. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis In case of the cured samples, the increase in the crosslink density was followed by a decrease in the tan delta height. The elastic properties were, therefore, improved. This effect is caused by the higher number of crosslinks connecting the polymer chains. The crosslinks serve to efﬁciently recover to the unstrained spatial network. The trend is the strongest for the TBzTD and Vulcuren® cured samples. Materials 2016, 9, 607 13 of 18 chains. The crosslinks serve to efficiently recover to the unstrained spatial network. The trend is the strongest for the TBzTD and Vulcuren® cured samples. Figure 8. Height of tan delta peak as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 8. Height of tan delta peak as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 8. Height of tan delta peak as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 8. Height of tan delta peak as a function of crosslink density for the uncrosslinked reference and all cured series of samples. The range of temperatures, in which the molecular mobility begins, was determined. For this, the width of the tan delta peak in the half of its height was calculated. Figure 9 shows values of this parameter, correlated with the crosslink density. An increase in the crosslink density resulted in a broadening of the tan delta peak. This is probably caused by more densely located structures on the chains formed by curatives. Additionally, the number and size of heterogenic microregions increased. These structures relaxed over a broader range of temperature. Changes in width of the tan delta peak are similar for most of the investigated series of samples, such as DCP, TMTD, S8, DPG/S8, CBS/S8, MBTS/S8, and ZDT/S8. However, in case of the TBzTD or Vulcuren® samples, this increase is The range of temperatures, in which the molecular mobility begins, was determined. For this, the width of the tan delta peak in the half of its height was calculated. Figure 9 shows values of this parameter, correlated with the crosslink density. An increase in the crosslink density resulted in a broadening of the tan delta peak. This is probably caused by more densely located structures on the chains formed by curatives. Additionally, the number and size of heterogenic microregions increased. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis These structures relaxed over a broader range of temperature. Changes in width of the tan delta peak 13 of 17 n a the 13 of 17 n a the Materials 2016, 9, 607 parameter, co broadening of are similar for most of the investigated series of samples, such as DCP, TMTD, S8, DPG/S8, CBS/S8, MBTS/S8, and ZDT/S8. However, in case of the TBzTD or Vulcuren® samples, this increase is much higher. This can be caused by the large amounts of TBzTD and Vulcuren® present in the samples. Consequently, the molecular movements during the glass transition process occurred over a broad range of temperature. increased. These structures relaxed over a broader range of temperature. Changes in width of the tan delta peak are similar for most of the investigated series of samples, such as DCP, TMTD, S8, DPG/S8, CBS/S8, MBTS/S8, and ZDT/S8. However, in case of the TBzTD or Vulcuren® samples, this increase is much higher. This can be caused by the large amounts of TBzTD and Vulcuren® present in the samples. Consequently, the molecular movements during the glass transition process occurred over a broad range of temperature. Figure 9. Width of tan delta peak in the half of height, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 9. Width of tan delta peak in the half of height, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 9. Width of tan delta peak in the half of height, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 9. Width of tan delta peak in the half of height, as a function of crosslink density for the uncrosslinked reference and all cured series of samples. e da pi g p ope ties at e evated te pe atu e, i.e., 0 C, we e eva uated by dete i atio of the tan delta. Figure 10 presents these values, correlated with the crosslink density. The dependence shows that an increase in the crosslink density resulted in a large decrease in damping. This is due to an increase in the number of crosslinks, which provide elastic response and lower The damping properties at elevated temperature, i.e., 70 ˝C, were evaluated by determination of the tan delta. Figure 10 presents these values, correlated with the crosslink density. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis The dependence shows that an increase in the crosslink density resulted in a large decrease in damping. This is due to an increase in the number of crosslinks, which provide elastic response and lower dissipation of energy. This dependence is similar in most of the investigated series of samples: DCP, TMTD, TBzTD, Vulcuren®, DPG/S8, CBS/S8, MBTS/S8, and ZDT/S8. However, in the case of samples cured with pure sulfur S8, the investigated ability to dissipate energy is slightly larger. This can be attributed to the fact that in addition to crosslinking, pure sulfur produced numerous modiﬁcations of the polymer chains. They are not elastically effective, hence the slightly larger dissipation of the energy. Additionally, the S8 series was cured at 180 ˝C, whereas all the other series at 160 ˝C. The higher temperature could result in increased chain scission in the sulfur-cured samples. This could contribute to different behavior of the S8 cured samples. Materials 2016, 9, 607 14 of 18 dissipation of energy. This dependence is similar in most of the investigated series of samples: DCP, TMTD, TBzTD, Vulcuren®, DPG/S8, CBS/S8, MBTS/S8, and ZDT/S8. However, in the case of samples cured with pure sulfur S8, the investigated ability to dissipate energy is slightly larger. This can be attributed to the fact that in addition to crosslinking, pure sulfur produced numerous modifications of the polymer chains. They are not elastically effective, hence the slightly larger dissipation of the energy. Additionally, the S8 series was cured at 180 °C, whereas all the other series at 160 °C. The higher temperature could result in increased chain scission in the sulfur-cured samples. This could contribute to different behavior of the S8 cured samples. Figure 10. Dependence between height of tan delta at 70 °C and the crosslink density for the uncrosslinked reference and all cured series of samples. Figure 10. Dependence between height of tan delta at 70 ˝C and the crosslink density for the uncrosslinked reference and all cured series of samples. Figure 10. Dependence between height of tan delta at 70 °C and the crosslink density for the uncrosslinked reference and all cured series of samples. Figure 10. Dependence between height of tan delta at 70 ˝C and the crosslink density for the uncrosslinked reference and all cured series of samples. 14 of 17 Materials 2016, 9, 607 As discussed above, the presence of crosslinks caused an increase in the Tg. 3.3.1. Density Determined by Buoyancy Method The density of the samples cured with various curatives was measured by buoyancy method. Figure 11 shows the density values as a function of crosslink density. Materials 2016, 9, 607 15 of 18 Figure 11. Density of samples as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 11. Density of samples as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 11. Density of samples as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 11. Density of samples as a function of crosslink density for the uncrosslinked reference and all cured series of samples. The presented dependence indicates the density caused by structural packing of the cured polymer chains. The uncrosslinked reference characterized itself with the lowest density. For all series of samples, the density linearly increased together with an increase in the crosslink density. The change in density varied for particular series of samples. The least dense packing was observed for DCP and TMTD series, increasing for CBS/S8, then DPG/S8, Vulcuren® and TBzTD. Further rise was observed for series cured with pure sulfur S8, followed by MBTS/S8. Finally, the highest increase was for the ZDT/S8 cured samples. The de ity ea u e e t i di ate that the ate ial be o e o e o a t a a e ult of The presented dependence indicates the density caused by structural packing of the cured polymer chains. The uncrosslinked reference characterized itself with the lowest density. For all series of samples, the density linearly increased together with an increase in the crosslink density. The change in density varied for particular series of samples. The least dense packing was observed for DCP and TMTD series, increasing for CBS/S8, then DPG/S8, Vulcuren® and TBzTD. Further rise was observed for series cured with pure sulfur S8, followed by MBTS/S8. Finally, the highest increase was for the ZDT/S8 cured samples. The density measurements indicate that the material becomes more compact as a result of curing. Furthermore, structures formed by curatives locate and pack tightly in areas between the polymer chains. In general, the larger the molecules of the curatives are, the denser the packing of polymer chains with crosslinks and grafted modifications is. 3.2.2. Glass Transition Process Monitored by Dynamic Mechanical Analysis This effect considerably differs between the samples cured with the various curatives. Therefore, other structural parameters than the total crosslink density contribute greatly to the changes in Tg. The obtained results show that the crosslink structure does not explain them. It can be concluded that this parameter is not of a crucial importance. The differences between the Tg values of particular samples are ascribed to the presence of the curatives. They chemically attach to the polymer chains and form intramolecular modiﬁcations. If the curatives substances are small in size, i.e., have a low molecular weight, they do not affect the ﬂexibility of the polymer chains. However, curatives having a high molecular weight, and bulky inﬂexible moieties, stiffen the polymer chains. It causes signiﬁcant decrease of mobility of the chains, especially in vinyl polymers [20], such as SBR. This rotational restriction of these macromolecules inﬂuences the glass transition process and increases the Tg values considerably. Furthermore, the chemical structures of the curatives are usually very different from the structure of the basic polymers. It can, therefore, be considered that as a result of curing, a copolymer is formed, as already proposed by Fox and Loshaek [17]. In this way, curatives grafted on the chains can be considered as functional groups, which modify the polymer chains. The Tg of the cured system is not only dependent on the crosslinks themselves. Additionally, the average composition of the system plays a role. 3.3. Effect of Curing on the Molecular Structure and Packing of the Polymer Chains 3.3. Effect of Curing on the Molecular Structure and Packing of the Polymer Chains 3.3.1. Density Determined by Buoyancy Method 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy PALS is a very capable experimental tool to study the structure of free volumes in polymers and related materials. In this context, free volume is deﬁned as an area of signiﬁcantly decreased electron density. Therefore, it is not occupied by any molecular structure. In the PALS spectrum of the polymers, the value of the longest lifetime component correlates with the size of the free volume. A direct relation between the value of this lifetime component and a radius of the free volume is given by the Tao-Eldrup equation [21,22]. This dependence is for a spherical symmetry of the free volume. Room-temperature PALS measurements were carried out to evaluate the effect of curing on the size of free volumes. Figure 12 shows changes in free volume size, expressed as a radius, as a function of crosslink density. PALS is a very capable experimental tool to study the structure of free volumes in polymers and related materials. In this context, free volume is defined as an area of significantly decreased electron density. Therefore, it is not occupied by any molecular structure. In the PALS spectrum of the polymers, the value of the longest lifetime component correlates with the size of the free volume. A direct relation between the value of this lifetime component and a radius of the free volume is given by the Tao-Eldrup equation [21,22]. This dependence is for a spherical symmetry of the free volume. Room-temperature PALS measurements were carried out to evaluate the effect of curing on the size of free volumes. Figure 12 shows changes in free volume size, expressed as a radius, as a function of crosslink density. Figure 12. Size of free volumes as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 12. Size of free volumes as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 12. Size of free volumes as a function of crosslink density for the uncrosslinked reference and all cured series of samples. Figure 12. Size of free volumes as a function of crosslink density for the uncrosslinked reference and all cured series of samples. 3.3.1. Density Determined by Buoyancy Method The density values show a direct correlation with Tg values, as investigated by DMA (Section 3.2.2). The density measurements indicate that the material becomes more compact as a result of curing. Furthermore, structures formed by curatives locate and pack tightly in areas between the polymer chains. In general, the larger the molecules of the curatives are, the denser the packing of polymer chains with crosslinks and grafted modiﬁcations is. The density values show a direct correlation with Tg values, as investigated by DMA (Section 3.2.2). Materials 2016, 9, 607 polymer chains w correlation with T 15 of 17 a direct 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy The size of free volume is the largest for the uncrosslinked reference. For all cured samples, an increase in the crosslink density resulted in a reduction of the free volume size. Moreover, the degree of this change is strongly dependent on the type of curatives used. The lowest decrease in free volume size, show the samples cured with DCP and TMTD—curatives of small size molecules. A visibly larger decrease in free volume size is observed for CBS/S8 and DPG/S8 series, in which both accelerator and sulfur are present. Therefore, the modiﬁcations with bulky side groups grafted on the chains inﬂuence the size of free volumes. They ﬁll them in to a higher extent and decrease their size. A further decrease occurred in case of the series cured with pure sulfur S8. Those crosslinked with TBzTD and Vulcuren® exhibited very similar dependence to sulfur-cured samples. The reduction of free volume size with the increase in crosslink density proceeded for MBTS/S8 series. Finally, the most evident drop occurred for the ZDT/S8 cured samples. The most rapid decrease in the measured structural parameter occurs therefore in case of curing systems, in which accelerators have bulky, inﬂexible moieties. When the crosslink structure is considered, it would be expected that the short crosslinks reduce the free volume size signiﬁcantly. Consequently, the longer crosslinks, such as disulﬁdic and polysulﬁdic, should result in larger size of free volume. However, the present results show the opposite dependencies. In case of DCP, TMTD, and TBzTD cured samples, the free volume size is larger than for the other samples with longer crosslinks. This proves that the crosslink structure does not inﬂuence the free volume size. For all the investigated series of cured samples, the decrease in free volume size is caused by the presence of crosslinks. They reduce the size of the unoccupied area between the polymer chains. The denser the network is, the smaller the free volumes are. Additionally, differences between particular series of samples indicate that the type of curatives greatly contribute to the observed changes. Part of the curatives grafted on the chains and ﬁlled up the free volumes. The higher the bulkiness of the modiﬁcations is, the more area is occupied and the free volume is reduced. Materials 2016, 9, 607 16 of 17 The presence of intermolecular crosslinks and modiﬁcations of the chains manifests itself via the studied properties. 4. Conclusions The present research reveals that the crosslink density and the crosslink structures characterize the elastomer network insufﬁciently. It is also the type and amount of the curatives that inﬂuence the microstructure and behavior of the polymers. In research, it is often overlooked that part of the curatives graft on the polymer chains. They do not connect the chains via an intermolecular crosslink, but form modiﬁcations. These modiﬁcations can be in a form of (i) pendant groups, as fragments of curatives, i.e., accelerators, sulfur, accelerator/sulfur complexes; or (ii) sulfur chains connected to different positions on the same polymer chain, forming cyclic structures. It is proved that besides the elastically-effective crosslinks, the modiﬁcations also inﬂuence the network parameters and the resulting properties. PALS measurements revealed that the free volume size is dependent on the crosslink density and the type of curatives used. Curatives with a high molecular weight and larger size form bulky and inﬂexible modiﬁcations. They signiﬁcantly reduce the free volume size. Density measurements showed that the larger the molecules of the curatives are, the denser the packing of cured polymer chains is. This conﬁrms that the curatives locate themselves in free volumes and cause a decrease in their size. The presence of the modiﬁcations manifests itself via stiffening of the chains and hindering their molecular mobility. This was proved by following the changes of the glass transition temperature with the use of DSC and DMA. The measurements showed that curatives with a large molecular weight, and present in a considerable amount, caused a signiﬁcant increase in Tg. In view of the results of this study, a straightforward way to correlate the curatives’ structure with properties is proposed. It is based on an evaluation of the curative size and structure. Furthermore, the presence of the chains’ modiﬁcations can be proved by DMA and PALS. For this, these techniques can be used independently or combined. Finally, a strong recommendation is made to express the amount of curatives in the number of moles, instead of phr. This will help to study the microstructure and resulting properties of elastomers in a more reliable way. Acknowledgments: This work was performed with support of Young Scientists’ Fund at the Faculty of Chemistry, Lodz University of Technology, Grants W-3/FMN/20G/2014 and W-3/FMN/35G/2015. 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy 3.3.2. Free Volume Size Determined by Positron Annihilation Lifetime Spectroscopy Among others, they inﬂuence the density of the samples, reduce the free volume size and stiffen the polymer chains. The present results stay in a good agreement with the theoretical predictions [17]. According to them, in crosslinked polymers the increase in Tg is followed by a decrease in the free volume size. This is due to the fact that crosslinks strongly connect the polymer chains and lead to a more compact material. The measured density of the samples, size of free volumes, and the Tg values, provide complementary information. They show the effect of various types of curatives on the structural parameters and network microstructure. These structural features are inﬂuenced to a great extent by the size of the curatives’ molecules. Due to that, the microstructure of cured elastomers can be predicted and basic structure-properties relations can be established. The chains’ modiﬁcations, “detected” by Tg and the size of free volumes, possibly affects also other properties of the studied materials, such as e.g., thermal stability and mechanical properties. Studies related to these issues will be a subject to our succeeding paper [23]. Acknowledgments: This work was performed with support of Young Scientists’ Fund at the Faculty of Chemistry, Lodz University of Technology, Grants W-3/FMN/20G/2014 and W-3/FMN/35G/2015. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Conclusions Author Contributions: Katarzyna Bandzierz, Louis Reuvekamp and Dariusz Bielinski conceived and designed the experiments; Katarzyna Bandzierz performed the crosslink density and crosslink structure analyses, density measurements, DSC and DMA experiments; Jerzy Dryzek performed the PALS measurements; Katarzyna Bandzierz analyzed the data and wrote the paper, with contribution of Louis Reuvekamp and Dariusz Bielinski. All authors have given approval to the ﬁnal version of the manuscript. 17 of 17 17 of 17 Materials 2016, 9, 607 References 1. Aleman, J.V.; Chadwick, A.V.; He, J.; Hess, M.; Horie, K.; Jones, R.G.; Kratochvil, P.; Meisel, I.; Mita, I.; Moad, G.; et al. Deﬁnitions of terms relating to the structure and processing of sols, gels, networks, and inorganic-organic hybrid materials (IUPAC Recommendations 2007). Pure Appl. Chem. 2007, 79, 1801–1829. [CrossRef] 2. Coran, A.Y. Vulcanization. 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Technol. 2012, 85, 350–386. [CrossRef] 7. Hertz, D.L. Theory and Practice of vulcanization. Elastomerics 1984, 116, 17–21. 8. Dogadkin, B.A. Chemistry of Elastomers, 1st ed.; WNT: Warsaw, Poland, 1976; pp. 201–311. 9. Saville, B.; Watson, A.A. Structural Characterization of Sulfur-Vulcanized Rubber Networks. Rubber Chem. Technol. 1967, 40, 100–148. [CrossRef] 10. Diez, J.; Bellas, R.; Lopez, J.; Santoro, G.; Marco, C.; Ellis, G. Study of the crosslink density, dynamo-mechanical behaviour and microstructure of hot and cold SBR vulcanizates. J. Polym. Res. 2010, 17, 99–107. [CrossRef] 11. Kiroski, O.; Sims, J.; Packham, D.E.; Gregory, A.L. The use of thiol-amine chemical probes in network characterisation of NBR vulcanizates. Kautsch. Gummi Kunstst. 1997, 10, 716–720. 12. Rader, C.P. Vulcanization of Rubber—A. Sulfur and Non-Peroxides. In Basic Elastomer Technology, 1st ed.; Baranwal, K.C., Stephens, H.L., Eds.; Rubber Division: Akron, OH, USA, 2001; pp. 165–191. 13. 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In Natural Rubber Science and Technology, 1st ed.; Roberts, A.D., Ed.; Oxford University Press: Oxford, UK, 1988; pp. 511–620. 19. Zaper, A.M.; Koenig, J.L. Solid-state 13C NMR studies of vulcanized elastomers, 4. Sulfur-vulcanized polybutadiene. Makromol. Chem. 1988, 189, 1239–1251. [CrossRef] 20. Pious, C.V.; Thomas, S. Polymeric Materials—Structure, Properties, and Applications. In Printing on Polymers: Fundamentals and Applications, 1st ed.; Izdebska, J., Thomas, S., Eds.; William Andrew: Norwich, NY, USA, 2016; pp. 21–40. 21. Tao, S.J. Positronium annihilation in molecular substances. J. Chem. Phys. 1972, 56, 5499–5510. [CrossRef] 21. Tao, S.J. Positronium annihilation in molecular substances. J. Chem. Phys. 1972, 56, 5499–5510. [CrossRef] 22 Eldrup M ; Lightbody D ; Sherwood N J The temperature dependency of positron lifetimes in solid pivalic 21. Tao, S.J. Positronium annihilation in molecular substances. J. Chem. Phys. 1972, 56, 5499–5510. [CrossRef] 22. Eldrup, M.; Lightbody, D.; Sherwood, N.J. The temperature dependency of positron lifetimes in solid pivalic acid Chem Phys 1981 63 51 58 [CrossRef] 22. Eldrup, M.; Lightbody, D.; Sherwood, N.J. The temperature dependency of positron lifetimes in solid pivalic acid. Chem. Phys. 1981, 63, 51–58. [CrossRef] 23. Bandzierz, K.; Reuvekamp, L.; Dierkes, W.; Blume, A.; Bielinski, D. Inﬂuence of network structure on properties of elastomers. 2016, under preparation. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4281675657	https://link.springer.com/content/pdf/10.1007/s11116-022-10287-8.pdf	English	null	A minute of your time: The impact of survey recruitment method and interview location on the value of travel time	Transportation	2,022	cc-by	16,643	Abstract Web-based stated preference (SP) surveys are widely used to estimate values of travel time (VTT) for cost–benefit analysis, often with internet panels as the source of recruit- ment. The recruitment method could potentially bias the results because (1) those who fre- quently participate in surveys may have a lower opportunity cost of time and (2) people who answer the survey at home or in the office may answer differently because the choice situation is less salient to them. In this paper, we investigate both mechanisms using data from a VTT choice experiment study where respondents were recruited from an internet panel, an alternative email register or on-board/on the station. Within all three groups, some complete the survey while making an actual trip. We find that respondents who were recruited from the internet panel or report being members of a panel have a significantly lower VTT, suggesting that internet panels are less representative in this respect compared to other recruitment methods. We also find that those who answer while traveling have a higher VTT, possibly because the benefits of saving travel time are more salient to them than to those who answer while not traveling. Keywords Value of travel time · Stated preference · Survey methods · Internet panel · Hypothetical bias Askill H. Halse1 · Stefan Flügel1 · Marco Kouwenhoven2 · Gerard de Jong2 · Hanne B. Sundfør1 · Nina Hulleberg1 · Guri N. Jordbakke1 · Henrik Lindhjem3,4 Accepted: 30 March 2022 / Published online: 26 May 2022 © The Author(s) 2022, corrected publication 2023 Introduction Survey-based stated preference (SP) studies are widely used to estimate values of travel time (VTT) for application in cost–benefit analysis (Wardman et al. 2016; Flügel and Halse 2021). This approach has at least two important potential drawbacks: One is that those who spend time answering a survey may have a lower opportunity cost of time than the typical * Askill H. Halse ash@toi.no 1 Institute of Transport Economics (TØI), Oslo, Norway 2 Significance, The Hague, The Netherlands 3 Menon Centre for Environmental and Resource Economics, Oslo, Norway 4 Norwegian Institute for Nature Research (NINA), Oslo, Norway A minute of your time: The impact of survey recruitment method and interview location on the value of travel time Askill H. Halse1 · Stefan Flügel1 · Marco Kouwenhoven2 · Gerard de Jong2 · Hanne B. Sundfør1 · Nina Hulleberg1 · Guri N. Jordbakke1 · Henrik Lindhjem3,4 Transportation (2023) 50:1553–1584 https://doi.org/10.1007/s11116-022-10287-8 Transportation (2023) 50:1553–1584 https://doi.org/10.1007/s11116-022-10287-8 * Askill H. Halse ash@toi.no 123456789) 1 3 456789) 1 3 Transportation (2023) 50:1553–1584 1554 traveler. This is an issue of representativeness that can only partly be accounted for by controlling for observed respondent characteristics. The other potential drawback is that people might choose differently in a hypothetical choice setting than when making actual travel choices. This issue relates to the choice context and interview situation.l The influence of these factors is likely to depend on the survey recruitment and inter- view method chosen. First, internet panels are increasingly used as a source of recruitment for surveys in social science (Callegaro et al. 2014), including in SP surveys (Sandorf et al. 2022). While all voluntary recruitment potentially implies self-selection, those recruited from an internet panel will typically have spent more time answering other surveys before, and a significant share of them may be motivated by the relatively small compensation paid per minute. This could imply that panel members are less representative in terms of time and/or cost preferences than respondents recruited from other sources. Using data from the 2009–2011 Dutch VTT study, Significance et al. (2013) and Kouwenhoven et al. (2014) found that panel members have substantially lower VTT. Second, respondents are often invited via email and complete the survey online. This applies to studies using internet panels as well as other studies based on e-mail recruit- ment. An alternative to this is to recruit and interview people while they are traveling. In both cases, the choice questions are hypothetical, but the answers people give could depend on how realistic they perceive the choice situation. Those who answer the survey while traveling are likely to feel closer to the choice situation than those who answer while not traveling, typically at home or in their office. The VTT estimated based on the latter group could suffer from a type of hypothetical bias that could potentially go in either direction. In light of the phenomenon that people perceive experiences as more positive in retrospect (Mitchell et al. 1997), one hypothesis is that those who answer the survey while not trave- ling are less able to relate to the discomfort of travel or and therefore put less weight on travel time. 1 Contrary to this hypothesis, Hanssen (2012) found evidence suggesting that those interviewed on board have a lower VTT in his study of ferry passengers. See Section "Survey and interview methods in VTT studies". 2 Those who answer while traveling will answer based on their current trip, while the rest will answer based on a recent trip. Hence, this effect can also be interpreted as an effect of choice context. We return to this in Section “Discussion”. 1 Contrary to this hypothesis, Hanssen (2012) found evidence suggesting that those interviewed on board have a lower VTT in his study of ferry passengers. See Section "Survey and interview methods in VTT studies". 2 Those who answer while traveling will answer based on their current trip, while the rest will answer ff 2 Those who answer while traveling will answer based on their current trip, while the rest will answer based on a recent trip. Hence, this effect can also be interpreted as an effect of choice context. We return to this in Section “Discussion”. 1 Contrary to this hypothesis, Hanssen (2012) found evidence suggesting that those interviewed on board have a lower VTT in his study of ferry passengers. See Section "Survey and interview methods in VTT studies". 3 * Askill H. Halse ash@toi.no This factor would also imply a downward bias in the VTT.1 We investigate both issues based on data from a large-scale SP survey which was con- ducted in Norway in 2018 with the purpose to estimate national unit values for the VTT. Our data has three important features: (1) Respondents were recruited from three different sources – an internet panel, an alternative email register, and intercept. (2) All respondents were asked whether they were members of an internet panel. This means that we can inves- tigate the effect of internet panel membership on VTT more generally, not just whether members of this particular panel have a different VTT. (3) Those who participated online were asked if they were currently traveling while answering the survey. Hence, we can estimate the effect of interview location2 (traveling or not traveling), controlling for recruit- ment method.ii We find that those who report to be active members of an internet panel have a signifi- cantly lower VTT than non-members, also among those who were not recruited from an internet panel for this particular survey. This suggests that internet panel members have a lower opportunity cost of time which negatively impacts their VTT, and that this may be a general characteristic of internet panels. Although our samples differ somewhat in 1 3 Transportation (2023) 50:1553–1584 1555 observable characteristics, the results are highly robust across different model specifica- tions with different sets of control variables. They are also in line with the evidence from the 2009–2011 Dutch study.ii observable characteristics, the results are highly robust across different model specifica- tions with different sets of control variables. They are also in line with the evidence from the 2009–2011 Dutch study.ii We also find that those who answer while not traveling have a significantly lower VTT compared to those who answer while traveling. This effect is also highly robust across specifications. One possible explanation for this finding is that when people are further away from the context that the choice situation mimics, this results in a downward hypo- thetical bias. This potential mechanism could be further explored in future studies using an experimental approach. Our study contributes to a small existing literature on the impact of survey recruitment method in VTT studies (Börjesson and Algers 2011; Hanssen 2012; Significance et al. 2013; Lu et al. * Askill H. Halse ash@toi.no 2018) as well as to the more general and much larger literature on sur- vey mode effects and the representativeness of internet panels in environmental economics (e.g. Lindhjem and Navrud 2011a; Boyle et al. 2016; Menegaki et al. 2016; Sandorf et al. 2020, 2022), and in the survey method literature more generally (e.g. Callegaro et al. 2014; 2015; Zhang et al. 2020). As internet panels and web surveys are widely used in SP sur- veys, partly for cost reasons, our results have potentially strong implications for research and practice related to the value of travel time. Not accounting for these issues could poten- tially bias the results of cost–benefit analysis of transport projects. i Our paper proceeds as follows: In Section "Previous literature", we review the litera- ture on hypothetical bias and survey methods in VTT studies as well as other SP studies, focusing particularly on the experiences from the 2009–2011 Dutch VTT study. In Sec- tion "Survey design and data collection", we describe the survey design of the 2018 Nor- wegian VTT study, and in Section "Data overview" we present some key characteristics of the data. Section "Empirical modeling" explains our empirical specification and shows the estimated effects of survey recruitment method and interview location on the VTT. Section “Discussion” gives a discussion of the findings and Section "Conclusion" concludes. 3 Measured in different ways for example completeness (e.g., item nonresponse), accuracy (comparison with external benchmark data, e.g., actual votes), reliability (e.g., psychometric scale properties) and more generally comparing response distributions of key constructs under study. In SP, indicators such as the ten- dency to choose status quo or other alternatives and the scale parameter (error variance) (for stated choice experiments) (Sandorf et al. 2020, 2022), zero, protest, ‘don’t know’ responses and variance in willingness to pay (contingent valuation) have been used (Lindhjem and Navrud 2011b). Hypothetical choice situations and bias in SP studies Stated preference (SP) studies are hypothetical in their nature, which means that there is no guarantee that respondents make the same choices in the survey as they would have done in a real-life setting. There is an extensive literature exploring possible biases in SP results due to this (Harrison 2014). In environmental economics, the typical concern is that respondents will overstate their willingness to pay for environmental goods (e.g., Loomis 2011). However, this could be mitigated by using various good practice principles, includ- ing consequentiality designs and stated choice questions including multiple attributes, forc- ing the respondent to make tradeoffs on more than just one margin (Johnston et al. 2017). f In SP studies of the VTT, the VTT is often estimated based on stated choice questions with only travel time and cost as attributes, but sometimes also on stated choice experi- ments with more than two attributes (Flügel and Halse 2021). Comparing with the results of revealed preference (RP) studies, the typical finding is that the VTT is lower in SP stud- ies (Small, 2012). Shires and de Jong (2009) find that SP values are at least 25 percent lower for non-business trips, while Wardman et al. (2016) find SP values to be about 10–15 percent lower than those from RP studies. A possible reason is that respondents pay more 1 3 3 Transportation (2023) 50:1553–1584 1556 attention to travel cost in the SP setting, where costs are explicitly presented, than they do when making real-life travel choices. Another concern is the perceived realism of the choice task. In many recent studies, the attribute values of travel time and travel costs are centered around actual travel time and cost of a recent reference trip reported by the respondent. This could be expected to increase realism but might lead to a higher degree of reference dependence in the responses (Hess et al. 2020). The impact of this on average estimated VTT is however ambiguous. Hence, while hypothetical bias has been thoroughly studied in the valuation of environ- mental and public goods, there is little research to build on to derive expectations related to the effect of hypothetical vs. actual choice situations in the valuation of travel time. 4 Since social desirability bias is more frequently observed in settings with interviewers and since the VTT is a relatively neutral topic (compared to e.g. valuing public goods such as protection of the environment), this effect is unlikely to be important in our context. Transportation (2023) 50:1553–1584 Transportation (2023) 50:1553–1584 1557 A recent study of internet panel data quality found that experienced opt-in internet pan- elists are more likely to report “for money” as their main motivation, indicating that they trade off their time for very small monetary benefits (Zhang et al. 2020). The same study also finds, somewhat surprisingly, that these more experienced (‘professional’) respondents tend to provide answers of at least as high quality as other panelists who are less motivated by money. In the SP literature, most studies to date have found small to moderate effects on data quality and estimated monetary measures when comparing internet panel data with other recruitment methods (e.g. Lindhjem and Navrud 2011a, b; Boyle et al. 2016). Recent and emerging SP research, however, show that data quality of internet panels may depend on survey experience and degree of ‘professionalism’ among respondents (Sandorf et al. 2020, 2022). This suggests that even if some concerns about the effects of internet panel recruitment in SP have been put to rest, such effects are still likely to be context dependent, as pointed out by the recent SP guideline by Johnston et al. (2017; p. 340). Furthermore, they might be of particular relevance when estimating a monetary measure such as the VTT, since this measure is so directly related to respondents’ opportunity cost of time.f Even if the internet panel effect on indicators of data quality and rational responses may be small or moderate in SP surveys, there may still be substantial and economically sig- nificant effects on monetary measures estimated based on such data. As hypothesized here, this can be due to unobservable differences in the underlying preferences of (professional) internet panelists compared to the general population (or more precisely: respondents recruited by other means). While other SP studies have found similar or somewhat lower welfare measures from internet panel data compared to other survey modes (e.g. Lindhjem and Navrud 2011a; Boyle et al. 2016), these are not directly comparable with VTT.5 Fur- ther, the general context-dependence of survey mode effects means that it is difficult to generalize regarding the relative magnitude of the different underlying mechanisms that are driving the effects. In the following, we will investigate indicators of both data quality and effects on VTT. 5 Lindhjem and Navrud (2011a) believe some of this effect may be due to more honest responses and less social desirability bias in internet panel surveys giving lower willingness to pay responses. 6 Note that ‘intercept’ could imply either that respondents answer the survey right away on the location where they are recruited (on-site) or that they leave their contact info or receive a link to the questionnaire and answer it later. 7 In the 2012 German study, the non-business respondents who were recruited by phone were asked if they wanted to complete the survey on-line. However, only 5.6 percent of the respondents choose to do so which does not allow for a sensible test regarding the effect of interview type (Dubernet and Axhausen 2020). In the 2014 UK study, no results on the effect of source of recruitment or interview type are reported (Batley et al. 2017). 5 Lindhjem and Navrud (2011a) believe some of this effect may be due to more honest responses and l social desirability bias in internet panel surveys giving lower willingness to pay responses. Effects of survey and interview methods There is a growing interest in the effects of survey method or mode on data quality3 and results in the general survey methodology literature, more recently also picked up by the SP research community (e.g. Lindhjem and Navrud 2011a; Boyle et al. 2016). Apart from potential differences in self-selection and nonresponse between different survey/recruit- ment modes, the main concern is related to measurement effects. If similar respondents provide systematically different answers to the same survey depending on whether answer- ing e.g. on or off-site, by email recruitment or through an internet panel survey, a so-called “pure survey mode effect” is present (Lindhjem and Navrud 2011a).f f The two main sets of factors through which this effect is believed to occur are related to (1) cognitive or psychological factors and (2) normative or sociological (Dillman et al. 2014). The former is particularly related to potential satisficing behavior, i.e. shortcutting the thought process and providing a suboptimal response. The latter factor is especially related to social desirability bias, i.e. the tendency of survey respondents to answer ques- tions in a manner that will be viewed favorably by others (e.g. an interviewer).4 The rise of internet panels in survey research has raised concerns over the extent of satisficing behavior and potentially lower quality of internet panel data. A key concern is “professional respondents” answering a large number of surveys fast and with little effort, primarily for the small monetary incentive they typically receive (Callegaro et al. 2014; Hillygus et al. 2014). It seems, however, from recent research that data quality may not be systematically lower in internet panels compared to other types of survey modes, at least for a large share of respondents, indicating that satisficing behavior may not be more prevalent among average internet panelists (Zhang et al. 2020). This is the case also when considering that many such respondents answer on smartphones or tablets while traveling, exposing themselves to potentially higher level of distractions (Wenz 2019; de Bruijne and Oudejans 2015; Skeie et al. 2019; Liebe et al. 2015; Sandorf et al. 2022). 1 3 Survey and interview methods in VTT studies Table 1 below gives an overview of recent national VTT studies in Europe, including infor- mation on how respondents were recruited and how interviews were carried out.6 As we can see, most studies only relied on one source of recruitment and one type of interview, at least per traveler segment. Also, source of recruitment and type of interview might be bundled.7 However, there are a few studies that exploit within-study comparisons in survey method.f Börjesson and Algers (2011) use data from different samples collected in the 2007–2008 Swedish VTT study and investigate the relationship between interview method and average 1 3 3 1558 Transportation (2023) 50:1553–1584 Table 1 Recruitment method in recent national value of travel time (VTT) studies in Europe Country (year of data collection) Type of recruitment Type of interview /questionnaire References Switzerland (2003) From another survey (KEP2) Paper–pencil Axhausen et al. (2006; 2008) Denmark (2004) Internet panel Web-survey Fosgerau et al. (2007) Phone panel Personal interview Sweden (2007, 2008) Number plate register (2007) Telephone interview Börjesson and Eliasson (2014) Population register (2008, car) Web-survey or telephone interview Intercept method (2008, public transport) Web-survey or telephone interview Norway (2009) Internet panel Web-survey Ramjerdi et al. (2010) Netherlands (2009, 2011) Internet panel (2009) Web-survey Significance et al. (2013), Kouwenhoven et al. (2014) Intercept method (2011) Web-survey Germany (2012) Phone (non-business) Web-survey or paper–pencil Ehreke et al. (2014), Dubernet and Axhausen (2020) Panel (business) Web-survey UK (2014) Intercept method and telephone Web-survey or telephone interview Batley et al. (2017) Norway (2018) Internet panel, email register, and intercept method Web-survey Flügel et al. (2020) 1559 Transportation (2023) 50:1553–1584 VTT as well as error variance in the choices made by respondents. They find that error variance is larger in the data from telephone interviews than in the data based on an online questionnaire. This seems to be related to characteristics of the interview situation itself, like time pressure. The authors also find a 45 percent lower VTT in the telephone sample, but the difference is only 9 percent when controlling for socio-economic characteristics. Hence, the authors conclude that this effect can largely can be attributed to differences in socioeconomic characteristics. We should have in mind that this study is from a time when internet use was probably more highly correlated with socioeconomic status. Among other VTT studies not considered national, Lu et al. (2018) use both internet recruitment and a roadside interception as methods of recruitment for overlapping travel purposes/groups (as well as recruitment by phone and through Facebook). In this study about the Copenhagen Harbour Tunnel (a new toll tunnel), little evidence is found that the survey recruitment methodology impacted the resulting VTT. Covariates are included in the models to investigate whether the VTT varied by the different survey data collection methods. These covariates are found to be insignificant in the final models. The internet panel used was that of Kantar Gallup. 3 The researchers hint at the possibility that the pan- el’s positive outcome may be a result of the high quality of the Kantar Gallup panel.f There is also one study that exploits differences in interview location, similar to our study. Hanssen (2012) analyzes data from an SP survey among ferry passengers in Norway, where some of those who were recruited onboard the ferry answered on-site and some answered later in another location. In addition, some were recruited via mail. Somewhat contrary to expectations, Hanssen finds that those who answered on-site have a 23 percent lower value of travel time, but a 31 percent higher value of service frequency than the two other samples. However, it is not clear to what extent this can be interpreted as an effect of interview location, or whether it also picks up an effect of recruitment method. Evidence from the Dutch VTT study In this section, we present evidence on the effect of internet panel membership from the 2009–2011 Dutch study (Significance et al. 2013; Kouwenhoven et al. 2014), includ- ing some evidence based on new analysis of the data. This study is of particular interest because of the substantial effect of recruitment mode that was identified and the adjust- ments that were made to the data collection and survey design to account for this.f There were two different recruitment methods in this study, giving variation also within travel mode and trip purpose. All respondents of the 2009 survey were members of the PanelClix internet panel, a large opt-in panel. Respondents received a reward equivalent to € 1,50 for the completion of a 15–20-min questionnaire. However, the resulting VTTs were implausibly low. Therefore, in 2011 a survey with intercept or ‘en-route’ recruitment was carried out, using the same recruitment methods used in the previous national VTT studies in 1988 and 1997. When these two datasets are analyzed using simple Multinomial Logit (MNL) models and correcting for inflation, the VTT from the internet panel (i.e. the 2009 respondents) is typically between 40 and 60 percent of the VTT from the respondents recruited in the field (i.e. the 2011 respondents), as can be seen from Table 2. When corrections are made for differences in socio-economic classes, the VTT-differences are reduced by about 10 percent. When more advanced MNL models (incorporating non-linear dependencies on the current travel time and cost and (especially) on the time and cost differences offered in the 1 3 1560 Transportation (2023) 50:1553–1584 SP experiments) are used, the VTT from the internet panel respondents is still 10 to 20 per- cent lower than from the respondents recruited by the intercept method.ii SP experiments) are used, the VTT from the internet panel respondents is still 10 to 20 per- cent lower than from the respondents recruited by the intercept method.ii These results are in line with another finding from the same survey (Significance et al. 2013), shown in Table 3: In the 2011 survey, respondents were asked an additional ques- tion (that was not present in the 2009 survey) of whether they were a member of an internet panel. This information was used as an interaction variable for the VTT. The model esti- mations indicate that commuter respondents in the 2011 survey (i.e. 8 The respondent must make a choice between the two before proceeding to the next question. If respond- ents were allowed to skip the question, there would be a risk that many would be tempted to do so even though they actually preferred one alternative over the other. Evidence from the Dutch VTT study recruited at intercept locations) who were members of an internet panel had on average a 20 percent lower VTT compared to commuter respondents that were not a member of any internet panel. For busi- ness respondents, VTT is 13 percent lower. For respondents traveling for other purposes, there is no significant difference. In our survey design (see Section “Recruitment method and data collection”), we replicate this feature of the Dutch 2011 survey, which allows us to identify the effect of internet panel membership among respondents not recruited from an internet panel. The Dutch study concluded that the 2009 internet panel survey led to substantially lower VTT than the 2011 intercept survey. Those for 2011 are much more in line with the values found based on the surveys in 1988 and 1997, which had always been regarded in The Netherlands as very plausible values by the various transport sectors, and are not con- sidered to be particularly high in an international perspective: The meta-analysis of Shires and de Jong (2009) found comparable or higher values for many other Western countries compared to the Dutch values from 1997. The most likely conclusion therefore is that the 2011 values are correct and that the 2009 values are biased downwards. In other words, that the sample from the internet panel is less representative in terms of VTT. Survey design and data collection Our main empirical contribution is based on data from the recent Norwegian valuation study on personal travel, which included a stated preference survey on the value of travel time. In this section, we give an overview of this data, focusing on the parts that are most relevant to the effects of recruitment method and interview location. For more documenta- tion, see the technical report from the project (Flügel et al. 2020). Survey and choice experiment design The survey consists of several stated choice experiments with various attributes. In this paper, we use data from the two-attribute experiment illustrated in Fig. 1, which only includes the attributes travel time and cost. Respondents using all transport modes (except walking and cycling) participate in this experiment. They are instructed to imagine two different travel options (routes), where in both options the mode of transport and all other trip characteristics are the same as on an actual reference trip reported by the respondent. In eight consecutive choice tasks, respondents choose between two alternatives with dif- ference values of travel time and costs.8 The attribute values are pivoted around reference 1 1 3 1 3 Transportation (2023) 50:1553–1584 1561 Table 2 The effect of recruitment method on VTT in the 2009–2011 Dutch VTT study Number of respondents Ratio VTT internet panel (2009-respondents) vs. intercept recruitment (2011-respondents) 2009 2011 Simple MNL (%) Simple MNL + socio- econ. interactions (%) Advanced MNL (%) Commute Car 1008 150 40 47 80 Train 699 105 64 72 88 Local PT 469 97 59 55 85 Business Car 349 235 41 52 88 Train 235 41 42 52 77 Local PT 61 11 52 54 77 Other Car 538 93 50 58 86 Train 249 79 94 98 95 Local PT 136 70 50 90 84 Table 2 The effect of recruitment method on VTT in the 2009–2011 Dutch VTT study Table 3 The effect of internet panel membership on VTT among respondents recruited via intercept in the 2011-survey of the Dutch VTT study Number of respondents Ratio VTT internet panel vs. non-internet panel members (2011-respondents) 2011—internet panel members 2011—non-internet panel members Advanced MNL specification Commute 70 282 80% Business 51 236 87% Other 42 200 Approx. 100% le 3 The effect of internet panel membership on VTT among respondents recruited via intercept in the 1-survey of the Dutch VTT study values of travel time and cost are from the reference trip. To avoid confounding factors, no further explanation is given regarding the reasons for differences in travel time and cost.9 values of travel time and cost are from the reference trip. 9 In the questionnaire, travel cost of car travelers is calculated based on travel distance and an estimate of variable driving costs, but the respondent is invited to correct this calculation and add additional costs like road tolls. 10 For long trips except air travel, the range is 10–1000 NOK/hour. For air travel, the range is 50–1250 NOK/hour. A restriction on travel cost ensures that this will never be lower than 1 NOK. 11 This experiment is either an experiment with three or more attributes or a version of the first experiment where the respondent is instructed to picture a different mode of travel. We leave these out for the following reasons: (1) With more than two attributes, we cannot use our preferred model specification to identify the effects of recruitment method while controlling for other factors. (2) When the travel mode is not the refer- ence mode, the interpretation of a potential hypothetical bias becomes less straightforward. Survey and choice experiment design To avoid confounding factors, no further explanation is given regarding the reasons for differences in travel time and cost.9 f Each respondent faces two choice tasks of each the following types: (I) Cost and travel time is equal to the reference value in one alternative, while the other alternative is more expensive and faster. (II) Cost and travel time is equal to the reference value in one alterna- tive, while the other alternative is cheaper and slower. (III) One alternative has cost equal to the reference value but is faster. The other alternative has travel time equal to the refer- ence value but is cheaper. (IV) One alternative has cost equal to the reference value but is slower. The other alternative has travel time equal to the reference value but is more expen- sive. There are no choice tasks in which one alternative is better in terms of both travel time and cost. The design is constructed such that all respondents face some situations in which the price of saving travel time is high and some in which it is low, and both small and large 1 3 Transportation (2023) 50:1553–1584 1562 Fig. 1 Example of choice card in the stated choice experiment (translated from Norwegian) Recruitment method and data collection Respondents were recruited from three sources: An internet panel, an alternative email reg- ister and field intercept. Originally, the email register was chosen as the main source of recruitment because it was relatively cheap. At the same time, it was decided that a part of the sample should be from an internet panel for comparability with the previous Norwe- gian VTT study (Ramjerdi et al. 2010). Field intercept was chosen as a supplement specifi- cally to test for the effect of recruitment method. f The data was collected in October and November 2018. For practical reasons, the sur- vey was not launched at the same time using all sources of recruitment, but the samples are overlapping in terms of interview date and time of day (see Section “Heterogeneous effects by recruitment method”). The samples contacted in the internet panel and email register were national without any particular stratification. i The internet panel was provided by Norstat, (norstatpanelet.no), one of the leading panel providers in Norway.12 Invitations were sent to a representative subsample among the members of this panel until the quota of 3000 interviews was reached. The response rate was 14 percent, which is relatively low but comparable to other surveys based on the same panel (e.g. Navrud et al. 2017; Dugstad et al. 2020). The alternative email register was provided by a subsidiary of the Norwegian postal service. It contains individuals who have submitted their contact information to the postal service (e.g. when changing address or pausing mail delivery) and agreed to receive invita- tions to surveys. 200,000 emails were sent, which means that despite a very low response rate (2.7 percent), this recruitment method still accounts for the largest part of our sample. Response rates from such general email sample frames are known to be relatively low (Bie- mer et al. 2017). Field recruitment was targeted at travelers by local public transport and passenger boat. The latter group was targeted because passenger boat is a somewhat marginal mode, which means recruiting from a national sample (e.g. an internet panel) will only result in a few boat passengers being included. Those recruited in the field were asked if they want to answer on site (on a tablet or their smartphone) or receive the questionnaire via email and answer later. 12 Norstat uses a combination of opt-in and random recruitment of respondents to their panel, much like many other internet survey providers. 13 The gift vouchers had a total value of 100,000 NOK and were allocated in a joint lottery among respond- ents of this survey and other surveys that belong to the same project. The expected reward was hence about 10 NOK per respondent. Fig. 1 Example of choice card in the stated choice experiment (translated from Norwegian) changes in travel time. To achieve this, eight different percentage changes in travel time are drawn from eight different intervals and combined randomly with eight different prices of travel time (‘bids’), which are also drawn from intervals. Based on each combination, the difference in travel cost is calculated. The change in travel time ranges from 10 to 30 percent, and the bid ranges from 10 NOK/hour to 750 NOK/hour.10 The resulting differ- ences in travel time and cost are allocated randomly across the choice tasks of type (I-IV) described above. The statistical design is identical to the design used in the previous Nor- wegian national value of time study (Ramjerdi et al. 2010), except that the bid range has been adjusted somewhat. For the respondents in our sample, the questionnaire contains the stated choice experi- ment described in the previous section followed by another experiment which we do not consider here.11 Before the stated choice experiments, the respondent is asked a number of questions regarding a reference trip which is used as input to the choice tasks. This includes questions about reference travel time and cost. In addition, respondents are asked to report some background characteristics of themselves and their reference trip. In Section 3.3, we explain how the reference trip is selected. Like in the Dutch study described in Section "Evidence from the Dutch VTT study", all respondents are also asked whether they are active members of a commercial inter- net panel. This means that we can identify panel members also among those that are not recruited from the panel. Transportation (2023) 50:1553–1584 1563 Reference trip and interview location One important feature of field interviews in transport research is that one can ask respondents about a trip that they are currently making or just finished. When contacting respondents via email, mail or phone, it is common to ask about a recent trip. A novel feature of our data is that also those who were contacted via email were asked if they were currently traveling. This implies that we have variation in interview location within all three sources of recruitment. This is illustrated in Fig. 2. Those who are recruited from the internet panel or email register are asked if they are currently on a trip when answering the survey. If they answer yes, this trip will be their reference trip and the interview context will also be “Traveling”. If they answer no, they will be asked to report their recent trips, and a reference trip will be picked from these. In this case, the interview context will be “at home, office etc.”i fi Those recruited in the field are asked if they want to answer on site or receive the ques- tionnaire via email. If they choose to answer on-site, the trip that they are currently making or just completed will be their reference trip and the interview context will be “Traveling”. If they choose to receive the question via email, they will be asked the same question as those recruited from the internet panel or email register.i Among those in the “Traveling” group, some will have finished their trip and some will still be underway. A potential concern could be that those in the latter group are not able to predict their travel time. (No explicit instructions are given regarding this in the question- naire.) Although we expect that most respondents would be able to give a reasonable esti- mate of this, we investigate in Section “Heterogeneous effects by recruitment method” how travel time differs by interview context. We also control for the difference in travel time between alternatives in the analysis in Section "Empirical modeling".if The interviews of those who are recruited in the field and answer while traveling differ slightly from those who chose to answer in another location in the sense that an interview- ing assistant is present in the recruitment, and in some cases also during the interview. 14 In the estimation sample in Section "Empirical modeling", we exclude respondents who always pick the alternative on one side and a few other respondents with unlikely input values or choice behaviour. In this section, these respondents are included in the sample. Recruitment method and data collection Field recruitment and interviews were undertaken in the cities of Oslo, Trondheim and Molde, which have populations of about 670,000, 190,000 and 27,000, respectively. Trave- lers were approached approximately at random both on board and at stops and stations. In the beginning of the data collection period, respondents were only approached at stops and stations. Later, respondents were also recruited on board, except for on trains, where permission for on-board recruitment was not granted by the train operator. 50 percent of those who were approached by an interviewer accepted to be recruited for the survey. Out of these, 48 percent completed the survey. This yields a total response rate of 24 percent in the field recruitment sample.i i Those recruited in the field and from the email register were informed that by participat- ing, they were eligible for a lottery where they could win a gift voucher.13 Those recruited 1 3 3 1564 Transportation (2023) 50:1553–1584 from the internet panel could not join the lottery, as this would interfere with the reward system of the panel provider. In practice, the expected payoff is of a similar order of magni- tude across sources of recruitment. Reference trip and interview location However, respondents receive little guidance on how to answer the choice questions and the rest of the questionnaire. In this sense, all interviews can be regarded as unassisted. A more important effect of the presence of an interviewer is probably that they influence who chooses to participate in the survey, which we will explore in more detail below. 15 A likely explanation for this difference is that receiving an e-mail invitation to a survey is similar to receiving an invitation to a panel or a survey invitation from a panel, and therefore partly attracts the same group of respondents. 16 We over-sampled long-distance trips (70 km or more) by asking respondents whether they had under- taken such a trip during the last two weeks. If the answer was yes, the majority of respondents would be assigned the last such trip as their reference trip. A large share of trip by airplane will be in this category. As a result of this, most of those answering questions about air travel will not currently be traveling. Recruitment method and interview location The total sample used in this paper contains 7160 respondents.14 Among these, 37 per- cent are from the internet panel, 55 percent are from the email register and 8 percent are recruited in the field. 14 In the estimation sample in Section "Empirical modeling", we exclude respondents who always pick the alternative on one side and a few other respondents with unlikely input values or choice behaviour. In this section, these respondents are included in the sample. 1 3 1565 Transportation (2023) 50:1553–1584 Fig. 2 Choice context for different sources of recruitment. Fig. 2 Choice context for different sources of recruitment. Figure 3 shows the answers to the question about whether the respondent is an active member of an internet panel. As expected, the share of panel members is by far highest among those recruited from the internet panel, although a few of these apparently do not consider themselves as members. More importantly, there is a non-negligible share of panel members also in the two other samples, particularly the sample recruited from the email register.15 There is also a notable share reporting that they do not know whether they are panel members, particularly in the field sample. This means that we can not only study differences in VTT between the three recruitment samples used in this survey, but also the effect of internet panel membership more generally. f Figure 4 shows how many are actually traveling while making their choices, which means that their current trip will be the point of departure for the stated choice experiment. Most of the respondents are not currently traveling, but instead answering questions based on a recent trip. The share who is currently traveling is largest among those recruited in the field, which is expected since some of these answer on-site. However, there is also a non- negligible share of currently traveling respondents in the two other samples. This means that we can estimate an effect of interview location, controlling for recruitment method. f Among those recruited from the internet panel or the email register, travelers by public transport are most likely to be on a trip when answering the survey, followed by air travel- ers,16 car passengers and car drivers (presumably not while driving). This is as expected, since those traveling by public transport are more likely to check their email. Recruitment method and interview location In the empiri- cal analysis in Section "Empirical modeling", this will be taken into account by controlling for travel mode as well as other characteristics of the reference trip. Respondent characteristics Before we estimate the effects of survey recruitment method, internet panel membership and interview location on the VTT, we explore whether respondents from the different 1 3 3 Transportation (2023) 50:1553–1584 1566 Fig. 3 Share of respondents who report that they are members of an internet panel, by recruitment method Fig. 4 Share of respondents who are currently traveling when answering the questionnaire, by recruitment method and transport mode used on the reference trip. Note: Car and air travelers who were recruited in the field are excluded due to very small samples Fig. 3 Share of respondents who report that they are members of an internet panel, by recruitment method Fig. 4 Share of respondents who are currently traveling when answering the questionnaire, by recruitment method and transport mode used on the reference trip. Note: Car and air travelers who were recruited in the field are excluded due to very small samples Fig. 3 Share of respondents who report that they are members of an internet panel, by recruitment method Fig. 4 Share of respondents who are currently traveling when answering the questionnaire, by recruitment method and transport mode used on the reference trip. Note: Car and air travelers who were recruited in the field are excluded due to very small samples Fig. 4 Share of respondents who are currently traveling when answering the questionnaire, by recruitment method and transport mode used on the reference trip. Note: Car and air travelers who were recruited in the field are excluded due to very small samples survey samples differ on average in terms of observable characteristics. This gives evi- dence on the relationship between survey methods and representativeness, and it will guide us regarding which factors need to be controlled for in the regression models in Sec- tion "Empirical modeling". Table 4 shows observable characteristics of respondents by source of recruitment. This includes time of interview, which was recorded in the survey software. For the sake of brevity, we only report average values. As expected, respondents recruited in the field are on average much more likely to be traveling in or from the two major cities Oslo and Trondheim, where field recruitment was carried out. This sample also has a substantially higher share of female respondents17 and shorter reference travel time. There is not much difference in average travel time between the panel and email register samples. 17 A partial explanation for this could be that field recruitment was targeted at public transport, which is more frequently used by women. However, the difference still seems large. Respondent characteristics f We also see that panel respondents are on average older and have lower income and education, also compared to those recruited from the email register. They are also much 1 3 1 3 Transportation (2023) 50:1553–1584 1567 Table 4 Respondent characteristics, by recruitment method National figures are from official statistics provided by Statistics Norway, excluding individuals aged below 18 years Based on a question where respondents place themselves in an income interval. Population share of the respective city (1) (2) (3) (4) (5) Panel Email Field Total National pop. (2018) Age 45.5 41.9 40.2 43.1 47.9 Age > 66 (%) 16.0 6.2 5.1 9.7 18.9 Male (%) 51.4 51.1 37.5 50.1 50.1 Income (NOK) 363,998 393,337 357,740 380,812 466,400 Higher education (%) 58.6 67.1 72.4 64.1 34.1 Trip in/from Oslo (%) 15.6 17.5 61.8 20.8 13.0 Trip in/from Trondheim (%) 5.4 3.9 18.5 5.7 3.7 Reference travel time (min.) 89.2 93.6 67.3 89.9 Week of response 45.0 44.6 45.7 44.8 Weekend (%) 11.2 2.3 4.9 5.8 Time of interview: 6–12 AM (%) 31.5 12.5 26.6 20.6 Time of interview: 12 AM–5 PM (%) 34.1 51.3 48.8 44.8 Time of interview: 5–11 PM (%) 30.6 31.7 21.0 30.5 Observations 2624 3968 568 7160 Table 4 Respondent characteristics, by recruitment method National figures are from official statistics provided by Statistics Norway, excluding individuals aged below 18 years Based on a question where respondents place themselves in an income interval. Population share of the respective city more likely to answer the survey during the weekend or in the morning.18 Apart from that, the interviews in the three samples are on average conducted during almost the same week, but with some differences as expected.19 more likely to answer the survey during the weekend or in the morning.18 Apart from that, the interviews in the three samples are on average conducted during almost the same week, but with some differences as expected.19 f Table 4 also compares the characteristics of our sample to the general population. We see that our sample is somewhat younger on average, which might be as expected as younger people travel more. However, income is also lower and education level substan- tially higher in our sample. 18 The differences in age, income, education and interview time are all statistically significant on the 1% level, based on a two-sided test. 19 As described in Section 3 there was some overlap; recruitment from the email register started first, fol- lowed by recruitment form the panel sample, and finally, field recruitment. 20 These differences are all statistically significant on the 1% level, based on a two-sided test. Respondent characteristics The share of trips that take place or start in Oslo is also higher than the corresponding population share, but this might be because many people from around Oslo travel in and out of the capital. In Table 5, we compare respondents based on whether they report to be members of an internet panel. Here, we do not include those who were recruited from the internet panel. We see that those who are active members of an internet panel are older, have lower educa- tion and are less likely to be traveling in or from Oslo, similar to the findings in Table 4.20 They also have somewhat longer travel time. However, they do not have substantially lower income on average. Passive panel members and non-members seem to be more similar. Active panel members also answer the survey slightly earlier (0.25 weeks difference). 1 3 3 1 Transportation (2023) 50:1553–1584 1568 Table 5 Respondent characteristics, by internet panel membership (respondents recruited from internet panel not included) Based on a question where respondents place themselves in an income interval (1) (2) (3) (4) (5) Active member Passive member Non-mem- ber Unknown Total Age 45.0 40.4 41.5 41.0 41.7 Age > 66 (%) 9.5 3.9 5.5 7.2 6.1 Male (%) 52.1 49.7 50.9 43.1 49.4 Income (NOK)* 380,817 392,586 396,109 347,214 389,942 Higher education (%) 62.7 68.9 68.5 67.2 67.6 Trip in/from Oslo (%) 14.7 19.8 24.2 31.9 23.6 Trip in/from Trondheim (%) 4.6 5.6 5.9 7.2 5.9 Reference travel time (min.) 99.1 88.7 87.6 92.6 90.3 Week of response 44.5 44.7 44.8 44.9 44.7 Weekend (%) 1.6 2.7 3.0 2.3 2.6 Time of interview: 6–12 AM (%) 14.5 13.8 14.5 14.1 14.3 Time of interview. 12 AM–5 PM (%) 51.0 50.8 50.2 53.4 51.0 Time of interview: 5–11 PM (%) 29.6 30.8 31.3 27.9 30.4 Observations 608 811 2282 835 4536 Respondent characteristics, by internet panel membership (respondents recruited from internet ot included) Apart from this, there are no apparent differences in when the interviews were carried out (day or time).f Apart from this, there are no apparent differences in when the interviews were carried out (day or time).f Finally, Table 6 shows how these characteristics differ between respondents that answered while traveling and those who did not (‘not traveling’), within the field recruit- ment sample and the remaining sample (internet panel and email register). 21 These differences are also statistically significant on the 1% level, based on a two-sided test. 22 This leaves out 1 percent of the observations. Excluding extreme cases is important since respondents can close the questionnaire whenever they want and then continue again after having been inactive for a very long time. Respondent characteristics 1 3 1 Transportation (2023) 50:1553–1584 1569 Table 6 Respondent characteristics, by interview location and recruitment mode Based on a question where respondents place themselves in an income interval (1) (2) (3) (4) Traveling, field Not traveling, field Traveling, panel + email Not traveling, panel + email Age 40.3 40.1 35.9 43.8 Age > 66 (%) 5.9 4.2 1.6 10.6 Male (%) 38.9 35.9 53.7 51.1 Income (NOK) 358,759 356,557 399,940 381,225 Higher education (%) 75.7 68.5 65.7 63.5 Trip in/from Oslo (%) 58.8 65.5 35.4 15.7 Trip in/from Trondheim (%) 21.2 15.3 4.3 4.5 Reference travel time (min.) 81.8 50.3 84.4 92.3 Week of response 45.5 46.0 44.8 44.8 Weekend (%) 4.6 5.3 2.3 6.1 Time of interview: 6–12 AM (%) 28.4 24.4 18.0 20.2 Time of interview: 12 AM–5 PM (%) 47.7 50.0 58.5 43.6 Time of interview: 5–11 PM (%) 20.3 21.8 22.7 31.8 Observations 306 262 383 6209 Table 6 Respondent characteristics, by interview location and recruitment mode Based on a question where respondents place themselves in an income interval Respondent characteristics In the field recruitment sample, those who are not currently traveling have considerably shorter travel time, but this is not the case in the remaining sample. A possible explanation is that those who are on a short trip and are intercepted by an interviewer are less likely to have time to answer on-site than those who are on a longer trip. We return to this mechanism in Sec- tion “Heterogeneous effects by recruitment method”.fi f Apart from this, there are no apparent differences in the field recruitment sample between those answering while traveling and not. In the remaining sample, those who answered while traveling are significantly younger and are also somewhat more likely to be traveling in or from Oslo. They are also somewhat less likely to answer during a weekend and in the evening, which might be because they answer the survey right away.21 (Email invitations were sent out during daytime on working days.) There is no apparent difference in income, and also not in reference travel time.if The above findings imply that when we estimate the effect of survey recruitment method, internet panel membership and interview location on the VTT, we need to evalu- ate to what extent our results are robust to controlling for observed characteristics of the respondents. In this respect, factors like age, income, education, geography and when the survey was answered seem to be relevant control variables. Choice behavior Before analyzing the effects of internet panel membership and interview location on the VTT, we have a closer look at the choice behavior of respondents in the stated choice experiment. As pointed out in Section "Previous literature", survey method could impact both the average VTT and the variance or degree of consistency in choice behavior. We therefore compare the following indicators across samples: (a) How much time it takes the respondent to complete the stated choice experiment (excluding those that spend more than 15 min22), as recorded by the survey software. (a) How much time it takes the respondent to complete the stated choice experiment (excluding those that spend more than 15 min22), as recorded by the survey software. (b) Wh h h d l h h l i di l d h l f h d id (b) Whether the respondent always chooses the alternative displayed on the left-hand side or always the alternative displayed on the right-hand side (c) Whether the respondent makes at least one ‘inconsistent’ choice in the sense that he or she accepts a bid which is higher than the lowest bid he or she rejects.f (d) The difference (in NOK/hour) between the highest accepted bid and the lowest rejected bid. If the difference is negative, it is defined as zero. fi (e) Whether the respondent always chooses the cheapest alternative (rejects all eight bids) (f) Whether the respondent always chooses the fastest alternative (accepts all eight bids) (f) Whether the respondent always chooses the fastest alternative (accepts all eight bids) 22 This leaves out 1 percent of the observations. Excluding extreme cases is important since respondents can close the questionnaire whenever they want and then continue again after having been inactive for a very long time. 1 3 1 3 1570 Transportation (2023) 50:1553–1584 While differences in (a)–(d) can be thought of as mainly reflecting differences in the ability or willingness to engage in the choice situation, differences (e) and (f) can reflect both such dif- ferences and differences in average VTT. Other things equal, a higher share that always rejects (e) implies a lower VTT, and a higher share that always accepts (f) implies a higher VTT. 23 Both of these differences are statistically significant on the 1% level based on a two-sided test. 24 This difference is statistically significant on the 5% level based on a two-sided test. 25 These differences are statistically significant on the 5% and 1% level, respectively, based on a two-sided test treating all respondents that answer while traveling as one group (regardless of recruitment mode). 26 If choice behaviour differs depending on travel mode used on the reference trip, this will also be picked up by the differences here, since traveling while answering is more common among users of some modes. Choice behavior While differences in (a)–(d) can be thought of as mainly reflecting differences in the ability or willingness to engage in the choice situation, differences (e) and (f) can reflect both such dif- ferences and differences in average VTT. Other things equal, a higher share that always rejects (e) implies a lower VTT, and a higher share that always accepts (f) implies a higher VTT. When considering indicators (c) and (d), one should have in mind that choices might also depend on the size and sign of differences in travel time and costs. Hence, a higher number of ‘inconsistent’ choices does not necessarily indicate low answer quality. Here, we are mainly interested in differences between samples, not measuring overall answer quality. f Again, we only show average values in each subsample. Table 7 shows that average completion time (a) is lower in the sample recruited from the internet panel. We also see that the share that rejects all eight bids (e) is higher in this sample.23 The differences in the other indicators are less notable. The share that always picks the alternative on one side is slightly higher in the panel sample, but the difference is not statistically significant.i fi In line with the above findings, those who are not recruited from the internet panel but report to be active panel members are also more likely to reject all eight bids (Table 8).24 Both active and passive panel members also have a slightly shorter completion time than non-members. Apart from this, panel members do not seem to differ so much from the rest of the sample with respect to other indicators of choice behavior. Finally, those who answer the survey while traveling are less likely to reject all eight bids and more likely to accept all eight bids, indicating a higher VTT (Table 9).25 The pat- tern with respect to other indicators of choice behavior is less clear.26 Interpreting these results in light of the literature discussed in Section "Survey and interview methods in VTT studies", we find no evidence that internet panels are associated with lower data quality. We do find, however, that those recruited from the internet panel have a shorter completion time. This might suggest that these respondents are more trained in answering on-line surveys, and therefore are able to complete the stated choice experi- ment faster without sacrificing precision in their answers. Empirical modeling In this section, we show the effects of recruitment method and interview location when controlling for other characteristics of the traveler and reference trip as well as design variables. 23 Both of these differences are statistically significant on the 1% level based on a two-sided test. 24 This difference is statistically significant on the 5% level based on a two-sided test. 25fi 23 Both of these differences are statistically significant on the 1% level based on a two-sided test. 24fi Estimation model As explained in Section “Recruitment method and data collection”, the choice situation always involves the choice between two travel options of which one is faster and more expensive. Following Fosgerau et al. (2007), we model the probability of choosing the faster and more expensive option (‘accepting’), ynt = 1 , as a function of the offered price 1 3 Transportation (2023) 50:1553–1584 1571 Table 7 Choice behavior in the stated choice experiment, by recruitment method (1) (2) (3) (4) Panel E-mail Field Total Completion time (seconds) 77.2 90.4 87.6 85.3 Always chooses left/right (%) 2.7 2.1 2.1 2.4 ’Inconsistent’ choice (%) 41.0 42.2 40.7 41.7 ‘Inconsistency’ (NOK/h) 99.3 95.8 70.8 95.1 Always rejects bid (%) 12.8 9.9 9.5 10.9 Always accepts bid (%) 2.2 2.6 2.6 2.4 Observations 2624 3968 568 7160 Table 7 Choice behavior in the stated choice experiment, by recruitment method le 8 Choice behavior in the stated choice experiment, by internet panel membership (respondents uited from internet panel not included) Table 8 Choice behavior in the stated choice experiment, by internet panel membership (respondents recruited from internet panel not included) (1) (2) (3) (4) (5) Active member Passive member Non- member Unknown Total Completion time (seconds) 86.0 85.8 92.6 90.0 90.0 Always chooses left/right (%) 2.1 1.5 2.5 1.9 2.1 ’Inconsistent’ choice (%) 39.5 40.0 42.7 44.1 42.0 ’Inconsistency’ (NOK/h) 89.6 79.8 91.7 110.1 92.7 Always rejects bid (%) 12.3 9.6 9.5 9.3 9.9 Always accepts bid (%) 3.3 1.7 2.6 2.9 2.6 Observations 608 811 2282 835 4536 Table 9 Choice behavior in the stated choice experiment, by interview location and recruitment mode (1) (2) (3) (4) Traveling, field Not trave- ling, field Traveling, panel + email Not traveling, panel + email Completion time (seconds) 83.5 92.4 87.7 85.0 Always chooses left/right (%) 2.6 1.5 1.6 2.4 ’Inconsistent’ choice (%) 40.2 41.2 44.6 41.6 ‘Inconsistency’ (NOK/h) 76.0 64.7 92.5 97.5 Always rejects bid (%) 5.9 13.7 9.9 11.1 Always accepts bid (%) 3.6 1.5 4.7 2.3 Observations 306 262 383 6209 Table 9 Choice behavior in the stated choice experiment, by interview location and recruitment mode per time unit saved (‘bid’), Bnt , and a set of covariates including other characteristics of the choice task, characteristics of the reference trip and characteristics of the respondent. Estimation model Hence, the decision rule is: (1) ynt = 1 if ln(VTTnt ) + 1 𝜇𝜀nt > ln(Bnt ), (1) 1 3 1 3 Transportation (2023) 50:1553–1584 1572 where Bnt is the offered trade-off (“bid”) defined as the ratio of the absolute difference of the cost and the time attribute: where Bnt is the offered trade-off (“bid”) defined as the ratio of the absolute difference of the cost and the time attribute: (2) Bnt = \|\|ΔCnt\|\| \|\|ΔTnt\|\| . (2) The error terms 휀nj are assumed to be independently and identically (iid) logistic distrib- uted random variables with mean value of zero and variance of 휋2∕3 The error terms 휀nj are assumed to be independently and identically (iid) logistic distrib- uted random variables with mean value of zero and variance of 휋2∕3 We further parameterize the log of VTT with explanatory variables. (3) ln(VTTnt) = 훼+ 훽1Panelnt + 훽2Emailnt + 훽3Activent + 훽4Passivent + 훽5Unknownnt + 훽6Nottravelnt + 훾xnt + un. (3) where Emailnt is a dummy variable for being recruited from the email register and Pan- elnt is a dummy variable for being recruited from the internet panel. Activent, Passivent and Unknownnt are dummy variables for whether the respondent reports to be an active internet panel member, a passive internet panel member or not knowing whether he or she is an internet panel member, respectively. Finally, Nottravelnt is a dummy variable for not being currently traveling when answering the questionnaire. xnt is a vector of control variables.i The logarithmic specification implies that the estimated parameters of each covariate approximates the percentage change in VTT resulting from a one unit increase in the value of the covariate. un represents unobserved taste heterogeneity across respondents yielding a lognormal distribution assumption of VTT. un is normally distributed over respondents with mean value 0 and standard deviation σ.f Our main interest is in the estimated parameters β1-β6, which capture the effects of recruitment method, internet panel membership and interview context. 27 Note that one of the alternatives always contains the reference value of travel time, and one alternative always contains the reference value of travel cost. See Section Recruitment method and data collection. 28 With eight choice tasks per respondent, 6,973 respondents equals 55,784 choice tasks. However, some choice tasks are excluded because the difference in travel time or travel cost is zero. Since the values pre- sented to the respondents are rounded, this will occur in some cases for respondents that have reported very low reference values of travel time or cost. Estimation model To reduce concern that the estimated parameters instead capture some other unobserved characteristics that are correlated with the variables of interest, we compare the result of models including dif- ferent sets of control variables in xnt: (a) Characteristics of the reference trip and the experimental design: Trip purpose, travel mode, reference travel cost, trip distance, sign of the changes in travel time and travel cost relative to the reference values and absolute value of the difference in travel time relative to the reference value.27 (b) Socioeconomic characteristics and time of interview: Gender, age, age squared, income, income not reported (dummy), higher education ≥ 3 years (dummy), higher education ≥ 5 years (dummy), education level not reported (dummy), week of inter- view, interview 6–12 AM (dummy), interview 0–5 PM (dummy), interview 5–11 PM (dummy), interview in the weekend (dummy). (c) Geographic variables: Dummys for Oslo, Trondheim, and missing information Note that by controlling for the sign of changes in travel time and cost, we take into account reference-dependency in the VTT (De Borger and Fosgerau 2008). However, our experimental design (see Section “Recruitment method and data collection”) also implies that time (cost) increases and decreases are equally represented in our sample. 1 3 Transportation (2023) 50:1553–1584 1573 Table 10 Estimated effects of recruitment method, internet panel membership and interview location on value of travel time (VTT) Reference case: Respondents who are recruited in the field, are not members of an internet panel and are traveling while answering the survey. Standard errors in parentheses p<0 1 p<0 05 * p<0 01 (1) (2) (3) (4) (5) From email reg. Effects of recruitment method, panel membership and interview location Table 10 shows the effects of recruitment method, internet panel membership and interview location based on different model specifications. All models are estimated on 54,060 stated choices from 6,973 respondents.28 The full model results are presented in the Appendix. Column (1) shows that those recruited from the internet panel have a substantially lower VTT than those recruited in the field, while those recruited from the email register are somewhere in between. However, when controlling for panel membership among all respondents as well as interview location (column 2), the effects of recruitment method in itself become much smaller and is no longer statistically significant.i i In line with the Dutch evidence, we find that active members of an internet panel have lower VTT (columns 2–5). The difference is about -11 percent relative to being a non- member ( 1 −e−0.12 = 0.113 .) and is robust to controlling for sociodemographic character- istics, geographic location and unobserved heterogeneity.if Perhaps most interestingly, we find a strong and negative effect of answering the ques- tionnaire while ‘not traveling’, i.e. not while (or directly after) making the reference trip which is the point of departure for the stated choice experiment. The estimated effect is about –17 percent ( 1 −e−0.184 = 0.168 ) and is highly robust to changes in the model spec- ification. One possible explanation is that respondents perceive the travel experience as more positive in retrospect than when they are currently making the trip (Mitchell et al. 1997).fi The effect of interview location is contrary to the finding of Hanssen (2012) for ferry passengers in Norway. Note however that the effect of interview location reported by Hans- sen also partly captures an effect of recruitment method, as most of those who were not interviewed on site (i.e. received the questionnaire via mail) also had not been recruited in the field. This means that there could be other unobserved differences between the samples. Estimation model (β1) − 0.140 (0.070) − 0.040 (0.075) − 0.064 (0.079) − 0.061 (0.081) − 0.066 (0.080) From panel (β2) − 0.299* (0.072) − 0.112 (0.086) − 0.096 (0.087) − 0.091 (0.088) − 0.098 (0.088) Active panel member (β3) − 0.120 (0.051) − 0.112 (0.051) − 0.112 (0.051) − 0.105 (0.051) Passive panel member (β4) − 0.027 (0.051) − 0.022 (0.050) − 0.020 (0.051) − 0.023 (0.050) Panel membership unknown (β5) 0.055 (0.0514 0.034 (0.074) 0.039 (0.074) 0.064 (0.074) Not traveling (β6) − 0.184* (0.062) − 0.179* (0.062) − 0.177* (0.062) − 0.187* (0.062) Model type MNL MNL MNL MNL Mixed logit Trip + design controls Yes Yes Yes Yes Yes Socioeconomic controls No No Yes Yes Yes Geographic controls No No No Yes Yes 1574 Transportation (2023) 50:1553–1584 The model results can also be used to simulate the VTT distribution of the estimation sample or an alternative sample. In this paper, we do not focus on the average VTT or the distribution, only on the effect of recruitment method, controlling for other factors. The model results can also be used to simulate the VTT distribution of the estimation sample or an alternative sample. In this paper, we do not focus on the average VTT or the distribution, only on the effect of recruitment method, controlling for other factors. Discussion Our results provide new evidence on the effect of survey recruitment and interview meth- ods on both data quality and estimated values of travel time (VTT). Regarding data qual- ity in itself, we do not find any evidence that this is lower in the sample recruited from an internet panel or among those who report to be internet panel members. This supports previous studies that do not generally find evidence of significantly lower data quality in SP studies based on internet panels (Zhang et al. 2020; Sandorf et al. 2020, 2022). Regarding the level of VTT, our results clearly indicate that both recruitment method and interview location can impact the estimated VTT based on SP data. Those who report to be active members of an internet panel have a significantly lower VTT than those who do not. A likely explanation is that those who are panel members and regularly answer sur- veys have a lower opportunity cost of time measured in monetary terms. This could reflect both differences in marginal utility of time and the marginal utility of income.i f Regarding interview location, we find that those who answer the survey while traveling have a higher VTT, both among those recruited in the field and in the remaining sample. It is less clear whether this effect should also be taken into account if the objective is to obtain representative values. First, since interview location is the result of choices made by the respondent, it could be that this effect captures some differences in unobserved charac- teristics between those who are answering while traveling and those who are not. Second, it is not necessarily the case that the hypothetical choices made by those experiencing the travel situation while answering are more in line with actual behavior. Since travel deci- sions are made in advance, it could be that travelers experience the travel time as cost- lier when they are making the trip than they did when choosing their travel option (time inconsistency). Our survey design has the feature that those who answer the questionnaire while trave- ling will always be answering based on their current trip, while those who are not trave- ling will be answering based on a previous (in most cases recent) trip. Heterogeneous effects by recruitment method In the previous section, the effect of interview location was assumed to be the same across all sources of recruitment. One difference between these samples is that in the field recruit- ment sample, answering ‘while traveling’ implies that an interviewer is present in the recruitment and in some cases, also during the interview. This could potentially have some effect on the survey process (e.g. West and Blom 2017), though, as mentioned in Sec- tion "Previous literature", social desirability bias as a result of this is not likely in a VTT setting. In the panel and email register samples, an interviewer is never present. Also, those who choose to answer on-site could be less busy than those who do not. Therefore, we 1 1 3 3 Transportation (2023) 50:1553–1584 1575 estimate separate models to investigate whether the effect of answering while ‘not trave- ling’, as well as the effect of panel membership, differs between the samples.fi estimate separate models to investigate whether the effect of answering while ‘not trave- ling’, as well as the effect of panel membership, differs between the samples.fi ff Table 11 shows that the ‘not traveling’ effect is negative and statistically significant both in the field recruitment sample and the sample based on the two other recruitment methods. Although the estimated effect is somewhat higher in absolute terms in the field recruitment sample, the difference is not statistically significant. This strengthens the hypothesis that this effect is somehow related to the fact that the respondent is currently not making a trip – and therefore relates less closely to the choice situation described in the questionnaire. In other words, the choice situation may appear more salient for the respondent who is cur- rently traveling. However, other explanations can still not be ruled out.f The effect of being an active panel member is also negative in both samples, but not sta- tistically significant in the field recruitment sample. Hence, we cannot conclude regarding potential differences in this effect. The lack of precision in the field sample might be due to the lower sample size combined with the fact that the share of active panel members is very low in this sample. However, the point estimate is still negative. 29 The weights were based on the assumption that the share of panel members in the sample recruited in the field was representative for the traveling population. In the model used in these simulations, interview location was not controlled for. Since panel membership is correlated with answering while not traveling, this means that those answering while traveling are also given a somewhat higher weight. Discussion Those who answer based on their current trip might exhibit a greater degree of reference dependency in their choices, which has an ambiguous effect on estimated VTT (see Section “Hypothetical choice situations and bias in SP studies”). However, the choice of reference trip could also 1 3 3 Transportation (2023) 50:1553–1584 1576 Table 11 Estimated effects of internet panel membership and interview location on value of travel time (VTT), by recruitment method Reference case: Respondents who are recruited in the field, are not members of an internet panel and are traveling while answering the survey. Standard errors in parentheses p < 0.01, p < 0.05, *p < 0.01 Field Panel + email From panel (β2) − 0.028 (0.050) Active panel member (β3) − 0.315 (0.332) − 0.100 (0.052) Passive panel member (β4) − 0.218 (0.234) − 0.002 (0.052) Panel membership unknown (β5) − 0.318 (0.250) 0.073 (0.078) Not traveling (β6) − 0.245 (0.114) − 0.149 (0.075) Number of observations 555 6,418 Model type MNL MNL Trip + design controls Yes Yes Socioeconomic controls Yes Yes Geographic controls Yes Yes have other effects. A possible extension in future studies would be to ask some of those who answer while traveling about a previous trip instead of their current trip. This would enable the researcher to identify the effect in itself of answering while traveling, independ- ent of choice context. In our case, these two treatments are bundled. have other effects. A possible extension in future studies would be to ask some of those who answer while traveling about a previous trip instead of their current trip. This would enable the researcher to identify the effect in itself of answering while traveling, independ- ent of choice context. In our case, these two treatments are bundled. Table 11 Estimated effects of internet panel membership and interview location on value of travel time (VTT), by recruitment method Conclusion Our results have potentially strong implications for studies that aim to obtain representative values of travel time for use in cost–benefit analysis. If it is the case that (especially expe- rienced) internet panelists have a lower opportunity cost of time, results that are primar- ily based on internet panel surveys would need to be adjusted for this panel effect. Based on the findings documented in this paper, the recommended values in the new Norwegian VTT study (Flügel et al. 2020) are based on simulations in which those who reported that they were active members of an internet panel were given a lower weight.29 If this adjustment had not been made, the new Norwegian VTT values would have been low compared to the values based on the 2009 study. This is the case even if we do not assume that the VTT increases over time at the same rate as the income level, but assume a more moderate growth as advocated by ITF (2019). Interestingly, internet panel was the main source of recruitment in the 2009 study. Since the two studies were methodologically very similar, it might seem like there is a negative effect of internet panel membership in 1 1 3 3 Transportation (2023) 50:1553–1584 1577 2018 relative to 2009. One possible reason for this is that the representativeness of internet panels has changed over time, at least with respect to this particular purpose of obtaining VTT. However, alternative explanations cannot be ruled out. 2018 relative to 2009. One possible reason for this is that the representativeness of internet panels has changed over time, at least with respect to this particular purpose of obtaining VTT. However, alternative explanations cannot be ruled out. If it is the case that internet panel members have a lower opportunity cost of time than the general population, it might also be the case that those who respond to the survey (regardless of recruitment method) have a lower opportunity cost of time than those who choose not to respond. This kind of non-response bias (Groves and Peytcheva 2008) would imply that all survey-based VTT estimates are likely to be conservative. The magnitude of this effect is of course not possible to assess by use of (any) survey data, but a potentially testable implication is that the bias increases as the response rate declines.f Regarding the effect of interview location, several explanations are possible. Conclusion We rec- ommend investigating this further based on an experimental approach, where respondents are allocated randomly into answering while traveling or not while traveling. The effect of other treatments that make the choice situation more salient could also be explored. In any case, the strong effect found in this paper means that the issue should be taken seriously and calls for more research on the topic.fif On the other hand, it is difficult to see how the effect of internet panel membership (or voluntary survey participation more generally) could be verified in an experimental frame- work. Here, our hypothesis is that the voluntary nature of panel membership is exactly what drives our result. Instead, we recommend to further investigate the relationship between survey response rate and VTT, more specifically whether a lower response rate implies a lower VTT due to self-selection. Meta-analysis would be a suitable approach for such an investigation, which could include both SP and RP surveys. However, one should be aware that in many studies, recruitment method (and possibly response rate) and inter- view location cannot be disentangled. It is too early to conclude on what the implications for studies that aim to estimate a representative VTT will be. If the panel effect is driven by self-selection, all survey-based VTT estimates (both SP and RP) could potentially be biased downwards, but methods that achieve higher response rates might mitigate this problem. If the higher VTT of those who are currently traveling reflects that SP choices made by respondents who are currently traveling are closer to RP choices, this calls for concern regarding the validity of SP sur- veys more generally. For researchers and practitioners, designing surveys that are less vulnerable to the issues discussed here might involve some costs. Field recruitment requires more manpower than email recruitment, but this could be worth the cost if it results in a less self-selected sam- ple. At the same time, our results illustrate that field recruitment is not strictly necessary for being able to interview people while they are traveling. In the smartphone era, travelers are only an e-mail or text message away. Appendix The following table reports the full estimation results of model (1)–(5) presented in short form in Table 8. It includes the constant term 훼 (Eq. 3), the scale parameter 휇 (Eq. 1), the parameters of the set of additional explanatory variables parameters x , as well as σ, i.e. the standard error of the term un(Eq. 3). The unit of the dependent variable, ln(VTTnt) , is in NOK/minute. The standard errors are estimated based on a robust variance-covariance matrix. 3 3 Transportation (2023) 50:1553–1584 1578 1 3 Model Number of parameters (1) 18 (2) 22 (3) 35 (4) 38 (5) 39 Final-LL − 27,419,458 − 27,396,727 − 27,223,458 − 27,217,535 − 24,790,711 adjusted rho square 0.268 0.268 0.273 0.273 0.337 Parameter Measurement of vari- able Value Std. err. Value Std. err. Value Std. err. Value Std. err. Value Std. err. Appendix Constant term (σ) 2.55 0.0917 2.65 0.101 − 1.39 0.946 − 1.35 0.948 − 0.986 0.929 Recruitment method: email register Dummy − 0.14 0.0698 − 0.0399 0.0752 − 0.0638 0.0791 − 0.0608 0.0806 − 0.0656 0.08 Recruitment method: Internet panel Dummy − 0.299 0.0724 − 0.112 0.0855 − 0.0962 0.0868 − 0.0912 0.0883 − 0.0978 0.0878 Panel member: unknown Dummy 0.0546 0.0514 0.0398 0.074 0.0394 0.0741 0.064 0.0738 Panel member: active Dummy − 0.12 0.0508 − 0.112 0.0505 − 0.112 0.0505 − 0.105 0.0507 Panel member: passive Dummy − 0.027 0.0512 − 0.0215 0.0504 − 0.02 0.0505 − 0.0234 0.0503 Off-site interview Dummy − 0.184 0.0617 − 0.179 0.0619 − 0.177 0.0619 − 0.187 0.0616 Trip purpose: business Dummy 0.405 0.0547 0.4 0.0547 0.318 0.055 0.317 0.0551 0.313 0.0548 Trip purpose: commuting Dummy 0.237 0.0398 0.226 0.0399 0.112 0.041 0.112 0.041 0.116 0.0407 Reference trip: Travel cost Continuous in NOK (log transformed) 0.373 0.02 0.372 0.02 0.369 0.0201 0.374 0.0202 0.336 0.0198 Reference trip: Travel distance Continuous in km (log transformed) − 0.00844 0.0183 − 0.00867 0,0183 − 0.00777 0.0183 − 0.0122 0.0184 − 0.0177 0.0183 Reference trip: By air Dummy 0.329 0.0752 0.32 0.0753 0.348 0.0746 0.344 0.0749 0.394 0.0754 Reference trip: By boat Dummy − 0.0471 0.0738 − 0.0478 0.0739 0.00375 0.0729 − 0.00606 0.0729 0.0124 0.0734 Reference trip: By bus Dummy − 0.144 0.0568 − 0.174 0.0578 − 0.0796 0.0585 − 0.0865 0.0592 − 0.0896 0.0581 Reference trip: By car (pas- senger) Dummy − 0.0781 0.0623 − 0.0869 0.0624 0.00177 0.0624 0.000856 0.0625 − 0.0356 0.0618 Reference trip: By ferry Dummy 0.162 0.0788 0.164 0.0789 0.217 0.0779 0.22 0.0779 0.261 0.0779 Transportation (2023) 50:1553–1584 1579 Model Number of parameters (1) 18 (2) 22 (3) 35 (4) 38 (5) 39 rip: By metro/tram Dummy − 0.181 0.0718 − 0.211 0.0723 − 0.161 0.0734 − 0.2 0.076 − 0.195 0.0748 rip: By train Dummy − 0.0481 0.0621 − 0.0773 0.0626 − 0.026 0.0626 − 0.0392 0.0629 − 0.0401 0.0626 able: sign of cost ta c) Dummy − 0.168 0.0103 − 0.168 0.0103 − 0.168 0.0103 − 0.168 0.0103 − 0.166 0.01 able: sign of time ta t) Dummy 0.151 0.0102 0.151 0.0102 0.151 0.0102 0.151 0.0102 0.147 0.00994 able: absolute size hange Continuous in minutes (log transformed) 0.00654 0.0207 0.00465 0.0207 0.0113 0.0205 0.0134 0.0206 0.0741 0.019 ble (μ) 1.02 0.0144 1.02 0.14 1.03 0.02 1.03 0.02 1.39 0.0211 ndents Dummy 0.112 0.0343 0.112 0.0344 0.114 0.0341 Continuous in NOK (log transformed) 0.298 0.0341 0.294 0.0342 0.282 0.0336 come Dummy 3.68 0.433 3.64 0.433 3.48 0.426 Continuous in age 0.00417 0.00671 0.00421 0.00671 0.00275 0.00668 0 Continuous in age (square and scaled) − 0.0752 0.0705 − 0.0759 0.0704 − 0.0601 0.07 higher education Dummy 0.0472 0.0418 0.0446 0.0419 0.0522 0.0417 higher education Dummy 0.056 0.0466 0.053 0.0468 0.0666 0.0465 ormation on Dummy 0.173 0.108 0.17 0.108 0.137 0.108 ay: week of year Continuous in weeks of year 0.00589 0.0188 0.00568 0.0188 0.00279 0.0185 me 06–12 o’clock Dummy − 0.00277 0.0874 − 0.00777 0.0874 − 0.00197 0.0874 me 12–17 o’clock Dummy − 0.0446 0.0831 − 0.0493 0.0831 − 0.0412 0.0832 me 17–23 o’clock Dummy − 0.0401 0.0841 − 0.0446 0.084 − 0.0374 0.0841 ay: weekend Dummy 0.00855 0.072 0.00593 0.0719 0.0123 0.0721 1 Transportation (2023) 50:1553–1584 1580 Model Number of parameters (1) 18 (2) 22 (3) 35 (4) 38 (5) 39 Geographic information: missing Dummy 0.0267 0.0378 0.0298 0.0376 Geographic information: Trondheim Dummy − 0.0681 0.0803 − 0.0936 0.0798 Geographic information: Oslo Dummy 0.097 0.0533 0.094 0.0529 Standard deviation of unob- served heterogeneity (σ) 1.07 0.0191 Model Number of parameters (1) 18 (2) 22 (3) 35 (4) 38 (5) 39 Geographic information: missing Dummy 0.0267 0.0378 0.0298 0.0376 Geographic information: Trondheim Dummy − 0.0681 0.0803 − 0.0936 0.0798 Geographic information: Oslo Dummy 0.097 0.0533 0.094 0.0529 Standard deviation of unob- served heterogeneity (σ) 1.07 0.0191 1581 Transportation (2023) 50:1553–1584 Acknowledgements We are grateful for comments from Oddbjørn Raaum and other participants at the 42nd Annual Meeting of the Norwegian Association of Economists in 2020, participants at the ITEA Annual Conference in 2019 and two anonymous reviewers. Declarations Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of inter- est. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp://creativecommons.org/licenses/by/4.0/. Appendix This paper is based on data from the Norwegian Valua- tion study 2018-2020, which is a contract research project carried out by the Institute of Transport Econom- ics (TØI) in co-operation with Menon Economics and Significance. The project was initiated and financed by the Norwegian Public Roads Administration, the Norwegian Railway Directorate, the Norwegian Coastal Administration, Avinor AS, Nye Veier AS and Bane Nor AS. Representatives from the abovementioned organizations and the expert panel of the project (Maria Börjesson, Kathrine Hjort and Mark Wardman) have provided valuable comments and input to the project. Author contributions All authors have contributed significantly to the study. AHH has been the lead author of the manuscript, coordinated survey design and data collection, contributed to the literature review, and conducted the analysis in Section 4. SF has contributed to survey design and literature review and con- ducted the analysis in Section 5. MK and GdJ have contributed to survey design and literature review and conducted the analysis in Section "Evidence from the Dutch VTT study". HBS has been in charge of data collection and contributed to survey design. NH has contributed to survey design and analysis. GNJ has contributed to survey design, data collection, and analysis. HL has contributed to the literature review, the theoretical framework, and interpretation of results. All authors have commented on previous versions of the collection and contributed to survey design. NH has contributed to survey design and analysis. GNJ has contributed to survey design, data collection, and analysis. HL has contributed to the literature review, the theoretical framework, and interpretation of results. All authors have commented on previous versions of the manuscript and have read and approved the final manuscript. Funding Open Access funding provided by Institute Of Transport Economics. 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Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. institutional affiliations. Askill H. Halse is chief research economist at the Institute of Transport Economics (TØI) in Oslo, Norway. His research interests include non-market valuation, travel demand analysis, and cost–benefit analysis, as well as public decision-making and voter behavior. Stefan Flügel is a senior research economist at the Institute of Transport Economics (TØI) in Oslo, Norway. His research interests include non-market valuation, discrete choice analysis, travel demand modelling, and agent-based traffic simulation. Marco Kouwenhoven is a Research Leader at Significance BV, an independent research agency in the Neth- erlands. During the research described in this paper, he also was a researcher at the Engineering Systems and Services department of Delft University of Technology. Marco’s work focuses on transport model- ling and the design and analysis of stated choice experiments. He holds a Ph.D. in astronomy from Utrecht University. Gerard de Jong is director of Significance BV in the Netherlands and Research Professor at the Institute for Transport Studies, University of Leeds. He has previously worked for Hague Consulting Group, RAND Europe, NEA, and CTS Stockholm. He has contributed to the development of mainly disaggregate model 3 3 1584 Transportation (2023) 50:1553–1584 systems at the international, national, and regional scale, both in passenger and freight transport. He has also worked on project appraisal and on SP value of time and reliability studies in passenger and freight transport in several countries. Hanne Beate Sundfør is a researcher and public health scientist at the Institute of Transport Economics (TØI) in Oslo, Norway. Her main areas of research include traffic safety of children and youth, active mobil- ity, e-bikes, travel behavior, health effects, and physical activity. She is experienced in designing surveys and organizing data collection. Nina Hulleberg is a researcher at the Institute of Transport Economics and research fellow at the University College of Molde, Norway. References Her research interest is on valuation, estimation, and optimization, particularly related to public transport. Her PhD dissertation project involves valuation of crowding and optimization of capacity. Guri Natalie Jordbakke is a research economist at the Institute of Transport Economics (TØI) in Oslo, Nor- way. Her research interest is environmental perspectives on transport and related consumer decisions, on which she conducts research using register and survey data. Henrik Lindhjem is the founder of and research director at Menon Centre for Environmental and Resource Economics (MERE) in Oslo, Norway, and scientific advisor at Norwegian Institute for Nature Research (NINA). One line of his research includes challenges in the use of survey methods in non-market valuation, including the use of internet survey panels. 1 3
https://openalex.org/W4225318134	https://www.researchsquare.com/article/rs-842110/latest.pdf	English	null	Tumor Microenvironment-Responsive Nanodrug for Clear-Cell Renal Cell Carcinoma Therapy via Triggering Waterfall-Like Cascade Ferroptosis	Journal of biomedical nanotechnology	2,022	cc-by	10,298	Tumor Microenvironment-responsive Nanodrug for Clear-cell Renal Cell Carcinoma Therapy via Triggering Waterfall-like Cascade Ferroptosis Clear-cell Renal Cell Carcinoma Therapy via Triggering Waterfall-like Cascade Ferroptosis Wenjun Ni Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Yongxiang Li Weifang People's Hospital Lingxia Liang Medical College of Guangxi University, Guangxi University Cuixia Lu Medical College of Guangxi University, Guangxi University Meixiao Zhan Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Ligong Lu Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Qun Xie Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Liewei Wen (  wenliewei@scnu.edu.cn ) Zhuhai People's Hospital, Zhuhai Hospital affiliated with Jinan University, Jinan University https://orcid.org/0000-0002-4657-6457 Research Keywords: Renal cell carcinoma, ferroptosis, metal organic frameworks, lipid peroxides Posted Date: September 1st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-842110/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International Licen Read Full License Wenjun Ni Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Yongxiang Li Weifang People's Hospital Lingxia Liang Medical College of Guangxi University, Guangxi University Cuixia Lu Medical College of Guangxi University, Guangxi University Meixiao Zhan Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Ligong Lu Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Qun Xie Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Jinan University Liewei Wen (  wenliewei@scnu.edu.cn ) Zhuhai People's Hospital, Zhuhai Hospital affiliated with Jinan University, Jinan University https://orcid.org/0000-0002-4657-6457 Research Keywords: Renal cell carcinoma, ferroptosis, metal organic frameworks, lipid peroxides Posted Date: September 1st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-842110/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Posted Date: September 1st, 2021 Conclusion MIL-101(Fe)@RSL3 could aggravate ferroptosis and be expected to be a promising nanodrug for ccRCC systemic therapy due to the targeted delivery and responsive release of RSL3 and iron ions. DOI: https://doi.org/10.21203/rs.3.rs-842110/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/25 Results In this study, iron-based metal-organic framework nanoparticles (MIL-101(Fe) NPs) delivered RSL3 to ccRCC tumors, and then released the iron ions and RSL3 accompanied by the degradation of MIL-101(Fe) NPs in the acidic tumor microenvironment. The MIL-101(Fe)@RSL3 as a pH-responsive nanodrug causes cellular iron overload and promotes the hydroxyl radical (•OH) generation by Fenton reaction to attack PUFAs, leading to the aberrant accumulation of lipid peroxides (L-OOH). Additionally, RSL3 directly inhibits glutathione peroxidase 4 (GPX4) to detoxify L-OOH, and ferrous ions further catalyze the irreversible conversion of highly reactive lipid alkoxyl radicals (L-O•) from L-OOH to triggering waterfall- like cascade ferroptosis. In contrast to the limited antitumor efficiency of free RSL3, MIL-101(Fe)@RSL3 with high encapsulation efficiency (88.7%) shows a significant ccRCC-specific antitumor effect and negligible side effects. In this study, iron-based metal-organic framework nanoparticles (MIL-101(Fe) NPs) delivered RSL3 to ccRCC tumors, and then released the iron ions and RSL3 accompanied by the degradation of MIL-101(Fe) NPs in the acidic tumor microenvironment. The MIL-101(Fe)@RSL3 as a pH-responsive nanodrug causes cellular iron overload and promotes the hydroxyl radical (•OH) generation by Fenton reaction to attack PUFAs, leading to the aberrant accumulation of lipid peroxides (L-OOH). Additionally, RSL3 directly inhibits glutathione peroxidase 4 (GPX4) to detoxify L-OOH, and ferrous ions further catalyze the irreversible conversion of highly reactive lipid alkoxyl radicals (L-O•) from L-OOH to triggering waterfall- like cascade ferroptosis. In contrast to the limited antitumor efficiency of free RSL3, MIL-101(Fe)@RSL3 with high encapsulation efficiency (88.7%) shows a significant ccRCC-specific antitumor effect and negligible side effects. Background The most common type of kidney tumor, clear-cell renal cell carcinoma (ccRCC) with relatively insidious development and easily metastatic characteristics is generally insensitive to cytotoxic chemotherapy. Advanced ccRCC lacks effective treatment and has a poor prognosis. Fortunately, ccRCC with the hallmark of abundant polyunsaturated fatty acids (PUFAs) content is regarded as intrinsically vulnerable to ferroptosis-based therapeutic strategies. Nonetheless, the classic ferroptosis agonist (RSL3) with low specificity for tumors, short half-life in the blood, poor water solubility and deficient accumulation at the tumor site prevents its reliable application in vivo. Introduction Renal cell carcinoma (RCC), one of the most common genitourinary malignant tumors, causes 170,000 deaths each year worldwide. Clear cell renal cell carcinoma (ccRCC) accounts for more than 75% of RCC cases, which is characterized by high invasiveness and is inclined to metastasize.[1, 2] However, the treatment of ccRCC remains a challenge in clinical setting, owing to the facts that it is easy to develop metachronous distant metastases after surgical resection, and insensitive to chemotherapy or radiotherapy.[3, 4] Therefore, it is still an urgent clinical need for advanced ccRCC to develop the new drugs and treatment strategies. As far as we know, metabolic reprogramming is an important feature of ccRCC, which is usually associated with mutations in VHL tumor suppressor genes, and about 90% of ccRCC have VHL Page 2/25 mutations.[5] Studies showed that mutations in the VHL gene can lead to the stable expression of hypoxia-inducible factors HIF-1α and HIF-2α.[6] In addition to the aberrant polyunsaturated fatty acids (PUFAs) elevation mediated by HIF-2α, the stable expression of HIF-1α and HIF-2α also reduce the catabolism of fatty acids through inhibiting β oxidation,[7] endowing the significantly higher level of PUFAs in ccRCC than that of normal kidney tissue.[8] It is worth noting that PUFAs containing diallyl groups are more likely to be oxidized to form lipid peroxides (L-OOH) compared with saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA).[9] Therefore, the changes of metabolic feature in ccRCC makes it inherently prone to a new form of regulated cell death, ferroptosis, which is characterized by the accumulation of iron-dependent reactive oxygen species (ROS) and LPO to lethal levels.[7, 8, 10, 11] Briefly, PUFAs in cells are more likely to be oxidized by ROS (e.g., hydroxyl radical (•OH)) to form L- OOH. Then, the L-OOH react with redox-active transition metals such as ferrous iron (Fe2+) to produce reactive accumulation of alkoxyl radicals (L-O•) that can deplete nucleic acids and proteins. Introduction Eventually, the extensive peroxidation of lipids including L-OOH and L-O• results in bio-membrane destabilization and micelle formation, driving cells to undergo ferroptosis.[12-15] In recent years, numerous studies have focused on the development of iron-based nanomaterials that catalyze •OH generation by the Fenton reaction between H2O2 and Fe3+/Fe2+ to attack PUFAs and initiate ferroptosis.[16-18] However, the intratumoral H2O2 concentration (50-100 × 10−6 M) is limited and insufficient to produce desirable and enough •OH for inducing satisfactory nanocatalytic-therapeutic efficacy via Fenton or Fenton-like reactions.[19] In addition, tumor cells have evolved powerful antioxidant systems to prevent lipid peroxidation and ferroptosis, resulting in limited antitumor efficiency when relying solely on Fenton reaction. Reduced glutathione (GSH), as a vital antioxidant molecule, can cycle between oxidized glutathione (GSSG) by participating in redox biochemical reactions, enabling GSH/GSSG to be an essential antioxidant defense conserved in eukaryotes.[20, 21] Cysteine is a building block for the biosynthesis of GSH, and system xc -, as a transmembrane cystine-glutamate antiporter, imports cystine into cells, which plays a key role in GSH synthesis and cellular redox regulation.[22, 23] On the other hand, glutathione peroxidase 4 (GPX4), as a major antioxidant enzyme can directly reduce lipid hydroperoxides to form lipid alcohols by using two GSH molecules as electron donors, which prevents the transition metal (e.g., Fe2+)-dependent formation of toxic lipid reactive oxygen species. [9] Therefore, to induce ferroptosis using iron-based nanomaterials, the Fe dose needs to be very high, even up to 75 mg kg−1 body weight, via intravenous injection,[17] and most of the reported iron-based nanomaterials need to either include an additional component that can contribute to ferroptosis, or be combined with other therapeutic approaches (e.g., chemotherapy).[19, 24] It is worth noting that erastin, sulfasalazine, ras-selective lethal small molecules (RSL3) etc. have been found to trigger accumulation of lipid hydroperoxides and ferroptosis by blocking the synthesis of GSH or directly inhibiting GPX4 activity. These small molecules have emerged as the most potent and selective compounds for killing ccRCC cells.[8, 25] However, their low specificity for tumors, short half-life in the blood, poor water solubility and deficient accumulation at the tumor site prevents their reliable application in vivo. Nanotechnology has been widely investigated to develop advanced drug delivery system for cancer therapy, with the intrinsic Page 3/25 Page 3/25 physicochemical properties of nanomaterials. Introduction Therefore, MIL- 101(Fe) is not only an RSL3 delivery platform for improving tumor specificity and prolonging the circulation half-life but also a ferric ion supplier for increasing the labile iron pool. MIL-101(Fe)@RSL3 is expected to provide a promising nanodrug for clear-cell renal cell carcinoma therapy by triggering waterfall-like ferroptosis based on “iron overload” and the antioxidative system suppression. Materials PEG-COOH (MW: 2000), 2-aminoterephthalic acid (H2BDC-NH2), chloroform and FeCl3 .6H2O were purchased from Aladdin Reagent (Shanghai, China). RSL3 was obtained from Meilun Biotechnology Co., Ltd. (Dalian, China). Cell Counting Kit-8 (CCK-8), Prussian Blue iron stain kit, and Calcein-AM/propidium iodide (PI) double stain kit was obtained from Solarbio Science & Technology Co, Ltd. (Beijing, China). LysoTracker Red, a cellular glutathione peroxidase assay kit, and 2',7'-dichlorofluorescin diacetate (DCF- DA) were purchased from Beyotime Biotechnology Co. (Beijing, China). ICG-NHS was purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Xi'an, China). The anti-GPX4 and β-actin antibodies were purchased from Abcam (CA, USA). BODIPY581/591-C11 was obtained from Thermo Fisher (Waltham, MA, USA). Introduction Metal-organic frameworks (MOFs) with a high loading capacity attributed to their large surface area and ultra-high porosity enable them to be promising platforms for hydrophobic small molecule drugs delivery.[26] Particularly, Iron based MOFs nanoparticles (MIL-101(Fe) NPs) with great biocompatibility and degradability provides the possibility of achieving stimuli-responsive controlled release of various pharmacological molecules.[27, 28] In this study, MIL-101(Fe) nanoparticles are loaded with RSL3 to form a new ferroptosis agonist (MIL- 101(Fe)@RSL3) in which MIL-101(Fe) released RSL3 and ferric ions (Fe3+) accompanying with the degradation in the acidic tumor environment. Fe3+, as a degradation product, can be reduced to ferrous ions (Fe2+) by ferric reductase and other reductive molecules in the cell, thereby triggering “iron overload”. [29] Fe2+ catalyzes the intrinsic nontoxic H2O2 to form highly reactive •OH by the Fenton reaction in cancer cells, which attacks PUFAs to form L–OOH and initiates the ferroptosis.[27] Meanwhile, RSL3 directly inhibits GPX4 to further induce the aberrated accumulation of L–OOH, triggering severe ferroptosis. Subsequently, Fe2+ continues to catalyze the generation of highly reactive lipid alkoxyl radicals (L-O•) from L-OOH, thereby aggravating ferroptosis-based cell death (Scheme 1). Therefore, MIL- 101(Fe) is not only an RSL3 delivery platform for improving tumor specificity and prolonging the circulation half-life but also a ferric ion supplier for increasing the labile iron pool. MIL-101(Fe)@RSL3 is expected to provide a promising nanodrug for clear-cell renal cell carcinoma therapy by triggering waterfall-like ferroptosis based on “iron overload” and the antioxidative system suppression. In this study, MIL-101(Fe) nanoparticles are loaded with RSL3 to form a new ferroptosis agonist (MIL- 101(Fe)@RSL3) in which MIL-101(Fe) released RSL3 and ferric ions (Fe3+) accompanying with the degradation in the acidic tumor environment. Fe3+, as a degradation product, can be reduced to ferrous ions (Fe2+) by ferric reductase and other reductive molecules in the cell, thereby triggering “iron overload”. [29] Fe2+ catalyzes the intrinsic nontoxic H2O2 to form highly reactive •OH by the Fenton reaction in cancer cells, which attacks PUFAs to form L–OOH and initiates the ferroptosis.[27] Meanwhile, RSL3 directly inhibits GPX4 to further induce the aberrated accumulation of L–OOH, triggering severe ferroptosis. Subsequently, Fe2+ continues to catalyze the generation of highly reactive lipid alkoxyl radicals (L-O•) from L-OOH, thereby aggravating ferroptosis-based cell death (Scheme 1). Analysis of drug and iron ions releasing in vitro Analysis of drug and iron ions releasing in vitro The release profiles of RSL3 and iron ions (Fe3+) were analyzed according to previous reports.[15, 31] PEG-MIL-101(Fe)@RSL3 NPs were dispersed in PBS at pH 7.4 and pH 5.0 containing 5% (w/v) sodium dodecyl sulfate for different durations. The mixtures were encapsulated in the regenerated cellulose membrane (MWCO: 1000 Da), and the buffer was stirred with a magnetic flea. At pre-determined time points, buffer was withdrawn for drug concentration analysis by HPLC. For Fe3+ analysis, PEG-MIL- 101(Fe)@RSL3 NPs (1 mg/mL) were re-suspended in PBS at pH 7.4 or 5.0. Then, the Fe3+ concentration was spectrophotometrically calculated in the o-phenanthroline reaction system at different times. Synthesis of MIL-101(Fe) NPs The synthetic procedure of MIL-101(Fe) NPs was consistent with a previous report.[30] Briefly, 2.48 mmol (0.4492 g) of H2BDC-NH2 and 5.0 mmol (1.35 g) of FeCl3 .6H2O were dissolved in 60 mL of DMF, and the mixture was packaged in a Teflon-lined stainless-steel autoclave and kept at 120 °C for 12 hours. After cooling to room temperature, the precipitate was obtained by centrifugation and washed with ethanol three times. Instruments The morphology and elemental mapping images were recorded with a transmission electron microscope (FEI Tecnai F20, acceleration voltage = 200 kV). The hydrodynamic sizes and zeta potentials of the NPs were detected with a Malvern Zetasizer Nano ZS (Malvern, UK). Confocal microscopy images were obtained with a confocal laser scanning microscope (Leica TCS SP8 STED, Germany), and the flow cytometry assay was performed with a flow cytometer (BD FACSAria™ III, USA). In vivo imaging was performed with an IVIS imaging system (PerkinElmer, USA). Page 4/25 Page 4/25 Synthesis of MIL-101(Fe) NPs Drug loading and surface modification Drug loading and surface modification MIL-101(Fe) NPs (25 mg) were dispersed into 10 mL of chloroform, and 1.5 mg of RSL3 was added to the solution under ultrasonication for 30 min. Then, the reaction system was continuously stirred overnight. After solvent evaporation, the obtained NPs were suspended in deionized water and dialyzed against distilled water to obtain the MIL-101(Fe)@RSL3 nanocomposites. PEG-COOH was used for the surface modification of MIL-101(Fe)@RSL3 nanocomposites. First, 20 mg of PEG-COOH (MW: 2000) was dissolved in water and 2 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.5 mg of NHS were added to react for 4 hours, and then the mixed solution was stirred with 25 mg of the MIL- 101(Fe)@RSL3 nanocomposites for 16 hours. The PEGylated MIL-101(Fe)@RSL3 NPs were recycled by centrifugation (8500 rpm, 10 min), and the supernatant was collected to determine the content and release of RSL3 by HPLC. Lipid peroxide detection Intracellular LPO was detected using BODIPY581/591-C11, which can insert into lipid membranes and be oxidized by LPO. 786-O cells were seeded in 20 mm glass-bottom cell culture dishes and incubated for 24 hours. Then, PBS, MIL-101(Fe), free RSL3 or MIL-101(Fe)@RSL3 (RSL3 dose: 1.4 μM) was added to the cells followed by incubation for another 3 hours. After that, the cells were washed with PBS and incubated in serum-free RPMI 1640 medium containing BODIPY581/591-C11 (5 μM) for 20 min. After washing with PBS, the cells were subjected to CLSM observation. The oxidized products display green fluorescence (Ex/Em: 485 nm/520 nm). Cellular uptake and subcellular localization in vitro Cellular uptake and subcellular localization in vitro 786-O cells were seeded in 6-well culture plates for 24 hours. Then, the ICG-labelled MIL-101(Fe)@RSL3 NPs were incubated with the cells for 1, 2, 4, and 8 hours, and the cell culture medium was removed. After washing with PBS twice, the cells were collected and re-suspended in 500 μL of PBS. Then, the cells of different groups were evaluated by flow cytometry. To evaluate the subcellular localization of MIL-101(Fe) NPs, 786-O cells were seeded in 20 mm glass- bottom cell culture dishes. After removing the cell culture medium, Lyso Tracker Red staining solution was incubated with cells for 20 min. Then, ICG-labelled MIL-101(Fe)@RSL3 was added followed by incubation for an additional 1 hour. The cells were observed by CLSM (Ex/Em: 543 nm/590 nm for LysoTracker Red and Ex/Em: 633 nm/780 nm for MIL-101(Fe)@RSL3-ICG). Intracellular iron ion release and ROS detection 786-O cells were seeded in 20 mm glass-bottom cell culture dishes and incubated for 24 hours. Then, PBS, MIL-101(Fe), free RSL3 or MIL-101(Fe)@RSL3 (RSL3 dose: 1.4 μM) was added to the cells followed by incubation for another 3 hours. After that, the cells were washed with Hanks’ balanced salt solution (HBSS) and incubated in HBSS containing Phen Green SK (20 μM) for 20 min. The cells were washed with HBSS before CLSM observation (Ex/Em: 488 nm/517 nm). Intracellular ROS was determined by using DCFH-DA. 786-O cells were treated with different samples, and then DCFH-DA (10 μM) in serum-free RPMI 1640 medium was added. After coincubation for 30 min, the cells were washed with PBS before CLSM observation (Ex/Em: 488 nm/529 nm). Cell and animal model All cell lines were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Human renal clear cell adenocarcinoma cell line 786-O cells were cultured in RPMI-1640 (Gibco, USA) media; human renal tubular epithelial cell line HK-2 and human hepatocyte cell line LO2 cells were cultured in DMEM (Gibco, USA) media. All media were supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin. All cells were cultured in a humidified incubator at 37℃ with 5% CO2. Female BALB/C nude mice were sourced from Guangdong Medical Laboratory Animal Center (Guangdong, China) and used according to the protocols approved by the Animal Ethics Committee of Page 5/25 Jinan University. The tumor model was established by subcutaneously injecting 4×106 786-O cells suspended in PBS (pH 7.4) into the dorsum of each mouse. When the tumor volumes reach to 50 mm3, the mice were applied for experiment. Jinan University. The tumor model was established by subcutaneously injecting 4×106 786-O cells suspended in PBS (pH 7.4) into the dorsum of each mouse. When the tumor volumes reach to 50 mm3, the mice were applied for experiment. Cytotoxic and anti-tumor effect in vivo The cytotoxicity measurement was quantified according to the standard CCK-8 approach. 786-O cells were seeded in 96-well plates at a density of 5×103 cells/well and incubated for 24 hours. Then, MIL- 101(Fe), free RSL3 or MIL-101(Fe)@RSL3 was added to each well at different concentrations. After 24 hours of incubation, CCK-8 solution was added to each well, and the cells were incubated for an additional 1 hour. Afterwards, the absorbance was measured with a microplate reader at 450 nm. Cell viability was calculated by the ratio of the absorbance of the sample to the absorbance of the control, and each test was repeated at least three times. Then, calcein-AM and PI were used for live/dead cell staining after different treatments. The cytotoxicity measurement was quantified according to the stan were seeded in 96-well plates at a density of 5×103 cells/well and i 101(Fe), free RSL3 or MIL-101(Fe)@RSL3 was added to each well a hours of incubation, CCK-8 solution was added to each well, and th additional 1 hour. Afterwards, the absorbance was measured with a viability was calculated by the ratio of the absorbance of the samp and each test was repeated at least three times. Then, calcein-AM a staining after different treatments. In vivo fluorescence imaging and biodistribution of nanodrugs To track the biodistribution of MIL-101(Fe)@RSL3, ICG-labelled MIL-101(Fe)@RSL3 was intravenously injected into mice. Fluorescence images of mice were recorded at different time points with an IVIS imaging system, and then the tumor and main organs were harvested for fluorescence imaging. Blood circulation was measured by drawing 10 µL of blood from the tail vein of the tumor-bearing mice at different times after intravenous injection of ICG-labelled PEG-MIL-101(Fe)@RSL3. Each blood sample was dissolved in 140 µL of lysis buffer (1% sodium dodecyl sulfonate, 1% Triton X-100, 40×10-3 M Tris acetate), and the fluorescence intensities were measured using a microplate reader (excitation wavelength: 789 nm, emission wavelength: 814 nm, n=3). The blank blood sample was also measured as the control to subtract blood autofluorescence. GPX4 activity and level assay GPX4 activity was measured with a commercial glutathione peroxidase assay kit. 786-O cells were treated with PBS, MIL-101(Fe), free RSL3 or MIL-101(Fe)@RSL3 (RSL3 dose: 1.4 μM). Twelve hours later, Page 6/25 the cells were harvested in PBS solution containing 0.2% (w/v) ethylenediaminetetraacetic acid (EDTA). Then, GPX4 activity was detected according to the manufacturer's protocol. The absorbance at 340 nm was recorded every 1 min with a SpectraMax microplate reader with the temperature maintained at 25 °C. the cells were harvested in PBS solution containing 0.2% (w/v) ethylenediaminetetraacetic acid (EDTA). Then, GPX4 activity was detected according to the manufacturer's protocol. The absorbance at 340 nm was recorded every 1 min with a SpectraMax microplate reader with the temperature maintained at 25 °C. We also employed a Western blotting approach to assess GPX4 levels. In brief, the lysate of formulation- treated 786-O cells was collected for analysis. The level of protein in these samples was quantified. Then, samples containing equal amounts of protein (20 μg) were loaded and subjected to standard SDS polyacrylamide gel electrophoresis. The proteins were separated at a constant voltage, followed by electrical transfer to a 0.45 μm poly (vinylidene fluoride) (PVDF) membrane. Upon membrane blocking with 5% milk phosphate buffered saline with Tween 20 (PBST) solution for 1 hour at room temperature, the target proteins were incubated with GPX4-specific antibody and β-actin antibody overnight at dilutions of 1:2500 and 1:5000, respectively, at 4 °C. Thereafter, HRP-conjugated anti-rabbit IgG H&L at a dilution of 1:5000 was applied as the secondary antibody, and the specific bands were developed using ECLTM Western blotting detection reagents. Cytotoxic and anti-tumor effect in vivo Anti-tumor effect in vivo BALB/c nude mice were randomly divided into four groups (n=6). The nanodrugs were intravenously injected into mice three times a week for a total of 5 injections. The tumor volume was measured every three days according to the formula: V=(L×W2)/2, where L is the tumor length and W is the tumor width. The relative tumor volume was calculated according to the equation: V/V0, where V0 is the initial tumor volume before the start of treatment. Then, 21 days later, the tumor tissues were excised for weighing and histological examination. Statistical analysis The data are presented as the mean±standard deviation (SD). The difference of each treatment group was statistically compared via either Student’s t test or analysis of variance integrated with Tukey’s post hoc analysis. The threshold P value was set at 0.05. Side effects evaluation Human normal hepatocyte LO2 and human renal tubular epithelial cell HK-2 cells were seeded into 96- well plates and treated with a series of MIL-101(Fe)@RSL3 concentrations (RSL3 dose 0, 0.35, 0.70, 1.40, Page 7/25 Page 7/25 Page 7/25 2.80, 5.60 μM). Cell viability was evaluated by CCK-8 assay, which was conducted according to the manufacturer's protocol. 2.80, 5.60 μM). Cell viability was evaluated by CCK-8 assay, which was conducted according to the manufacturer's protocol. Whole blood was collected from the mice and used for haemolytic analysis. After centrifugation at 800 rpm, the upper serum was discarded, and the lower blood cells were washed with cold PBS three times and mixed with a certain amount of MIL-101(Fe)@RSL3. Twenty-four hours later, the mixture was centrifuged at 800 rpm, and the absorbance of the supernatant was measured at 350 nm to calculate the haemolysis rate. To evaluate side effects in vivo, the mice were sacrificed seven days after treatment. Approximately 0.8 mL of blood was collected from each mouse to conduct complete blood panel analysis and serum biochemistry assays at the Laboratory of Comparative Medicine, Guangdong Medical Laboratory Animal Center. The major organs of the mice from each group were collected and stained with H&E according to standard techniques to evaluate toxicity. Characterization of MIL-101(Fe)@RSL3 nanoparticles (NPs) In contrast, when exposed to acidic conditions (pH 5.0), the structure of MIL- 101(Fe) NPs showed a tendency to degrade and collapse (Figure S2B), which was consistent with previous reports.[27, 31] Moreover, whether the MIL-101(Fe) NPs could release iron ions were colourimetrically determined with the products of o-phenanthroline and ferrous ions. After incubation with MIL-101(Fe) NPs in PBS pH 7.4 (Figure S3C) and pH 5.0 (Figure S3D) for 1, 2, 4, 8, 12, and 24 hours, the supernatants became reddish-orange in color, which was attributed to the iron ion release into solution. It could be clearly observed that the number of iron ions released into the acidic solution increased significantly. According to calculations from the standard curve (Figure S3A, B), the iron ion concentrations in the supernatants were 0.21 mM at pH 7.4 and 0.50 mM at pH 5.0 (Figure 1G). Hydroxyl radicals (•OH) were generally considered to be the most toxic ROS, which could be generated by the Fenton reaction between H2O2 and Fe3+/Fe2+ to result in the lethal lipid peroxidation and initiate ferroptosis.[23, 33] To investigate whether MIL-101(Fe) NPs could produce •OH in the presence of H2O2, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was employed as a trapping probe to monitor the •OH generation by electron paramagnetic resonance (ESR). It was found that the characteristic 1:2:2:1 signal in the ESR spectrum attributing to •OH was observed for the MIL-101(Fe) NPs incubated with H2O2 (1 mM) at acidic (pH 5.0) and neutral (pH7.4) solution (Figure 1H). Particularly, the stronger •OH signals were observed at acidic solution, indicating the acidic microenvironment promoted the release of iron ions and thus increased the production of •OH. Moreover, the production of •OH was further detected by methylene blue (MB) decolorization experiments More obvious color fading of MB was observed in acidic solution after 101(Fe) had been successfully modified with PEG. It is well known that MOF NPs possess tunable porous structures and high specific surface areas, which facilitate drug loading and release. Herein, a hydrophobic small molecular (1S,3R-RSL3) was loaded into MIL-101(Fe) to form a drug delivery composite (MIL-101(Fe)@RSL3). Energy-dispersive X-ray spectroscopy (EDX) elemental mapping of MIL- 101(Fe)@RSL3 showed C, O, Fe and Cl, which confirmed that RSL3 was loaded into MIL-101(Fe) (Figure 1E). Fourier transform infrared spectroscopy (FTIR) was used to analyze the PEGylated MIL- 101(Fe)@RSL3 (Figure S1). Characterization of MIL-101(Fe)@RSL3 nanoparticles (NPs) The vibrational band at approximately 585 cm-1 belonged to the Fe-O vibration, and the peaks at 3363 cm-1 and 3466 cm-1 were ascribed to the asymmetrical and symmetrical stretching vibrations of the amine groups, respectively.[32] After loading with RSL3, the characteristic C-Cl peak at 742 cm-1 appeared in PEG-MIL-101(Fe)@RSL3. Additionally, the band at 2883 cm-1 attributed to - OH disappeared, and the bands from the amine groups at 3466 cm-1 weakened, suggesting the formation of amide bonds between the -COOH of PEG and the -NH2 of MIL-101(Fe). Hereafter, these PEGylated nanoparticles were abbreviated as MIL-101(Fe) and MIL-101(Fe)@RSL3. The RSL3 content in MIL- 101(Fe) was further analyzed by HPLC, and the loading efficiency was 2.23% with an encapsulation efficiency of 88.7%, indicating a highly efficient drug loading properties. Apart from their drug delivery capability, MIL-101(Fe) NPs showed a pH-dependent drug release profile in vitro (Figure 1F), and the RSL3 release rate in the simulated lysosomal microenvironment (pH 5.0) was much faster than that in neutral phosphate buffer (pH 7.4). The MIL-101(Fe) NPs remained stable in pH 7.4 solution even after incubation for 12 hours (Figure S2A). In contrast, when exposed to acidic conditions (pH 5.0), the structure of MIL- 101(Fe) NPs showed a tendency to degrade and collapse (Figure S2B), which was consistent with previous reports.[27, 31] Moreover, whether the MIL-101(Fe) NPs could release iron ions were colourimetrically determined with the products of o-phenanthroline and ferrous ions. After incubation with MIL-101(Fe) NPs in PBS pH 7.4 (Figure S3C) and pH 5.0 (Figure S3D) for 1, 2, 4, 8, 12, and 24 hours, the supernatants became reddish-orange in color, which was attributed to the iron ion release into solution. It could be clearly observed that the number of iron ions released into the acidic solution increased significantly. According to calculations from the standard curve (Figure S3A, B), the iron ion concentrations in the supernatants were 0.21 mM at pH 7.4 and 0.50 mM at pH 5.0 (Figure 1G). Hydroxyl radicals (•OH) were generally considered to be the most toxic ROS, which could be generated by the Fenton reaction between H2O2 and Fe3+/Fe2+ to result in the lethal lipid peroxidation and initiate ferroptosis.[23, 33] To investigate whether MIL-101(Fe) NPs could produce •OH in the presence of H2O2, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was employed as a trapping probe to monitor the •OH generation by electron paramagnetic resonance (ESR). Characterization of MIL-101(Fe)@RSL3 nanoparticles (NPs) Characterization of MIL-101(Fe)@RSL3 nanoparticles (NPs Iron-based MOF NPs, MIL-101(Fe), were prepared by the solvothermal method with simple materials (FeCl3·6H2O and 2-aminoterephthalic acid). The MIL-101(Fe) morphology was visualized by transmission electron microscopy (TEM) and the particles showed good dispersibility and a uniform size of approximately 150 nm (Figure 1A). After modification with polyethylene glycol (PEG)-COOH, a nearly transparent thin layer of PEG appeared on the surface of MIL-101(Fe) (Figure 1B). Additionally, the average hydrodynamic diameter of MIL-101(Fe) changed from 174 nm to 190 nm (Figure 1C), and the zeta potential reversed from 34.5±0.6 to -23.7±0.5 after modification (Figure 1D), which was measured by dynamic light scattering (DLS). The increased size and reversed surface charge suggested that MIL- Page 8/25 Page 8/25 101(Fe) had been successfully modified with PEG. It is well known that MOF NPs possess tunable porous structures and high specific surface areas, which facilitate drug loading and release. Herein, a hydrophobic small molecular (1S,3R-RSL3) was loaded into MIL-101(Fe) to form a drug delivery composite (MIL-101(Fe)@RSL3). Energy-dispersive X-ray spectroscopy (EDX) elemental mapping of MIL- 101(Fe)@RSL3 showed C, O, Fe and Cl, which confirmed that RSL3 was loaded into MIL-101(Fe) (Figure 1E). Fourier transform infrared spectroscopy (FTIR) was used to analyze the PEGylated MIL- 101(Fe)@RSL3 (Figure S1). The vibrational band at approximately 585 cm-1 belonged to the Fe-O vibration, and the peaks at 3363 cm-1 and 3466 cm-1 were ascribed to the asymmetrical and symmetrical stretching vibrations of the amine groups, respectively.[32] After loading with RSL3, the characteristic C-Cl peak at 742 cm-1 appeared in PEG-MIL-101(Fe)@RSL3. Additionally, the band at 2883 cm-1 attributed to - OH disappeared, and the bands from the amine groups at 3466 cm-1 weakened, suggesting the formation of amide bonds between the -COOH of PEG and the -NH2 of MIL-101(Fe). Hereafter, these PEGylated nanoparticles were abbreviated as MIL-101(Fe) and MIL-101(Fe)@RSL3. The RSL3 content in MIL- 101(Fe) was further analyzed by HPLC, and the loading efficiency was 2.23% with an encapsulation efficiency of 88.7%, indicating a highly efficient drug loading properties. Apart from their drug delivery capability, MIL-101(Fe) NPs showed a pH-dependent drug release profile in vitro (Figure 1F), and the RSL3 release rate in the simulated lysosomal microenvironment (pH 5.0) was much faster than that in neutral phosphate buffer (pH 7.4). The MIL-101(Fe) NPs remained stable in pH 7.4 solution even after incubation for 12 hours (Figure S2A). Intracellular iron ion release and ferroptosis of ccRCC cells. To further investigate the intracellular iron ion released from MIL-101(Fe)@RSL3, the NPs were pre- labelled with indocyanine green (ICG) on the surface and the cellular uptake was evaluated by fluorescence-activated cell sorting (FACS). After the MIL-101(Fe)@RSL3-ICG NPs were incubated with human RCC cells (786-O cells), the fluorescence intensity of ICG was significantly enhanced with prolonged incubation time, which indicated the cellular accumulation of fluorescently labelled NPs (Figure S5). Then, the intracellular localization of MIL-101(Fe)@RSL3-ICG NPs was determined by confocal laser scanning microscopy (CLSM). Notably, the fluorescence of MIL-101(Fe)@RSL3-ICG matched that of LysoTracker Red (Figure 2A), which suggested that MIL-101(Fe)@RSL3-ICG was internalized by endosomes or lysosomes. The extracellular microenvironment of solid tumors is acidic (pH 6.5~7.0) and lower than that of normal tissues (pH 7.4), and the tumor endosomes/lysosomes are severely acidic, reaching pH values as low as 4.5~5.5.[34] The acidic microenvironment of endosomes/lysosomes easily triggers the degradation of MIL-101(Fe) and the release of Fe3+ ions. Moreover, the presence of reducing agents such as ferrireductase, superoxide anion radicals (O2 .–), cysteine and glutathione can reduce the Fe3+ ions into Fe2+ ions, and then Fe2+ ions can be transported to the labile iron pool in the cytoplasm by divalent metal transporter 1 (DMT1).[35, 36] The intracellular Fe2+ ion fluorescent probe Phen Green SK (PGSK) was applied to detect changes in the Fe2+ ion levels. The fluorescence of PGSK was remarkably reduced in the MIL-101(Fe) and MIL-101(Fe)@RSL3 incubation groups owing to Fe2+ ion chelation (Figure 2B), which manifested the degradation of MIL-101(Fe) in the endosomes/lysosomes and enhanced the intracellular labile iron pool level. Next, to investigate whether the MIL-101(Fe) would catalyze the •OH production via the Fenton reaction, the aminophenyl fluorescein (APF), an •OH-specific fluorescent probe, was used to verify the intracellular •OH level. After incubated with MIL-101(Fe), the fluorescence of •OH was slightly enhanced. Interestingly, fluorescence signal was significantly enhanced in the MIL-101(Fe)@RSL3 group, suggesting a burst of •OH in 786-O cells (Figure 2C). It suggested that MIL-101(Fe) could catalyze the •OH production via the Fenton reaction attributing to the much higher content of H2O2 in cancer cells (up to 100×10-6 M) than in normal tissues (≈20×10-9 M). The •OH would cause the peroxidation of polyunsaturated fatty acid-containing phospholipids (PUFAs) to form lipid peroxides (L–OOH) and propagate the radical process to adjacent lipid molecules. Characterization of MIL-101(Fe)@RSL3 nanoparticles (NPs) It was found that the characteristic 1:2:2:1 signal in the ESR spectrum attributing to •OH was observed for the MIL-101(Fe) NPs incubated with H2O2 (1 mM) at acidic (pH 5.0) and neutral (pH7.4) solution (Figure 1H). Particularly, the stronger •OH signals were observed at Page 9/25 Page 9/25 confirmed the acidic environment accelerated the •OH-production. Overall, with excellent biocompatibility and biodegradable properties, MIL-101(Fe) NPs serve as an ideal drug delivery platform and continuously provide iron ions for •OH-production through the Fenton reaction. confirmed the acidic environment accelerated the •OH-production. Overall, with excellent biocompatibility and biodegradable properties, MIL-101(Fe) NPs serve as an ideal drug delivery platform and continuously provide iron ions for •OH-production through the Fenton reaction. Intracellular iron ion release and ferroptosis of ccRCC cells. Intracellular iron ion release and ferroptosis of ccRCC cells. [22, 37] Although there had been reported that an increase in the labile iron pool caused by intracellular iron overload would trigger ferroptosis, cellular iron overload was not sufficient to produce enough intracellular •OH by solely depending on the Fenton reaction.[17, 38] The strong antioxidant system in cells would block the propagation process of L–OOH, and prevented the generation of highly reactive l d lk l d l ( ) h b 2+ d [ ] h f To further investigate the intracellular iron ion released from MIL-101(Fe)@RSL3, the NPs were pre- labelled with indocyanine green (ICG) on the surface and the cellular uptake was evaluated by fluorescence-activated cell sorting (FACS). After the MIL-101(Fe)@RSL3-ICG NPs were incubated with human RCC cells (786-O cells), the fluorescence intensity of ICG was significantly enhanced with prolonged incubation time, which indicated the cellular accumulation of fluorescently labelled NPs (Figure S5). Then, the intracellular localization of MIL-101(Fe)@RSL3-ICG NPs was determined by confocal laser scanning microscopy (CLSM). Notably, the fluorescence of MIL-101(Fe)@RSL3-ICG matched that of LysoTracker Red (Figure 2A), which suggested that MIL-101(Fe)@RSL3-ICG was internalized by endosomes or lysosomes. The extracellular microenvironment of solid tumors is acidic (pH 6.5~7.0) and lower than that of normal tissues (pH 7.4), and the tumor endosomes/lysosomes are severely acidic, reaching pH values as low as 4.5~5.5.[34] The acidic microenvironment of Page 10/25 Page 10/25 expected to be an effective way to induce ferroptosis by producing the •OH and inhibiting the antioxidant system to disrupt the redox balance of cells. As an antioxidant enzyme, GPX4 is essential to maintain lipid homeostasis in cells, preventing the accumulation of lipid peroxidation and blocking ferroptosis. RSL3, a direct inhibitor of GPX4, was expected to be a small molecule drug that induces L–OOH in tumors, but its insolubility and poor pharmacokinetic properties have limited its application. Fortunately, in addition to providing iron ions to cancer cells, MIL-101(Fe) NPs can also serve as an RSL3 delivery platform. To evaluate the inhibition efficiency of RSL3, nicotinamide adenine dinucleotide phosphate (NADPH) levels, which indirectly reflect GPX4 activity, were detected. MIL-101(Fe) did not cause a loss of activity of GPX4, showing activity analogous to that of the control group. In contrast, intracellular NADPH remained almost constant in the free RSL3 and MIL-101(Fe)@RSL3 treatment groups, indicating a decrease in GPX4 activity (Figure 3A). Intracellular iron ion release and ferroptosis of ccRCC cells. Moreover, GPX4 expression showed a remarkable decrease after RSL3 and MIL-101(Fe)@RSL3 treatment (Figure 3B), which can be easily explained by the mechanism of action of RSL3 as a powerful GPX4 inhibitor. This result implied that RSL3 could be released from MIL-101(Fe) NPs and exert an inhibitory effect. Then, BODIPY-C11, a membrane-targeted fluorescence probe of L–OOH, was used for staining the various treatment groups.[39] MIL-101(Fe) NPs only slightly increased the oxidized BODIPY-C11 signal, whereas RSL3 and MIL-101(Fe)@RSL3 strongly enhanced the fluorescence of oxidized BODIPY-C11 (Figure 3C), indicating a breakdown of the redox equilibrium. In addition, the intracellular oxidative stress of 786-O cells after various treatments was carefully studied by utilizing DCFH-DA as the intracellular total ROS probe. Both the fluorescence images (Figure S6A) and quantitatively analyst results (Figure S6B) showed that MIL-101(Fe)@RSL3 triggered ROS burst and accumulation, which was owing to the iron overload and the GPX4 inhibition. Therefore, the accumulation of lipid ROS induced by MIL- 101(Fe)@RSL3 is expected to trigger more violent ferroptosis. Cell viability assays indicated that MIL-101(Fe)@RSL3 showed significant cytotoxicity against ccRCC cells compared to RSL3 treatment. Even at a low concentration of loaded RSL3 (1.4 μM), the cell survival rate in the MIL-101(Fe)@RSL3 group was only ~28% (Figure 3D). In contrast, the high concentration of MIL-101(Fe) NPs showed negligible cytotoxicity. Furthermore, live/dead cell fluorescence staining showed that MIL-101(Fe)@RSL3 induced evident cytotoxicity compared with the other treatment groups (Figure 3E). However, normal cells, such as human hepatocyte cells (LO2) and human renal proximal tubular cells (HK-2), maintained a high cellular survival rate even at 11.2 μM MIL-101(Fe)@RSL3 NPs, which was 8 times higher than the concentration for ccRCC treatment (Figure S7). Interestingly, MIL-101(Fe)@RSL3 did not cause severe cytotoxicity in normal human hepatocytes or renal cells, and it is well known that the liver and kidney are vital organs that metabolize NPs. In vivo antitumor efficiency of MIL-101(Fe)@RSL3 against ccRCC Good compatibility is a prerequisite for the clinical application of nanodrugs. The negligible cytotoxicity of MIL-101(Fe)@RSL3 in normal cells inspired us to further investigate the antitumor effects of MIL- 101(Fe)@RSL3 in ccRCC. As we expected, after incubation with a high concentration of MIL- Page 11/25 Page 11/25 101(Fe)@RSL3 (2 mg/mL) for 24 hours, the haemolysis rate was only 0.5%, which can almost be ignored (Figure S8). Then, the nanodrugs were labelled with ICG to analyze their tissue distribution and tumor accumulation in vivo. After intravenous injection of ICG-MIL-101(Fe)@RSL3, the whole bodies of tumor- bearing mice showed bright fluorescence, and the tumor region showed an obvious fluorescence enhancement (Figure 4A). This result suggested that ICG-MIL-101(Fe)@RSL3 accumulated to a great extent in tumors, especially one-hour post-injection. Subsequently, the fluorescence gradually faded away; however, the tumor region remained bright up to 12 hours later (Figure 4B). The blood circulation behavior of ICG-MIL-101(Fe)@RSL3 was determined by evaluating the ICG fluorescence from the blood samples collected via the tail vein at various time points, and the half-life of MIL-101(Fe)@RSL3 was calculated to be ~2.5 hours (Figure 4C). Then, the main organs and tumor were isolated for fluorescence imaging. Similar to the fluorescence imaging in vivo, the fluorescence intensity of the tumor was significantly higher than that of the heart, lung, etc. (Figure 4D), while the high-intensity fluorescence in the liver, intestine, etc. suggested that the MIL-101(Fe)@RSL3 NPs would be cleared by the liver and finally excreted in bile and faeces (Figure 4E). Antitumor effects from the MIL-101(Fe)@RSL3 NPs were detected after intravenous administration. Tumor growth was constantly recorded over 21 days (Figure S9), and the changes in the tumor volumes indicated that MIL-101(Fe)@RSL3 remarkably inhibited tumor growth (Figure 5A). It is worth noting that the RSL3 treatment group showed only limited tumor suppression, which can probably be attributed to the insolubility of RSL3 in water and its deficient accumulation at the tumor site (Figure 5B, C). Moreover, haematoxylin and eosin (H&E) and Ki-67 staining of tumor tissue sections from the MIL-101(Fe)@RSL3 group also showed significantly fewer cancer cells and abundant karyorrhectic debris than the other groups, indicating more obvious damage to the cancer cells (Figure 5D). In vivo antitumor efficiency of MIL-101(Fe)@RSL3 against ccRCC Although both the MIL-101(Fe) and MIL-101(Fe)@RSL3 treatment groups showed extensive blue spots from Prussian blue staining, indicating the accumulation, degradation and release of iron ions from MIL-101(Fe) NPs into the tumors, the MIL-101(Fe) NPs still did not show obvious antitumor effects (Figure S10). In RSL3 and MIL- 101(Fe)@RSL3 groups, the immunohistochemical staining of tumor sections showed the level of GPX4 decrease, suggesting the RSL3 released from MIL-101(Fe) and inhibited the GPX4 (Figure 5E). These results suggested that solely causing iron overload proved to be unsuccessful in triggering severe ferroptosis and unable to have a satisfactory antitumor effect because of the powerful intracellular antioxidant network. Synchronous destroying the antioxidant network and causing iron overload could remarkably inhibit tumor growth by aggravating ferroptosis, while simply inhibiting the antioxidant network or triggering iron overloading showed only moderate or negligible antitumor effects in vivo. Side effects of MIL-101(Fe)@RSL3 The potential side effects of the MIL-101(Fe)@RSL3 nanoagents were evaluated after systemic administration. Haematological and blood biochemical analyses were performed one day post-treatment. Standard haematology markers, including white blood cells (WBCs), red blood cells (RBCs), haemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets (PLTs) and haematocrit (HCT), were measured (Figure 6A). Page 12/25 Page 12/25 Page 12/25 Compared with the control group, all parameters in the treatment groups appeared to be normal and did not show a significant difference (P value>0.05). This result indicated that neither RSL3 nor MIL-101(Fe) NPs caused obvious inflammation in mice. In addition, blood biochemical analyses were carried out, and various parameters, including alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CRE) and blood urea nitrogen (BUN), were examined (Figure 6B). There was no meaningful difference between the groups, suggesting that MIL-101(Fe)@RSL3 did not cause obvious hepatic or kidney toxicity to mice. During the treatment process, the body weights of the mice stably increased (Figure 6C). Then, histological analysis of the major organs, including the heart, liver, spleen, lung, and kidney, was conducting by slicing for H&E staining after 21 days (Figure 6D). No apparent histological abnormalities or lesions could be observed in any of the treatment groups throughout the whole treatment period. Especially, there was no noticeable damage in the liver or kidneys, which confirmed the biocompatibility of MIL-101(Fe)@RSL3. Acknowledgments This work is supported by the National Key Research and Development Program of China (2017YFA0205200), Guangdong Basic and Applied Basic Research Foundation (2020A1515011101, 2021A1515011703, 2021A1515010697), Natural Science Foundation of Heilongjiang Province of China (LC2017038), National Natural Science Foundation of China (61805049). We would like to acknowledge the Open Fund of MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science. Discussion It was well known that cancer cells have a higher demand for iron than the normal cells to support their rapid proliferation.[40, 41] In addition, most of cancer cells showed upregulated expression of transferrin receptor 1 and hepcidin along with downregulated expression of ferroportin, enabling cancer cells to accumulate iron highly efficiently via transferrin receptors.[42, 43] However, the intracellular concentration of iron ions still lies at a low level, making it insufficient to induce ferroptosis by catalyzing the Fenton reaction due to the massive demand of rapid growth cancer cells.[15, 31] In this study, iron-based MOFs nanoparticles with pH-responsive degradation properties could be uptake via endocytosis and release Fe3+ ions in lysosomes for remarkably increasing the intracellular content of Fe3+ ions. Subsequently, the intracellular Fe2+ referred to as the labile iron pool (LIP) also increase accordingly, because Fe3+ ions reduced to Fe2+ by the intracellular iron reductase (e.g., STEAP3) and superoxide.[44, 45] However, the intracellular •OH produced by Fe2+ together with H2O2 still remained low level only administrated with MIL-101(Fe) NPs. Even the high concentration of MIL-101(Fe) NPs (up to 106 μg/mL) administration could not induce plenty of lipid peroxidation and hardly cause cell death attributed to the strong anti- oxidation system. On the other hand, although the RSL3 as a small molecule drug effectively triggered ferroptosis in vitro evaluation, the in vivo anti-tumor efficacy was unsatisfactory, because of the low tumor specificity, short circulating half-life in blood, poor water solubility and deficiency accumulation at tumor site. In contrast, MIL-101(Fe) NPs not only served as the drug delivery platform but also continuously provided the iron ions for cancer cells, thereby enhancing the anti-tumor effect by aggravating ferroptosis. It could not be ignored that in this study, the ferroptosis was initiated by the Fenton reaction between intracellular Fe2+ and H2O2, which generate •OH to attack PUFAs. While, the limited contents of intracellular H2O2 (100 μM~1 mM) were difficult to produce sufficient •OH and highly efficiently trigger ferroptosis, making it is undesirable to induce satisfactory therapeutic efficacy. Therefore, it is reasonable to believe that further elevating the intracellular oxidative stress by combining Page 13/25 with chemotherapy, PDT and radiotherapy was expected to aggravate ferroptosis and achieve better curative effect.[46-48] with chemotherapy, PDT and radiotherapy was expected to aggravate ferroptosis and achieve better curative effect.[46-48] Declarations The author reports no conflicts of interest in this work. Conclusion In this study, MIL-101(Fe) nanoparticles rich in iron were used to load a ferroptosis inducer (RSL3) for targeted delivery and responsive release. MIL-101(Fe) showed high encapsulation efficiency and the tumor-targeted delivery of RSL3. Under the acidic tumor microenvironment, MIL-101(Fe)@RSL3 with gradual degradation released iron ions and RSL3, thereby aggravating ferroptosis in ccRCC cells by triggering iron overload and inhibiting GPX4. Therefore, compared with free RSL3, MIL-101(Fe)@RSL3 showed more remarkable antitumor efficacy against ccRCC, which displays an abnormal PUFAs metabolic state, while these nanodrugs did not cause obvious cytotoxicity in normal cells even at very high concentrations. In addition, MIL-101(Fe)@RSL3 caused negligible side effects, enabling it to be a promising nanodrug for systemic ccRCC therapy. Author contributions Conceptualization, Ni W., Lu C., and Wen L.; methodology, Ni W., Yuan X., Liang L.; data curation, Ni W., Liang L., Yuan X., Wen L.; writing and original draft preparation, Ni. W., Yuan X., Wen L.; writing and review and editing, Zhan M., Lu L., Xie Q., Wen L.; supervision, Lu L., Xie Q.; funding acquisition, Ni W., Lu C., Lu L., Wen L.. References [1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. 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[46] Hadi, Ranji-Burachaloo, Paul, Andrew, Gurr, Dave, Dunstan, Greg, Cancer Treatment Through Nanoparticle Facilitated Fenton Reaction, Acs Nano (2018). [47] Z. Shen, T. Liu, Y. Li, J. Lau, Z. Yang, W. Fan, Z. Zhou, C. Shi, C. Ke, V.I. Bregadze, S.K. Mandal, Y. Liu, Z. Li, T. Xue, G. Zhu, J. Munasinghe, G. Niu, A. Wu, X. Chen, Fenton-Reaction-Acceleratable Magnetic Nanoparticles for Ferroptosis Therapy of Orthotopic Brain Tumors, Acs Nano 12(11) (2018) 11355-11365. [48] T. Zhu, L. Shi, C. Yu, Y. Dong, F. Qiu, L. Shen, Q. Qian, G. Zhou, X. Zhu, Ferroptosis Promotes Photodynamic Therapy: Supramolecular Photosensitizer-Inducer Nanodrug for Enhanced Cancer Treatment, Theranostics 9(11) (2019) 3293-3307. Figures Page 18/25 Page 18/25 Page 18/25 Figure 1 Characterization of MIL-101(Fe), PEG-MIL-101(Fe) and PEG-MIL-101(Fe)@RSL3. TEM images of (A) M 101(Fe) NPs and (B) PEG-MIL-101(Fe) NPs. (C) DLS of MIL-101(Fe) and PEG-MIL-101(Fe) NPs. (D) Zet potentials of MIL-101(Fe) and PEG-MIL-101(Fe) NPs. (E) Elemental mapping of C, Pt, O, Fe and Cl of P MIL-101(Fe)@RSL3 NPs. (F) Release of RSL3 and (G) iron from PEG-MIL-101(Fe)@RSL3 NPs under Figure 1 Figure 1 Characterization of MIL-101(Fe), PEG-MIL-101(Fe) and PEG-MIL-101(Fe)@RSL3. TEM images of (A) MIL- 101(Fe) NPs and (B) PEG-MIL-101(Fe) NPs. (C) DLS of MIL-101(Fe) and PEG-MIL-101(Fe) NPs. (D) Zeta potentials of MIL-101(Fe) and PEG-MIL-101(Fe) NPs. (E) Elemental mapping of C, Pt, O, Fe and Cl of PEG- MIL-101(Fe)@RSL3 NPs. (F) Release of RSL3 and (G) iron from PEG-MIL-101(Fe)@RSL3 NPs under different pH conditions (7.4 and 5.0). (H) ESR spectra of different sample. Page 19/25 Figure 2 Intracellular iron ion release from MIL-101(Fe)@RSL3. (A) Intracellular localization of ICG-labelled MIL- 101(Fe)@RSL3 NPs in 786-O cells. (B) Fluorescence images of PGSK-stained 786-O cells with different treatments. (C) DCF fluorescence of cells. Figure 2 Figure 2 Intracellular iron ion release from MIL-101(Fe)@RSL3. (A) Intracellular localization of ICG-labelled MIL- 101(Fe)@RSL3 NPs in 786-O cells. (B) Fluorescence images of PGSK-stained 786-O cells with different treatments. (C) DCF fluorescence of cells. Page 20/25 Figure 3 MIL-101(Fe)@RSL3 triggered ferroptosis in ccRCC cells. (A) GPX4 activ represent the standard error of the mean of three replicates. P values: P levels in 786-O cells. (C) CLSM observations of cellular LPO with BODIP and the corresponding fluorescence intensity. (D) In vitro cytotoxicity of concentrations determined by CCK-8 proliferation assay. The error bars Page 21/25 Figure 3 MIL-101(Fe)@RSL3 triggered ferroptosis in ccRCC cells. (A) GPX4 activity in 786-O cells. The error bars represent the standard error of the mean of three replicates. P values: P <0.05. (B) Western blots of GPX4 levels in 786-O cells. (C) CLSM observations of cellular LPO with BODIPY581/591-C11 probe detection and the corresponding fluorescence intensity. (D) In vitro cytotoxicity of nanodrugs at different concentrations determined by CCK-8 proliferation assay. The error bars represent the standard error of the Figure 3 Page 21/25 MIL-101(Fe)@RSL3 triggered ferroptosis in ccRCC cells. (A) GPX4 activity in 786-O cells. The error bars represent the standard error of the mean of three replicates. P values: P <0.05. (B) Western blots of GPX4 levels in 786-O cells. (C) CLSM observations of cellular LPO with BODIPY581/591-C11 probe detection and the corresponding fluorescence intensity. (D) In vitro cytotoxicity of nanodrugs at different concentrations determined by CCK-8 proliferation assay. The error bars represent the standard error of the Page 21/25 Page 21/25 mean of six replicates. P values: P <0.05. (E) Fluorescence images of 786-O cells co-stained with calcein- AM (live cells, green) and PI (dead cells, red) after different treatments. Figure 4 Biodistribution of ICG-MIL-101(Fe)@RSL3. (A) Fluorescence images of tumor-bearing mice at different time points post-injection of ICG-MIL-101(Fe)@RSL3. The circles indicate the tumor regions. (B) Corresponding fluorescence intensity of the tumor regions. (C) Blood circulation curve of ICG-MIL- 101(Fe)@RSL3 determined by measuring the ICG fluorescence in the blood at different time points post- injection (n=3). (D) The distribution of ICG-MIL-101(Fe)@RSL3 in the main organs and tumors of tumor- bearing mice (I: intestine, H: heart, LU: lung, LI: liver, K: kidney, SP: spleen, and T: tumor) and (E) the corresponding fluorescence intensity of the organs (n=3). Figure 4 Biodistribution of ICG-MIL-101(Fe)@RSL3. (A) Fluorescence images of tumor-bearing mice at different time points post-injection of ICG-MIL-101(Fe)@RSL3. The circles indicate the tumor regions. (B) Corresponding fluorescence intensity of the tumor regions. (C) Blood circulation curve of ICG-MIL- 101(Fe)@RSL3 determined by measuring the ICG fluorescence in the blood at different time points post- injection (n=3). (D) The distribution of ICG-MIL-101(Fe)@RSL3 in the main organs and tumors of tumor- bearing mice (I: intestine, H: heart, LU: lung, LI: liver, K: kidney, SP: spleen, and T: tumor) and (E) the corresponding fluorescence intensity of the organs (n=3). Page 22/25 Figure 5 Figure 5 In vivo therapeutic efficacy of MIL-101(Fe)@RSL3 NPs. (A) Representative photographic images of mic with different treatments. (B) Relative tumor volumes after different treatments (n=6). (C) Photographic images of tumors and (D) corresponding tumor weights in different treatment groups at 21 days post- treatment (n=6). (E) H&E stained images of tumor tissue slices from different treatment groups. P < 0. Figure 5 In vivo therapeutic efficacy of MIL-101(Fe)@RSL3 NPs. (A) Representative photographic images of mice with different treatments. (B) Relative tumor volumes after different treatments (n=6). (C) Photographic images of tumors and (D) corresponding tumor weights in different treatment groups at 21 days post- treatment (n=6). (E) H&E stained images of tumor tissue slices from different treatment groups. P < 0.05. Page 23/25 Page 23/25 gure 6 de effects evaluation. (A) Haematological data of the mice after different treatments llows: white blood cell (WBC), red blood cell (RBC), haemoglobin (HGB), mean corpus MCV) l h l bi (MCH) l h l bi Page 24/25 Figure 6 Side effects evaluation. (A) Haematological data of the mice after different treatments. The terms are as follows: white blood cell (WBC), red blood cell (RBC), haemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet (PLT) and haematocrit (HCT). (B) Blood biochemical analysis. (C) The body weights of the mice were measured during the 21-day evaluation period under different conditions. (D) Representative H&E- Figure 6 Page 24/25 Side effects evaluation. (A) Haematological data of the mice after different treatments. The terms are as follows: white blood cell (WBC), red blood cell (RBC), haemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet (PLT) and haematocrit (HCT). (B) Blood biochemical analysis. Figure 4 (C) The body weights of the mice were measured during the 21-day evaluation period under different conditions. (D) Representative H&E- Page 24/25 Page 24/25 stained sections of the major organs (heart, liver, spleen, lung, and kidney). Data presented as the mean ± SD (n=6). stained sections of the major organs (heart, liver, spleen, lung, and kidney). Data presented as the mean ± SD (n=6). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. scheme1.jpg si.docx scheme1.jpg Page 25/25
W2895302935.txt	http://aminbiol.com.ua/20183pdf/4.pdf	uk		The condition of the prooxidant-antioxidant system in skeletal muscles of experimentally diabetic rats under vanadium citrate effect	Bìologìâ tvarin/Biologìâ tvarin	2,018	cc-by	3,813	Біологія тварин, 2018, т. 20, № 3 УДК 546.881:678.048:614.449 http://doi.org/10.15407/animbiol20.03.030 СТАН ПРООКСИДАНТНО-АНТИОКСИДАНТНОЇ СИСТЕМИ У СКЕЛЕТНИХ М’ЯЗАХ ЩУРІВ З ЕКСПЕРИМЕНТАЛЬНИМ ДІАБЕТОМ І ЗА ДІЇ ЦИТРАТУ ВАНАДІЮ Р. Я. Іскра, Г. В. Климець, О. О. Сушко, Л. І. Понкало, О. З. Сварчевська klimets.halyna@gmail.com Інститут біології тварин НААН, вул. В. Стуса, 38, м. Львів, 79034, Україна Досліджували вплив цитрату ванадію на вміст продуктів пероксидного окиснення ліпідів, відновленого глутатіону, активність ензимів антиоксидантної системи та глюкозо-6-фосфатдегідрогенази у скелетних м’язах стегна щурів з алоксан-індукованим діабетом. Тварини були поділені на п’ять груп: I група — контрольна, II, III, IV та V — дослідні. Щурам І та ІІ груп давали пити чисту воду, III, IV та V групам впродовж місяця до питної води додавали розчин цитрату ванадію у концентраціях 0,125, 0,5 і 2,0 мкг V/мл води. У тварин усіх дослідних (II–V) груп експериментально викликали цукровий діабет внутрішньоочеревинним введенням алоксану (150 мг/кг маси тіла). У м’язах тварин ІІ групи з експериментальним діабетом спостерігалось зростання вмісту гідропероксидів і ТБК-активних продуктів та зниження активності ензимів антиоксидантного захисту (супероксиддисмутази, каталази і глутатіонредуктази), глюкозо-6-фосфатдегідрогенази та вмісту відновленого глутатіону, тоді як активність глутатіонпероксидази підвищувалася порівняно з тваринами контрольної групи. За випоювання щурам цитрату ванадію у різних концентраціях спостерігалося зниження вмісту гідропероксидів (в ІІІ, IV та V групах) та ТБК-активних продуктів (у ІІІ групі), однак зростання активності супероксиддисмутази та каталази (в V групі), глутатіонредуктази (в ІІІ, IV та V групах), глюкозо-6фосфатдегідрогенази (в IV та V групах) та вмісту відновленого глутатіону (в V групі), тоді як активність глутатіонпероксидази знижувалася (в ІІІ, IV та V групах) порівняно з показниками у м’язах тварин ІІ групи з експериментальним діабетом. Цитрат ванадію у досліджуваних концентраціях здійснює дозо залежний стабілізуючий вплив на стан прооксидантно-антиоксидантної системи у скелетних м’язах щурів з експериментальним діабетом. Ключові слова: ЦИТРАТ ВАНАДІЮ, АНТИОКСИДАНТНА СИСТЕМА, ДІАБЕТ, СКЕ ЛЕТНІ М’ЯЗИ, ЩУРИ THE CONDITION OF THE PROOXIDANT-ANTIOXIDANT SYSTEM IN SKELETAL MUSCLES OF EXPERIMENTALLY DIABETIC RATS UNDER VANADIUM CITRATE EFFECT R. Iskra, H. Klymets, O. Sushko, L. Ponkalo, O. Svarchevska klimets.halyna@gmail.com Institute of Animal Biology NAAS, 38 V. Stus str., Lviv 79034, Ukraine The effect of vanadium citrate on the content of lipid peroxide oxidation products, reduced glutathione, the activity of the antioxidant system enzymes and glucose-6-phosphate dehydrogenase in the thigh skeletal muscles of alloxan-induced diabetic rats was studied. Animals were divided into five groups: I group — control, II, III, IV and V — experimental. The rats of I and II groups were given pure water to drink, whereas a solution of vanadium citrate at concentrations of 0.125, 0.5 and 2.0 μg V/ml of water was added to drinking water of III, IV and V groups within a month. The animals of all experimental (II–V) groups were induced diabetes by intraperitoneal injection of alloxan (150 mg/kg body weight). In muscles of the II group experimentally diabetic animals, the increase in the content of hydroperoxides and TBA-active products and the decrease in the activity of antioxidant protection enzymes (superoxide dismutase, catalase and glutathione reductase), glucose-6-phosphate dehydrogenase and the content of reduced glutathione were observed, while the activity of glutathione peroxidase increased as compared to control animals. Under watering vanadium citrate at different concentrations, there was a decrease in the content of hydroperoxides (in III, IV and V groups) and TBA-active products (in group III), but the increase in the activity of superoxide 30 The Animal Biology, 2018, vol. 20, no. 3 dismutase and catalase (in group V), glutathione reductase (in III, IV and V groups), glucose-6-phosphate dehydrogenase (in IV and V groups), and the content of reduced glutathione (in group V), while glutathione peroxidase activity decreased (in III, IV and V groups), as compared to the muscle indexes in experimentally diabetic animals of the II group. Vanadium citrate at the concentrations under investigation has a dose-dependent stabilizing effect on the condition of the prooxidant-antioxidant system in skeletal muscles of experimentally diabetic rats. Keywords: VANADIUM CITRATE, ANTIOXIDANT SYSTEM, DIABETES, SKELETAL MUSCLES, RATS СОСТОЯНИЕ ПРООКСИДАНТНО-АНТИОКСИДАНТНОЙ СИСТЕМЫ В СКЕЛЕТНЫХ МЫШЦАХ КРЫС С ЭКСПЕРИМЕНТАЛЬНЫМ ДИАБЕТОМ И ПОД ДЕЙСТВИЕМ ЦИТРАТА ВАНАДИЯ Р. Я. Искра, Г. В. Климец, О. А. Сушко, Л. И. Понкало, О. З. Сварчевская klimets.halyna@gmail.com Институт биологии животных НААН, ул. В. Стуса, 38, г. Львов, 79034, Украина Исследовали влияние цитрата ванадия на содержание продуктов перекисного окисления липидов, восстановленного глутатиона, активность ферментов антиоксидантной системы и глюкозо-6фосфатдегидрогеназы в скелетных мышцах бедра крыс с аллоксан-индуцированным диабетом. Животные были разделены на пять групп: I группа — контрольная, II, III, IV и V — опытные. Крысам I и II групп давали пить чистую воду, III, IV и V — в течение месяца к питьевой воде добавляли раствор цитрата ванадия в концентрациях 0,125, 0,5 и 2,0 мкг V/мл воды. У животных всех опытных (II–V) групп экспериментально вызывали сахарный диабет внутрибрюшинным введением аллоксана (150 мг/кг массы тела). В мышцах животных II группы с экспериментальным диабетом наблюдалось повышение содержания гидропероксидов и ТБК-активных продуктов и снижение активности ферментов антиоксидантной защиты (супероксиддисмутазы, каталазы и глутатионредуктазы), глюкозо-6-фосфатдегидрогеназы и содержания восстановленного глутатиона, в то время как активность глутатионпероксидазы повышалась по сравнению с животными контрольной группы. Под влиянием цитрата ванадия в различных концентрациях наблюдалось снижение содержания гидропероксидов (в III, IV и V группах) и ТБК-активных продуктов (в III группе), повышение активности супероксиддисмутазы и каталазы (в V группе), глутатионредуктазы (в III, IV и V группах), глюкозо-6-фосфатдегидрогеназы (в IV и V группах) и содержания восстановленного глутатиона (в V группе), в то время как активность глутатионпероксидазы снижалась (в III, IV и V группах) по сравнению с показателями в мышцах животных II группы с экспериментальным диабетом. Цитрат ванадия в исследуемых концентрациях осуществляет дозозависимое стабилизирующее влияние на состояние прооксидантно-антиоксидантной системы в скелетных мышцах крыс с экспериментальным диабетом. Ключевые слова: ЦИТРАТ ВАНАДИЯ, АНТИОКСИДАНТНАЯ СИСТЕМА, ДИАБЕТ, СКЕЛЕТНЫЕ МЫЩЦЫ, КРЫСЫ Цукровий діабет (ЦД) — захворюван ня, яке характеризується метаболічними по рушеннями, гіперглікемією та недостатньою секрецією і дією ендогенного інсуліну. Низка експериментальних та клінічних досліджень вказують на важливу роль оксидативного стре су в патогенезі ЦД [2, 18, 15, 25]. Надмірне утворення вільних радикалів за діабету у ре зультаті окиснення глюкози, глікозилювання протеїнів і послідовної їх деградації, а також порушення механізмів оксидативного захисту призводить до руйнування внутрішньоклітин них органел, зростання пероксидного окис нення ліпідів та резистентності до інсуліну [6]. Наслідки оксидативного стресу спричиняють розвиток дисфункцій різних тканин [15]. Зокрема у скелетних м’язах за ЦД відбуваєть ся порушення β-окиснення жирних кислот та процесів мітохондріального дихання, зростає рівень прооксидантних сполук, тоді як рівень антиоксидантних ензимів знижується. Крім того, погіршується синтез м’язової креатин кінази та обох ланцюгів міозину (важкого та легкого). Є припущення, що оксидативний стрес запускає каскад, який призупиняє онов лення м’язової тканини [2, 25]. 31 Біологія тварин, 2018, т. 20, № 3 Ванадій та його сполуки мають інсуліно міметичні та інсулінопідсилюючі властивості у моделях цукрового діабету як 1-го, так і 2-го типу [16, 17]. Є чимало праць, які характеризу ють Ванадій як ефективний засіб глікемічно го контролю у хворих на діабет завдяки його глюкозознижуючій властивості [19]. Солі ванадію знижують гіперглікемію і підсилюють дію інсу ліну зростанням кількості глюкозних транспор терів через субстрати інсулінових рецепторів (IRS1/2) та фосфатидилінозитол-3-кіназу (PI3-kinase) [24]. Ванадій також активує серин- та треонінкінази, включаючись у внутрішньоклі тинні інсулінові сигнали в дистальних ділянках інсулінових рецепторів, запобігаючи дефосфо рилюванню протеїну гальмуванням тирозин фосфатаз [13]. Він може мати позитивний або негативний вплив на антиоксидантний захист загалом. Його механізм залежить від викорис таної дози, типу ліганду тощо. Споживання Ванадію пришвидшує кровообіг у м’язах, по ліпшує васкуляризацію цієї тканини, зменшує синтез та накопичення глікогену, збільшує масу тіла, ріст скелету і м’язову масу [12]. Цей мікро елемент призупиняє надмірне утворення віль них радикалів у результаті посилення природ ного антиоксидантного захисту організму [23]. Скелетні м’язи є основною тканиною, в якій відбувається поглинання глюкози з крові (близько 75 %) за індукованого збільшення се креції інсуліну [22]. Метаболізм у клітинах ске летних м’язів як інсулінозалежній тканині за діа бету істотно знижується, що може спричинити розвиток значних діабетичних ускладнень [2]. Тому метою досліджень було з’ясувати дію різних концентрацій цитрату ванадію, отриманого способом нанотехнології [11], на активність ензимів антиоксидантної системи, глюкозо-6-фосфатдегідрогенази, вміст віднов леного глутатіону та продуктів пероксидного окиснення ліпідів у скелетних м’язах щурів із алоксан-індукованим діабетом. світлових умов. Щури були розділені на п’ять груп: І група — контрольна, ІІ, ІІІ, ІV і V — до слідні. Тваринам І і ІІ груп давали пити чисту воду без добавок, а ІІІ, ІV та V групам впродовж місяця до питної води додавали розчин цитра ту ванадію в кількостях, відповідно, 0,125, 0,5 і 2,0 мкг V/мл води. Враховуючи кількість води, випитої щурами щодобово (15–20 мл), вони споживали 15,625, 62,5 і 250,0 мкг V/кг маси тіла. У тварин усіх чотирьох дослідних груп на тлі 24-годинного голодування індукували екс периментальний ЦД у результаті внутрішньо очеревинного введення 5 % розчину алоксан моногідрату («Синбіас») з розрахунку 150 мг/кг маси тіла [4]. Гіперглікемію контролювали ви мірюванням концентрації глюкози у крові, віді браної з хвоста тварин. На 40 добу досліджень тварин виводили з експерименту декапітацією за легкої анестезії. Маніпуляції з тваринами здійснено відповідно до Європейської конвен ції про захист хребетних тварин, що використо вуються для дослідних та інших наукових цілей (Страсбург, 1986). Матеріалом для дослідження були гомо генати м’язової тканини стегна щурів, у яких визначали активність антиоксидантних ензимів та глюкозо-6-фосфатдегідрогенази, вміст від новленого глутатіону і продуктів пероксидного окиснення ліпідів. 10 % гомогенат стегнового м’язу готували способом подрібнення та гомоге нізації (Homogenizer type 302) в буфері трис-НСІ (pH 7,4; 5мМ). Активність глутатіонпероксида зи (ГП, КФ 1.11.1.9) визначали за швидкістю окиснення відновленого глутатіону; глутатіон редуктази (ГР, КФ 1.6.4.2) — за швидкістю відновлення глутатіону за наявності НАДФН; глюкозо-6-фосфатдегідрогенази (Г-6-ДГ, КФ 1.1.1.49) — спектрофотометричним мето дом, який базується на використанні спряжених систем окиснення нікотинамідних коензимів; вміст відновленого глутатіону (ВГ) — за рівнем утворення тіонітрофенільного аніону в резуль таті взаємодії SH-груп глутатіону з 5,5-дитіобіс, 2-нітробензойною кислотою; вміст гідропер оксидів визначали шляхом екстракції ліпідів етанолом з наступною взаємодією досліджу ваних екстрактів з тіоціанатом амонію; вміст ТБК-активних продуктів визначали за реакцією між малоновим диальдегідом і тіобарбітуровою Матеріали і методи Дослідження проведені на білих лабо раторних щурах з масою тіла 100–120 г, яких утримували в умовах віварію Інституту біо логії тварин за відповідних температурних та 32 The Animal Biology, 2018, vol. 20, no. 3 кислотою [26]. Одержані цифрові дані обро бляли статистично за допомогою комп’ютерної програми Statistica. Для визначення вірогідних відмінностей між середніми величинами вико ристовували критерій Стьюдента. тлі зниження активності СОД та КАТ у тварин цієї ж групи спостерігається компенсаторне зростання активності ГП в 3,6 разу (P<0,001), порівняно з контрольною, що зазвичай спри чинене посиленням оксидативного стресу [20], оскільки відомо, що ГП каталізує відновлення Н2О2 та органічних гідропероксидів, таким чином захищаючи клітини від дії реактивних форм Оксигену. Також слід відзначити знижен ня вмісту ВГ на 66,67 % (P<0,005) та активності ГР — на 47,3 % (P<0,01) у м’язах тварин ІІ до слідної групи стосовно аналогічних показників у тварин контрольної групи. Зниження актив ності ензимів антиоксидантного захисту часто спостерігається в скелетних м’язах за діабету, що може бути зумовлено пригніченням функ ції мітохондрій або зниженням їх вмісту у цій тканині [7]. За умов споживання щурами розчину цитрату ванадію у м’язовій тканині тварин змі нюються показники антиоксидантної системи. Зокрема відзначено зниження активності ГП у м’язах тварин ІІІ, ІV, V груп (P<0,001), по рівняно з ІІ групою з наближенням їхнього рів ня в V групі до показників контрольної групи. Водночас у м’язах тварин ІІІ і ІV дослідних груп за дії цитрату ванадію в кількості 0,125 і 0,5 мкг V/мл виявлено зростання рівня ВГ, відповідно, на 13,3 і 15,6 % та активності ГР — на 50,6 та 84,4 % (P<0,001). Встановлено також зниження активності КАТ у ІІІ групі на 24,1 % (P<0,05) та СОД у тварин ІV групи — на 42,7 % (P<0,001) порівняно з їх величинами у тварин ІІ групи. Водночас у м’язах тварин V дослідної групи спостерігалося підвищення вмісту ВГ на 35,6 % (P<0,001), активності ГР — на 79,5 % (P<0,01), СОД — на 47 % і КАТ — на 40,2 % (P<0,005), що вказує на нормалізацію ензимів антиокси дантного захисту за впливу цитрату ванадію в кількості 2 мкг V/мл. Також слід відзначити, що активність ензимів антиоксидантного захис ту певною мірою залежить від кількості цитрату ванадію, який, очевидно, стимулює збільшення синтезу їх ензимних молекул. Пригнічення глутатіонредуктазної ре акції за ЦД може бути зумовлене низьким рів нем НАДФН, які утворюються в глюкозо-6фосфатдегідрогеназній реакції пентозофосфат ного шляху. М’язові клітини за ЦД зі зниженим Результати й обговорення Пероксидне окиснення ліпідів (ПОЛ) відіграє важливу роль у патогенезі ЦД, зокрема руйнування мембранних структур вільними радикалами створює токсичний кінцевий про дукт — TБК-активні продукти, які використову ють як маркер прооксидантно-антиоксидантного балансу у пацієнтів з діабетом [1]. Аналізуючи результати досліджень (табл.), було встановлено, що розвиток гіпер глікемії (15,14 ммоль/л) після експеримен тально викликаного діабету супроводжувався підвищенням вмісту як проміжних, так і кін цевих продуктів пероксидного окиснення лі підів майже у всіх дослідних групах порівняно з контрольною. Це може бути зумовлене під вищенням концентрації вільних радикалів, які утворюються в результаті ішемії або реперфузії скелетного м’язу за ЦД [18], що, у свою чергу, призводить до пошкодження цієї тканини та зумовлює зниження антиоксидантного захисту в скелетних м’язах й організмі загалом [25]. За впливу цитрату ванадію відбувалося вірогідне зниження концентрації гідроперокси дів ліпідів у м’язах тварин III, IV та V груп, від повідно на 27,7, 30,2 і 27,1 % і ТБК-активних продуктів у тварин ІІІ групи — на 25,9 % порів няно з ІІ дослідною групою. Очевидно, Ванадій як інсулін-міметик та антиоксидант має здат ність виступати акцептором вільних радикалів і, відповідно, зменшувати оксидативний стрес у м’язовій тканині. Отримані в експерименті дані свідчать, що у м’язовій тканині тварин усіх дослідних груп за умов експериментально індукованого діабету відбувалося зниження активності основ них ензимів антиоксиданного захисту порівняно з тваринами контрольної групи. Так, у скелет них м’язах тварин ІІ дослідної групи виявлено зниження активності КАТ на 33,3 % та СОД на 4,03 %, що може бути зумовлене їх неензима тичним глікозилюванням [21]. Крім цього, на 33 34 1,747±0,111 29,136±1,426 9,780±0,392 72,638±1,436 0,135±0,032 1,237±0,088 412,756±6,1 ТБК-активні продукти, нмоль/мл TBA-active substances, nmol/ml СОД, U/мг протеїну SOD, U/mg of protein КАТ, мкмоль/хв×мг протеїну CAT, μmol/min×mg of protein ГП, мкмоль/хв×мг протеїну GPx, μmol/min×mg of protein ВГ, ммоль/л GSH, mmol/l ГР, мкмоль/хв×мг протеїну GR, μmol/min×mg of protein Г-6-ФДГ, мкмоль/хв×мг протеїну G-6-PDH, μmol/min×mg of protein 305,969±6,1* 0,797±0,069 0,045±0,002* 262,789±11,324* 6,426±0,511 27,963±0,32 2,323±0,113 0,358±0,025* ІІ 243,6635±5,68 ### 1,208±0,04### 0,051±0,002 138,595±5,675*### 4,879±0,108# 26,728±3,145 1,721±0,098## 0,259±0,028# ІІІ 387,575±7,57### 1,470±0,146### 0,052±0,005 171,593±7,372*### 6,879±0,33* 16,006±1,386*### 2,161±0,162 0,25±0,02## ІV Дослідні / Experimental 394,3914±4,91 ### 1,431±0,16## 0,061±0,002### 64,209±7,621### 9,011±0,794# 41,111±6,812 2,122±0,124 0,261±0,019**## V Таблиця ### Примітки: різниці статистично вірогідні: — P<0,05; — P<0,01; * — P<0,001 у тварин дослідних груп порівняно з контролем; # — P<0,05; ## — P<0,01; — P<0,001 різниці статистично вірогідні порівняно з ІІ дослідною групою з ЦД. Note: the difference is statistically significant: * — P<0,05; — P<0.01; * — P<0.001 in animals of experimental groups compared to the control; # — P<0,05; ## — P<0.01; ### — P<0,001 differences are statistically significant compared to the 2nd experimental group with DM. 0,153±0,01 І Контрольна / Control ГПЛ, о.о.г./мл LPO, OE/ml Показники Parameters Група тварин Animal group Вміст продуктів пероксидного окиснення ліпідів, відновленого глутатіону, активність ензимів антиоксидантної системи та глюкозо-6-фосфатдегідрогенази в скелетних м’язах щурів з експериментальним діабетом (ІІ) та за дії цитрату ванадію в концентраціях 0,125 мкг V/мл (ІІІ), 0,5 мкг V/мл (ІV) і 2 мкг V/мл (V) (М±m, n=8) The content of lipid peroxide oxidation products, reduced glutathione, the activity of antioxidant system enzymes and glucose-6-phosphate dehydrogenase in skeletal muscles of experimentally diabetic rats (II) under vanadium citrate effect at concentrations of 0.125 μg/ml (III), 0.5 μg V/ml (IV) and 2 μg V/ml of water (V) (M±m, n=8) Біологія тварин, 2018, т. 20, № 3 The Animal Biology, 2018, vol. 20, no. 3 рівнем глюкозо-6-фосфатдегідрогеназної активності особливо чутливі до оксидативно го стресу [10], оскільки відомо, що НАДФН виконує функцію важливого антиоксиданту, який здійснює детоксикацію підвищеного рівня активних форм Оксигену (АФО) [9]. Утворення АФО у процесі окисного метабо лізму за ЦД завдають шкоди всім клітинам організму, зокрема м’язовим, і можуть при звести до їхньої загибелі. Варто відзначити, що активність Г-6ФДГ у м’язах тварин ІІ, ІІІ, IV та V груп стосов но І групи знижувалась, відповідно, на 25,87 % (P<0,001), 40,97 % (P<0,001), 6,1 % (P<0,01) і 4,45 % (P<0,05). Інгібування активності Г-6ФДГ в експериментальних тварин з моделю ванням ЦД, можливо, спричинене активацією протеїнкінази А, яка, фосфорилюючи Г-6-ФДГ, знижує її активність [27]. Зниження активності цього ензиму також може відбуватися внаслі док його глікозилювання [5]. Існує припущен ня, що Г-6-ФДГ може бути мішенню, яка збіль шує інсуліннезалежне поглинання глюкози в скелетних м’язах за умов несприйнятливості до глюкози та інсулінорезистентності [14]. За умов випоювання розчину цитрату ванадію активність Г-6-ФДГ у скелетних м’язах щурів ІІІ групи знижувалась на 20,67 % (P<0,001), а в IV та V груп — підвищувалася, відповідно, на 27 і 29 % (P<0,001) стосовно ІІ групи. Очевидно, що за впливу цитрату ванадію в кількості 0,5 і 2 мкг V/мл підвищується транскрипція мРНК глюкозного транспортера GLUT4, що спричи няє інтенсифікацію надходження глюкози в клі тину і перетворення її в пентозофосфатному шляху, про що свідчить підвищення активності Г-6-ФДГ у цій тканині [3]. Цитрат ванадію у концентраціях 0,125, 0,5 і 2 мкг V/мл води має дозозалежний стабілізуючий вплив на стан прооксидантноантиоксидантної системи у скелетних м’язах щурів з експериментальним діабетом. Перспективи подальших досліджень. Оскільки цитрат ванадію, порівняно з неорганіч ними солями, є безпечнішим і екологічнішим, слід встановити оптимальні кількості його ви користання з метою запобігання виникненню цукрового діабету. 1. Altomare E., Vendemiale G., Chicco D., Procacci V., Cirelli F. Increased lipid peroxidation in type 2 poorly controlled diabetic patients. Diabetes & Metabolism, 1992, vol. 18, no. 4, pp. 264–271. 2. Aragno M., Mastrocola R., Catalano M. G., Brignardello E., Danni O., Boccuzzi G. Oxidative stress impairs skeletal muscle repair in diabetic rats. Diabetes, 2004, vol. 53, no. 4, pp. 1082–1088. DOI: 10.2337/diabetes.53.4.1082. 3. Askar M., Vijay S., McNeill J. H. Vanadium in creases GLUT4 in diabetic rat skeletal muscle. Molecular and Cellular Biochemistry, 2002, vol. 233, issue 1–2, pp. 139–143. DOI: 10.1023/A:1015558328757. 4. Etuk E. U. Animals models for studying diabetes mellitus. Agric. Biol. J. N. Am., 2010, vol. 1, no. 2, pp. 130–134. 5. Ganea E., Harding J. J. Molecular chaperones protect against glycation-induced inactivation of glu cose-6-phosphate dehydrogenase. Eur. J. Biochem., 1995, vol. 231, issue 1, pp. 181–185. DOI: 10.1111/ j.1432-1033.1995.tb20684.x. 6. Giacco F., Brownlee M., Schmidt A. M. Oxidative stress and diabetic complications. Circ. Res., 2010, vol. 107, no. 9, pp. 1058–1070. DOI: 10.1161/ CIRCRESAHA.110.223545. 7. He J., Watkins S., Kelley D. E. Skeletal mus cle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity. Diabetes, 2001, vol. 50, no. 4, pp. 817–821. DOI: 10.2337/diabetes.50.4.817. 8. Jacob S., Machann J., Rett K., Brechtel K., Volk A., Renn W., Maerker E., Matthaei S., Schick F., Claussen C. D., Häring H. U. Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetc offspring of type 2 diabetic sub jects. Diabetes, 1999, vol. 48, no. 5, pp. 1113–1119. DOI: 10.2337/diabetes.48.5.1113. 9. Jiang P., Du W., Wu M. Regulation of the pentose phosphate pathway in cancer. Protein Cell, 2014, vol. 5, issue 8, pp. 592–602. DOI: 10.1007/s13238-014-0082-8. 10. Khavrona O. P. Disturbances of function of the glutathione antioxidant system in gastric mucosa, liver and erythrocytes of rats with experimental gas tric lesions. Experimental and clinical physiology and biochemistry, 2015, no.1, pp. 26–31. (in Ukrainian) Висновки За алоксан-індукованого діабету змі нювався стан прооксидантно-антиоксидантної системи в скелетних м’язах щурів, зокрема посилювалося пероксидне окиснення ліпідів, що підтверджувалося зростанням рівня ГПЛ і ТБК-активних продуктів, знижувалася актив ність ензимів антиоксидантної системи, зокрема СОД, КАТ, ГР, та активність ензиму пентозо фосфатного шляху — Г-6-ФДГ. 35 Біологія тварин, 2018, т. 20, № 3 11. Kosinov M. V., Kaplunenko V. G. Ukrainian patent for utility model number 38391. IPC (2006): C07C 51/41, C07F 5/00, C07F 15/00, C07C 53/126 (2008.01), C07C 53/10 (2008.01), A23L 1/00, B82B 3/00. Method metal carboxylates “Nanotechnology receiving metal carboxylates”. Publish. 12.01.2009, Bull. no. 1. (in Ukrainian) 12. Kreider R. B. 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Ramachandran B., Kandaswamy M., Narayanan V., Subramanian S. Insulin mimetic effects of macrocyclic binuclear oxovanadium complexes on streptozotocin-induced experimental diabetes in rats. Diabetes Obes. Metab., 2003, vol. 5, issue 6, pp. 455– 461. DOI: 10.1046/j.1463-1326.2003.00302.x. 17. Rehder D. Biological and medicinal aspects of vanadium. Inorganic Chemistry Communications, 2003, vol. 6, issue 5, pp. 604–617. DOI: 10.1016/ S1387-7003(03)00050-9. 18. Shilpashree Y. D., Tejaswi H. L. Lipid peroxida tion and superoxide dismutase activities in patients with type 2 diabetes complicated with peripheral neuropathy. Int. J. Res. Med. Sci., 2016, vol.4, no. 2, pp. 457–460. DOI: 10.18203/2320-6012.ijrms20160295. 19. Smith D. M., Pickering R. M., Lewith G. T. A systematic review of vanadium oral supplements for glycaemic control in type II diabetes mellitus. QJM, 2008, vol. 101, issue 5, pp. 351–358. DOI: 10.1093/ qjmed/hcn003. 20. Srivastava P., Saxena A. K., Kale R. K., Baquer N. Z. Insulin like effects of lithium and vanadate on the altered antioxidant status of diabetic rats. Res. Comon. Chem. Pathol. Pharmacol., 1993, vol. 80, no. 3, pp. 283–293. 21. Stefanov A. V., Derimedved L. V., Churilova I. V. Clinicoexperimental justification of using of superoxide dismutase in medicine. NFaU “Zolotye. Stranitsy”, 2004, 288 p. (in Russian) 22. Stump C. S., Henriksen E. J., Wei Y., Sowers J. R. The metabolic syndrome: role of skeletal muscle metab olism. Ann. Med., 2006, vol. 38, issue 6, pp. 389–402. DOI: 10.1080/07853890600888413. 23. Subramanian S. P., Pillai S. I., Kandaswamy M. Evaluation of antioxidant efficacy of vanadium-3-hy droxyflavone complex in streptozotocin-diabetic rats. Chemico-Biological Interactions, 2013, vol. 204, issue 2, pp. 67–74. DOI: 10.1016/j.cbi.2013.04.012. 24. Tsiani E., Bogdanovic E., Sorisky A., Nagy L., Fantus I. G. Tyrosine phosphatase inhibitors, vanadate and pervanadate, stimulate glucose transport and Glut translocation in muscle cells by a mechanism inde pendent of phsophatidylinositol-3-kinase and protein kinase C. Diabetes, 1998, vol. 47, no. 11, pp. 1676–1686. DOI: 10.2337/diabetes.47.11.1676. 25. Tsutsui H., Kinugawa S., Matsushima S., Yokota T. Oxidative stress in cardiac and skeletal muscle dysfunction associated with diabetes melli tus. J. Clin. Biochem. Nutr., 2011, vol. 48, issue 1, pp. 68–71. DOI: 10.3164/jcbn.11-012FR. 26. Vlizlo V. V. (ed.), Fedoruk R. S., Ratych I. B. Laboratory methods of research in biology, veterinary medicine. A guide. Lviv, Spolom, 2012, 764 p. (in Ukrainian) 27. Xu Y., Osborne B. W., Stanton R. C. Diabetes causes inhibition of glucose-6-phosphate dehydroge nase via activation of PKA, which contributes to oxi dative stress in rat kidney cortex. AJP Renal. Physiol., 2005, vol. 289, issue 5, pp. F1040–F1047. DOI: 10.1152/ajprenal.00076.2005.
https://openalex.org/W2806296929	https://europepmc.org/articles/pmc5991654?pdf=render	English	null	Ensuring no one is left behind: Urgent action required to address implementation challenges for NTD control and elimination	PLoS neglected tropical diseases	2,018	cc-by	2,202	VIEWPOINTS a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Krentel A, Gyapong M, Ogundahunsi O, Amuyunzu-Nyamongo M, McFarland DA (2018) Ensuring no one is left behind: Urgent action required to address implementation challenges for NTD control and elimination. PLoS Negl Trop Dis 12(6): e0006426. https://doi.org/10.1371/journal. pntd.0006426 Editor: Zulfiqar A. Bhutta, The Hospital for Sick Children, CANADA We argue that it is time to bring these issues to the table and give them the attention they merit. We propose five principle areas for consideration by the NTD community. First, let’s rephrase these “social science” issues as implementation challenges that can be addressed by social science as well as epidemiological and other implementation research methods. Many disciplines such as anthropology, psychology, sociology, behavioral science, health economics, health services research, and other public health disciplines have the methods and tools to respond to these questions, which until now have been lumped together as “social science” questions. We need to acknowledge the strengths that each of these academic traditions can bring to our understanding of NTD elimination and control. Alison Krentel1, Margaret Gyapong2, Olumide Ogundahunsi3, Mary Amuyunzu- Nyamongo4, Deborah A. McFarland5 Alison Krentel1, Margaret Gyapong2, Olumide Ogundahunsi3, Mary Amuyunzu- Nyamongo4, Deborah A. McFarland5 1 Bruyère Research Institute, Ottawa, Canada, 2 Centre for Health Policy and Implementation Research, Institute for Health Research, University of Health and Allied Sciences, Ho, Ghana, 3 Research Capacity Strengthening, Special Programme for Research and Training in Tropical Diseases, WHO Geneva, Switzerland, 4 African Institute for Health and Development, Nairobi, Kenya, 5 Rollins School of Public Health, Emory University, Atlanta, Georgia, United States of America * akrentel@bruyere.org Since the ambitious goals to eliminate and control neglected tropical diseases (NTDs) were launched, the crucial role of partnerships has been emphasized as a pathway to ensure success. With multiple drug donations, we have a good supply of medicines necessary to eliminate parasites from the body; with donors, key funding for research and implementation; with researchers, the capability to create the evidence base for recommendations; with nongovern- mental development organizations (NGDOs), support for implementation; and with national programs, the willingness and impetus to accomplish these ambitious goals. A country’s health system is almost always invoked as crucial to NTD implementation, with the claim that NTD programs contribute to the strengthening of health systems. Key partners, often missing at the table, are the endemic communities themselves. Yet we acknowledge that both communities and local health systems are the “backbone” of our programs. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Some key implementation questions frequently arise in NTD programs: sustaining the motivation of community drug distributors, appropriateness of timing of mass drug admi- nistration (MDA) activities, the coverage–compliance gap [1], social mobilization, human resource constraints in low- and middle-income countries, inefficient or weak health systems, multiple reporting requirements and different funding cycles from donors, and many more (see Fig 1). While these issues continue to plague our NTD community, we have not commit- ted the necessary levels of research funds, expertise, or priority to adequately answer these questions. Often termed “social science questions,” these questions have been relegated to a category of research that is too difficult to conduct, too time-consuming, too costly, and seem- ingly less important than studies of drug efficacy or of the sensitivity of diagnostic tools. Fig 1. Examples of implementation barriers and bottlenecks inhibiting success in NTD programs. https://doi.org/10.1371/journal.pntd.0006426.g001 members of the research team. Implemention research provides a framework for implemen- ters and researchers to address implementation bottlenecks, identify optimal approaches for a particular setting, and promote the uptake of research findings. Ultimately, this process leads to improved healthcare and its delivery [2]. As we identify the implementation barriers and challenges that NTD programs face, we need to determine if they are global, regional, or coun- try-specific issues. There is the urgent need for us to find out if these challenges are related to the community drug distributors, the community, the health system, the NTD program itself, or the funders of the program and try to address them in a more holistic way. When we spend time conducting implementation research to understand why barriers occur, we get the infor- mation we need to begin constructive dialogue with health personnel to understand the perti- nent issues and to craft feasible solutions. Once interventions are put into place, we see evidence of improved programmatic outcomes [3,4,5]. Secondly, we should not shy away from the time and financial commitment that is needed to properly address these implementation questions. Getting the right answer today may ulti- mately save us time and money tomorrow. Within the context of the elimination program for lymphatic filariasis (LF), taking the time to understand why there are differences between reported coverage and actual compliance or why directly observed treatment is not imple- mented will provide the necessary information to adjust MDA, ensuring distributed drugs are consumed drugs. The time and expense of investigating these issues in a timely manner can translate into a savings of expensive additional MDA rounds if transmission assessment sur- veys fail. We do not develop new diagnostic tools overnight, and we should not expect to develop program diagnostic tools overnight, either. Both take time, resources, and periods of reiteration to optimize and determine the generalizability of the tool. Thirdly, we should be more specific about the impact NTD programs have on health sys- tems. There is a paucity of research into the interface between health systems and global health initiatives, like NTD control and elimination [6]. There is potential for enhancement in some elements of the health systems, such as drug procurement, health workforce, and community volunteers [6]. Published: June 7, 2018 Copyright: © 2018 Krentel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this work. In addition, we need to ensure that the program implementers rather than the researchers are in the driver’s seat in the identification of the programmatic challenges and are valued Competing interests: The authors have declared that no competing interests exist. 1 / 4 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006426 June 7, 2018 Addressing implementation challenges in NTD programs Addressing implementation challenges in NTD programs strategies to integrate it into other public health programs and structures [7,8]. We must also be cognizant that NTD programs can have negative effects on the health system [9]. For exam- ple, district health systems bear a considerable portion of the weight of an MDA campaign through coordination of training, social mobilization, drug distribution, supervision, mopping up, and reporting. These activities are implemented in addition to regular service delivery activities and sometimes with only limited supplementary budgets. Implementation research provides an appropriate framework from which to understand the effects of NTD programs on the health system, as the research is situated within the health system itself [2]. By conduct- ing research in this manner, the outcome is more likely to lead to feasible, targeted recommen- dations with high potential to improve delivery of NTD activities in a sustained manner as well as influence policy revision and change. Within the current context of universal healthcare (UHC) and the Sustainable Development Goals (SDGs) [10], the formal inclusion of NTDs into health system planning and programming can ensure more equitable access to medica- tions as well as continued management of the morbidities and disabilities resulting from pro- longed infection. Including some NTD indicators in district health management information systems (DHIMSs) can provide sustainable monitoring. Fourthly, we note that cost per treatment will likely increase as elimination programs begin to exhaust economies of scale and scope and move toward harder-to-reach populations. In the context of LF elimination, a recent WHO Weekly Epidemiological Report highlights some of these persistently challenging situations: cross-border areas, highly populated districts, and areas that have not yet begun MDA [11]. Activities and resource inputs will certainly change in the end game and will require that we are flexible, realistic, and adept at moving resources to the districts that may get left behind [12]. Lastly, as we dedicated ourselves to ensuring that no one is left behind in the Geneva com- mitment signed at the end of the NTD summit a year ago in April 2017 [13], let us mobilize our efforts to understand why some people, communities, and ethnic groups are left behind. As many national programs stop MDA in some districts, many others continue to have chal- lenging areas where deeper understanding and direction are needed if these programs are to be successful. Disclaimer The views expressed in this article are those of the authors and do not represent the positions of their affiliated institutions. We acknowledge the systematic noncompliers as the potential reservoirs of infection [14], but we have done little to understand who these people are, why they have been left out, and what we need to do to reach them. Ignoring these kinds of implementation chal- lenges risks maintaining them in perpetuity. We need the skill and creativity of many disci- plines to respond to these challenges. The 2020 goals are less than 30 months away. Now is the time to act. We are committed to those living in NTD-endemic communities and those working in local health systems, elevat- ing their role as equal partners in our goal to effectively control and eliminate these diseases. Such respectful and equitable partnerships are a necessary pathway to success in 2020. Let us commit ourselves to advocating for the resources and political will to answer these implemen- tation challenges correctly and sufficiently. 1. Babu BV, Babu GR. Coverage of, and compliance with, mass drug administration under the programme to eliminate lymphatic filariasis in India: a systematic review. Trans R Soc Trop Med Hyg. 2014; 108 (9):538–49. Epub 2014/04/15. https://doi.org/10.1093/trstmh/tru057 PMID: 24728444 We know that NTD programs have mobilized and trained a large community workforce. These community drug distributors (CDDs) provide the critical link between the communities and health systems to ensure NTD program goals are met. Implementation research should focus on sustaining the gains of this workforce and help to develop key 2 / 4 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006426 June 7, 2018 Addressing implementation challenges in NTD programs 2. World Health Organization on behalf of the Special Programme for Research and Training in Tropical Diseases (TDR). Implementation Research Toolkit, 2nd edition. 2017. Available from: http://adphealth. org/irtoolkit/. Accessed March 27, 2018. 3. Njomo DW, Mukoko DA, Nyamongo NK, Karanja J. Increasing Coverage in Mass Drug Administration for Lymphatic Filariasis Elimination in an Urban Setting: a Study of Malindi Town, Kenya. PLoS ONE. 2014 9(1): e83413. https://doi.org/10.1371/journal.pone.0083413 PMID: 24454703 4. Biritwum N-K, Garshong B, Alomatu B, de Souza DK, Gyapong M, Kyelem D. Improving drug delivery strategies for lymphatic filariasis elimination in urban areas in Ghana. PLoS Negl Trop Dis. 2017 11(5): e0005619. https://doi.org/10.1371/journal.pntd.0005619 PMID: 28493966 5. Krentel A, Damayanti R, Titaley CR, Suharno N, Bradley M, Lynam T. Improving Coverage and Compli- ance in Mass Drug Administration for the Elimination of LF in Two ‘Endgame’ Districts in Indonesia Using Micronarrative Surveys. PLoS Negl Trop Dis. 2016 10(11): e0005027. https://doi.org/10.1371/ journal.pntd.0005027 PMID: 27812107 6. Marchal B, Van Dormael M, Pirard M, Cavalli A, Kegels G, Polman K. Neglected Tropical Diseases (NTD) control in health systems: The interface between programmes and general health services. Acta Tropica 2011 120S: S177–S185. https://doi.org/10.1016/j.actatropica.2011.02.017 PMID: 21439930 7. Hooper PJ, Zoerhoff KL, Kyelem D, Chu B, Flueckiger RM, Bamani S et al. The Effects of Integration on Financing and Coverage of Neglected Tropical Disease Programs. Am J Trop Med Hyg. 2013 Sep 4; 89 (3): 407–410. https://doi.org/10.4269/ajtmh.13-0018 PMID: 23836563 8. Mwingira U, Means AR, Chikawe M, Kilembe B, Lyimo D, Corwley K et al. Integrating neglected tropical disease and immunization programs: the experiences of the Tanzanian Ministry of Health. Am J Trop Med Hyg. 2016; 95(3):505–507. https://doi.org/10.4269/ajtmh.15-0724 PMID: 27246449 9. Cavalli A, Bamba SI, Traore MN, Boelaert M, Coulibaly Y, et al. Interactions between Global Health Ini- tiatives and Country Health Systems: The Case of a Neglected Tropical Diseases Control Program in Mali. PLoS Negl Trop Dis. 2010; 4(8): e798. https://doi.org/10.1371/journal.pntd.0000798 PMID: 20808908 10. Bangert M, Molyneux DH, Lindsay SW, Fitzpatrick C, Engels. The Cross-cutting Contribution of the End of Neglected Tropical Diseases to the Sustainable Development Goals. Infectious Diseases of Poverty 2017; 6(73). https://doi.org/10.1186/s40249-017-0288-0 PMID: 28372566 11. World Health Organization. Weekly Epidemiological Record, 6 October 2017; 92(40): 589–608. PMID: 28984120 12. Turner HC, Toor J, Hollingsworth D, Anderson RM. Economic evaluations of mass drug administration: the importance of scale and scope. Clin Infect Dis. 2017;cix1001. https://doi.org/10.1093/cid/cix1001. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006426 June 7, 2018 References 3 / 4 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006426 June 7, 2018 Accessed 28 March 2018. 13. Uniting to Combat Neglected Tropical Diseases [Internet]. Reaching the Unreached: Fourth Progress Report of the London Declaration. 2017. Available from: http://unitingtocombatntds.org/reports/4th- report/. Accessed 28 March 2018. 14. El-Setouhy M, Abd Elaziz KM, Helmy H, Farid HA, Kamal HA, et al. The Effect of Compliance on the Impact of Mass Drug Administration for Elimination of Lymphatic Filariasis in Egypt. 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https://openalex.org/W2524804654	https://europepmc.org/articles/pmc5124036?pdf=render	English	null	An ultrasound-assisted procedure for fast screening of mobile fractions of Cd, Pb and Ni in soil. Insight into method optimization and validation	Environmental science and pollution research international	2,016	cc-by	10,202	Introduction Abstract A fast ultrasound-assisted sequential extraction (UASE) procedure for the determination of cadmium, lead and nickel fractions in soil was developed and fully validated. The working parameters of an ultrasound probe were opti- mized by comparing the content of metals in soil extracts obtained by the UASE procedure with that obtained by the conventional (with the aid of a vertical rotor) modified Community Bureau of Reference (BCR) procedure. The con- tent of metals in soil fractions was determined by electrother- mal atomic absorption spectrometry. The total time of extrac- tion of metals from soil was shorten from 48 h to 27 min (total sonication time). The trueness of the developed method was confirmed by analysis of the certified reference material BCR- 701. In order to indicate critical points of the developed UASE method, uncertainties of fractionation results were calculated and compared with those calculated for conventional modified BCR procedure. The method usefulness was tested for the determination of metal fractions in different types of soil col- lected in the Podlasie Province (Poland). The proposed proce- dure could be used for fast screening of mobile fractions of several heavy metals in soil. The contamination of sediments, soils and plants by heavy metals is of major concern due to their toxicity and bioaccumulative nature. Many anthropogenic activities (e.g. combustion of oil and coal, chemical industry, ferrous and non-ferrous metal production, waste incineration) have resulted in the redistribution of cadmium, lead and nickel from the earth’s crust to the soil and other environmental compartments. Lead and cadmium are considered as toxic metals for plants and humans (Andresen and Küpper 2013; Pourrut et al. 2011). They can be absorbed from soil solution mainly through the roots and thereby may enter the food chain. Nickel, in low concentrations, fulfils a variety of essential roles in living or- ganisms, e.g. as a constituent of several metal enzymes. However, excessive amount of nickel in soil and in nutrient solution is toxic to most plant species, affecting, e.g. nutrient absorption by roots, inhibiting photosynthesis and transpiration and causing ultrastructural modifications (Ahmad and Ashraf 2011). Total emission of these metals in 2013 in Poland was 15 t for Cd, 561 t for Pb and 148 t for Ni, while in the European Union was 63 t for Cd, 1836 t for Pb and 697 t for Ni (EEA Technical report no 8/ 2015). * Barbara Leśniewska blesniew@uwb.edu.pl Environ Sci Pollut Res (2016) 23:25093–25104 DOI 10.1007/s11356-016-7745-3 Environ Sci Pollut Res (2016) 23:25093–25104 DOI 10.1007/s11356-016-7745-3 RESEARCH ARTICLE Barbara Leśniewska1 & Marta Krymska1 & Ewelina Świerad1 & Józefa Wiater2 & Beata Godlewska-Żyłkiewicz1 Received: 22 April 2016 /Accepted: 18 September 2016 /Published online: 27 September 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com 1 Institute of Chemistry, University of Bialystok, K. Ciołkowskiego 1K, 15-245 Białystok, Poland 2 Faculty of Civil and Environmental Engineering, Bialystok University of Technology, Wiejska 45E, 15-351 Białystok, Poland Responsible editor: Zhihong Xu An ultrasound-assisted procedure for fast screening of mobile fractions of Cd, Pb and Ni in soil. Insight into method optimization and validation Barbara Leśniewska1 & Marta Krymska1 & Ewelina Świerad1 & Józefa Wiater2 & Beata Godlewska-Żyłkiewicz1 Introduction Only a few papers have demonstrated achievement of metal amounts extracted by ultrasounds equivalent to those obtained by conventional standardized protocol, such as sequential extraction scheme. So far, the best results using ultrasonic bath (Kazi et al. 2006) or probe (Pérez- Cid et al. 1998) were obtained for sewage sludge. Kazi et al. (2006) has observed that, except of copper, the recoveries of Cd, Cr, Ni, Pb and Zn in steps 1–3 were in the range 95– 117 %. The recoveries in the range 96–100 % were obtained for Cu, Cr, Ni, Pb and Zn in all fractions by Pérez-Cid et al. (1998), but it must be mentioned that the content of metals in some fractions (Cr and Pb in fractions I and II and Ni in fraction II) was not detectable by flame atomic absorption spectrometry (FAAS). The main sources of errors influencing analytical results were not identified so far. The aim of this work was to develop a universal ultrasound-assisted sequential extraction (UASE) procedure for fractionation of several trace metals in soil. Therefore, natural soil samples of different physicochemical properties have been used within optimization of working parameters of the ultrasound probe. During optimization of the procedure, the modified BCR conventional sequential extraction (CSE) protocol (with the aid of a vertical rotor) was used for com- parison. The developed UASE method of fractionation of Cd, Pb and Ni in soil with electrothermal atomic absorption spec- trometric (ETAAS) detection was fully validated according to the international guidelines ISO/IEC 17025 (2005) and uncer- tainty budget was estimated. The certified reference material of lake sediment BCR-701 was used for trueness control. The method was applied for the determination of heavy metal frac- tions in soil collected from an arable layer in the province of Podlasie (Poland). The most beneficial effect of ultrasounds is the particle fragmentation and the micro-cracks that facilitate and acceler- ate many physicochemical processes, such as dissolution, di- gestion and extraction or leaching (Bendicho et al. 2012; Kazi et al. 2009). One should consider that during a sonication process, some properties of a sample may be changed and different fractionation patterns may be obtained in comparison to conventional shaking. However, such accelerated extrac- tion process has been already used for fast monitoring the mobility, bioavailability and the eventual impact of anthropo- genic heavy metals in environmental solid samples (Vaisanen et al. 2002). Introduction The excessive metal exposures result in reduced yields of agricultural crops. Keywords Ultrasound probe . Modified BCR sequential extraction . Environmental analysis . Uncertainty budget Total metal content in polluted environmental samples is a poor indicator of its bioavailability, mobility and toxicity, as its environmental behaviour depends critically on its form. Metals can form various complexes with soil components, but only some of them are bioavailable. The behaviour of heavy metals in soil, and uptake by plants, is controlled by element speciation and by soil properties, such as pH, particle size, cation-exchange capacity, content of organic matter, con- tent and type of clay minerals and Al, Fe and Mn oxides, and redox potential (Fijałkowski et al. 2012; Łukowski et al. 2013). Responsible editor: Zhihong Xu * Barbara Leśniewska blesniew@uwb.edu.pl 1 Institute of Chemistry, University of Bialystok, K. Ciołkowskiego 1K, 15-245 Białystok, Poland 2 Faculty of Civil and Environmental Engineering, Bialystok University of Technology, Wiejska 45E, 15-351 Białystok, Poland Responsible editor: Zhihong Xu * Barbara Leśniewska blesniew@uwb.edu.pl 25094 Environ Sci Pollut Res (2016) 23:25093–25104 The sequential chemical extraction allows for operational fractionation of metals in solid samples, thus differentiates metal forms bound to different soil fractions. In the classic work of Tessier et al. (1979), the five-stage procedure was proposed to fractionate metals in river sediments. A harmo- nized, three-stage sediment sequential extraction protocol was established by the Community Bureau of Reference (BCR) of the Commission of the European Communities in 1993 (Quevauviller et al. 1994), while a modified BCR procedure was developed in 1999 (Rauret et al. 1999). Even this protocol is widely accepted and often used for fractionation of metals in various matrices, it still has some limitations (e.g. lack of specificity or species redistribution) as was discussed else- where (Bacon and Davidson 2008; Gleyses et al. 2002; Pérez et al. 2008). Moreover, this standardized protocol is very time consuming. In order to eliminate these shortcom- ings, sample treatment with microwaves (Castillo et al. 2011; Arain et al. 2008; Canepari et al. 2005; Garcia-Casillas et al. 2014; Reid et al. 2011; Ipolyi et al. 2002; Relić et al. 2013) or ultrasounds (Arain et al. 2008; Canepari et al. 2005; Garcia- Casillas et al. 2014; Krasnodębska-Ostręga et al. 2006; Rusnak et al. 2010; Relić et al. 2013) was proposed for single-step or, less frequently, for sequential extraction of metals from soil, sediment or sewage sludge samples. Rusnak et al. 2010). Introduction Different sources of ultrasounds, such as ultra- sonic baths (Davidson and Delevoye 2001; Kazi et al. 2006; Krasnodębska-Ostręga et al. 2006) and probes (Davidson and Delevoye 2001; Greenway and Song 2002; Pérez-Cid et al. 1998), have been used for metal fractionation. Generally, the probe system, carried out by direct insertion of an ultrasonic probe into a suspension of the powdered material, provides more efficient extraction of analytes in shorter time (Davidson and Delevoye 2001). However, samples are treated by the ultrasound probe one by one, while in the ultrasound bath or microwave oven, many samples can be treated simultaneously. Instrumentation and methods An ultrasound processor, VCX 130 model (Sonics and Materials, USA) (max power 130 W, max frequency 20 kHz) equipped with titanium probe, was used in a pulsed mode (on/off, 15 s/15 s). In order to keep the constant temper- ature during the sonication process (T = 25 ± 5 °C), the system was cooled down with flowing tap water as described previ- ously (Leśniewska et al. 2016). The pH of soil was measured in 1 mol L−1 KCl by a poten- tiometric method. In order to classify collected soil samples into proper agricultural type, the content of organic carbon was determined by the modified Tiurin’s method (Bednarek et al. 2004). The aqua regia procedure ISO 11466 (1995) was used for the determination of pseudo-total content of metals in soil. Electrothermal atomic absorption spectrometer (Solaar M6, Thermo Electron Corporation) equipped with a Zeeman-effect background correction and graphite tubes with integrated Lvov’s platform were used for the determination of metal content. Hollow cathode lamps were operated as fol- lows: for Cd (Thermo Scientific, UK) at 5 mA, for Pb (Photron, Australia) at 4 mA and for Ni (Narva, Germany) at 15 mA. The absorbance of metals in soil fractions was mea- sured at 228.8 nm for Cd and at 217.0 nm for Pb with a spectral bandpass of 0.5 nm, while at 232.0 nm for Ni with a spectral bandpass of 0.2 nm. A palladium modifier (10 μL of 0.5 mg mL−1) was used for the determination of Cd in fraction I and fraction III, and a phosphate modifier (10 μL of 0.1 mol L−1 NH4H2PO4) was used for the determination of Reagents and materials Acetic acid, hydroxylammonium chloride, ammonium acetate and hydrogen peroxide (30 %) (pure for analysis) were ob- tained from POCh (Poland). Nitric acid and hydrochloric acid (Suprapur) were obtained from Merck (Germany). Standard solutions were prepared by gravimetric dilution of stock solu- tions of cadmium, lead and nickel (1000 μg mL−1, Fluka, Germany). Magnesium nitrate(V), palladium and ammonium dihydrogen phosphate, used as matrix modifiers, were obtain- ed from Fluka (Germany). Ultrapure water was obtained from Milli-Q system (Millipore, USA). The literature review reveals that the effect of ultrasound- assisted extraction was different for different solid samples and each element, and the recoveries of metals were often non-quantitative when compared to classical method or certi- fied values of reference materials (Davidson and Delevoye 2001; Greenway and Song 2002; Pérez-Cid et al. 1999; Soil samples, various in terms of physicochemical proper- ties (agricultural type, pH, content of organic matrix), were collected from the arable layer in the province of Podlasie (Poland). All soil samples, air-dried, were homogenized and sieved using a 1-mm sieve. Samples of mineral soil, light Environ Sci Pollut Res (2016) 23:25093–25104 25095 Cd in fraction II. Magnesium nitrate (10 μL of 0.5 mg mL−1) was used as a chemical modifier for Pb determination in all fractions. Due to very corrosive properties of hydroxylamine chloride towards graphite tubes, the extracts of fraction II were evaporated and the residues were dissolved in 0.1 mol L−1 nitric acid. soil—L, medium soil—M and heavy soil—H, were used for optimization of the UASE procedure. Seven samples of light (L) and medium (M) mineral soil and 3 samples of organic (O) soil, used as arable land, were analysed using developed pro- cedure. The physicochemical characteristics of collected soils are presented in Table 1. The following optimized heating programs were used for the determination of metals in soil fractions FI/FII/FIII: Cd— drying at 110 °C for 25 s, ashing at 1000/1200/1000 °C for 5 s and atomization at 1500/1800/1500 °C for 3 s; Pb—drying at 110 °C for 25 s, ashing at 800/900/900 °C for 20 s and atom- ization at 1600/2300/1900 °C for 3 s; and Ni—drying at 110 °C for 25 s, ashing at 1100/1300/1300 °C for 10 s, and atomization at 2450/2700/2600 °C for 3 s. Certified reference material of lake sediment BCR-701 (IRMM, Belgium) was used for trueness control within vali- dation of the UASE procedure. Table 1 Characteristic of soil samples collected in the Podlasie Province (agricultural type of soil: L—light, M—medium, H— heavy, O—organic) Optimization of the UASE procedure The extraction efficiency of metals from solid samples depends on the type of ultrasound processor, its power or frequency and sonication time, as well as the type of sample. In order to devel- op the UASE procedure useful for simultaneous fractionation of Cd, Pb and Ni in soil, the working conditions of ultrasound probe were optimized individually for each step of procedure. The extraction was performed at least in triplicate. Moreover, different types of mineral soil (light—L, medium—M and heavy—H) were used during the optimization process. The re- sults obtained by accelerated procedure were always compared with the results obtained in the same soil by CSE modified BCR procedure using reagents recommended in the original protocol, and the recoveries of metals were calculated. During the selec- tion of optimal sonication conditions, the parameters chosen previously for fractionation of Cu (Leśniewska et al. 2014) in soil were also taken into consideration. On the basis of our previous results (Leśniewska et al. 2014), only the second step of releasing of metals from oxidazible fraction, namely the sonication time required for efficient re-extraction of mineralization products with CH3COONH4, was optimized in this work. The power of ultrasound probe (15 W) applied in this step was the same as used before. The influence of sonication time on the recovery of metals is presented in Fig. 1c. The best recovery of all metals in FIII was achieved using 6 min sonication. The influence of a power of ultrasound probe on the recov- ery of metals in fraction I and fraction II was studied in the range from 10 to 26 W (amplitude from 50 to 100 %) at sonication time of 5 min (Fig. 1). In fraction I, the recoveries of metals from all studied samples were in the range 63– 114 %, except of the low recovery of Pb from light soil (22– 98 %). The recoveries approaching 100 % for all metals (mean recovery equal to 93.5 ± 7.7 %) were obtained using the power of ultrasound probe of 15 W. The same power was used pre- viously for fractionation of Cu in soil (Leśniewska et al. 2014). Hence, in the next step, the sonication time was altered in the range 1–12 min using ultrasound power of 15 W. As can be seen in Fig. 1a, the sonication for 1 or 3 min was too short to extract all metals from soil samples. Optimization of the UASE procedure The effective recovery of Cd was obtained using 5 min sonication time (with mean value for all soil samples of 102.5 %), while for Pb and Ni, more efficient recoveries were obtained using 7 min sonica- tion (97 % for Pb and 94 % for Ni). Longer ultrasonic treat- ment of samples, 10 min for Pb (L, M and H soil) and 10 and 12 min for Ni (L and M soil), resulted in partial re-adsorption of Pb and Ni on soil particles, what was also demonstrated by Pérez et al. (2008). For the next study, the treatment of soil The important advantage of UASE procedure is short ex- traction time necessary for fractionation of Cd, Pb and Ni in soil, which is only 87 min (including 27 min of sonication), especially when compared to 51 h (48 h of extraction) of the original time of extraction according to CSE modified BCR procedure. The additional benefit of the developed method is that the same protocol is also suitable for fractionation of Cu. The comparison of results obtained for Cd, Pb and Ni by conventional and UASE procedures in various soil samples (L, M and H), expressed as recovery, is outlined in Table 2. Even some differences between light, medium and heavy soils have been observed; the recoveries of all metals were in the range of 81–113 %. In order to confirm the accuracy of results obtained by means of the above procedures, they were applied to the analysis of certified reference material BCR-701 (lake sediment). As can be seen in Table 3, the results obtained for all metals by conventional procedure are generally in good agreement with certified values (recoveries in the range 92– The important advantage of UASE procedure is short ex- traction time necessary for fractionation of Cd, Pb and Ni in soil, which is only 87 min (including 27 min of sonication), especially when compared to 51 h (48 h of extraction) of the original time of extraction according to CSE modified BCR procedure. The additional benefit of the developed method is that the same protocol is also suitable for fractionation of Cu. The comparison of results obtained for Cd, Pb and Ni by conventional and UASE procedures in various soil samples (L, M and H), expressed as recovery, is outlined in Table 2. Ultrasound-assisted sequential extraction procedure The organically bound metal fraction (FIII) was released by oxidation of the organic matter using 10 mL of 30 % H2O2 (pH 2) and sonication of the suspension for 4 min at 15 W, next heating for 1 h at 85 °C and re- extraction of mineralization products with 50 mL of 1 mol L−1 CH3COONH4 (pH 2) and sonication for 6 min at 15 W. The suspension was always centrifuged at 3000 rpm for 15 min. Before the next extraction step, the remaining solid residue was washed with 20 mL of ultrapure water. The highest recoveries of Cd, Pb and Ni in fraction II were obtained using the ultrasound probe working at 15 W (Fig. 1b). Under these conditions, the recoveries of Ni and Pb were in the range of 93–117 % (with mean recovery value of 102 ± 9 %). More complex situation was observed for extraction of Cd; as for sample L, the recovery was low, in the range 60–80 %, while for sample H, the recovery was very high, in the range 120–160 %. Such high results obtained for sample H were probably an effect of re-adsorption of Cd in the first step of extraction (Penilla et al. 2005; Pérez et al. 2008). The reason for low recovery of Cd in sample L is unknown, especially in view of the fact that the affinity of Cd to ≡FeOH is low (log β = −2.22). Optimization of sonication time has shown that the highest recoveries of all metals were obtained using 10 min sonication (Fig. 1b). Therefore, for extraction of metals bound to reducible fraction (FII), the sonication of samples for 10 min at 15 W was chosen. Such conditions provided quantitative recovery and good repeatability of the results for Pb, Ni and Cu and Zn. The recovery of Cd was lower and dependent on the type of soil sample. Ultrasound-assisted sequential extraction procedure The modified BCR procedure, conventional and ultrasound- assisted, was used for fractionation of metals in soil. In brief, for ultrasonic extraction of water-, acid-soluble, and ex- changeable metal fraction (FI), 40 mL of 0.11 mol L−1 CH3COOH was added to 1 g of soil sample and sonicated with the ultrasound probe for 7 min at 15 W. For extraction of reducible metal fraction (bound to iron and manganese hydroxides) (FII), 40 mL of 0.5 mol L−1 NH2OH·HCl (pH 1.5) solution was added to the soil residue and sonicated Sample Type of soil pHKCl CCorg, % Pseudo-total content of metal ± SD, mg kg−1 Cd Pb Ni L Brown 4.3 2.1 0.20 ± 0.01 7.3 ± 0.3 3.1 ± 0.1 M Brown 7.1 2.0 0.40 ± 0.01 6.9 ± 0.2 6.9 ± 0.2 H Brown 4.3 4.3 0.40 ± 0.01 13.9 ± 0.3 10.2 ± 0.5 L1 Brown 4.3 1.5 1.60 ± 0.04 11.9 ± 0.4 5.6 ± 0.2 L2 Brown 4.6 1.3 0.20 ± 0.01 9.8 ± 0.4 3.0 ± 0.1 M1 Brown 4.7 1.5 2.20 ± 0.06 8.5 ± 0.3 7.6 ± 0.3 M2 Brown 4.8 1.6 1.60 ± 0.05 7.3 ± 0.3 9.6 ± 0.2 M3 Podzols 6.0 2.8 0.70 ± 0.02 9.3 ± 0.2 7.0 ± 0.2 M4 Black 7.2 4.1 7.90 ± 0.31 8.8 ± 0.3 8.7 ± 0.2 M5 Black 7.3 3.0 0.40 ± 0.01 7.8 ± 0.3 9.8 ± 0.3 O1 Peat 5.2 24.8 2.10 ± 0.04 18.8 ± 0.9 8.1 ± 0.2 O2 Mud-and-peat 5.6 36.4 1.60 ± 0.07 28.6 ± 1.1 12.1 ± 0.3 O3 Muck-and-mineral 6.2 27.3 1.60 ± 0.05 22.5 ± 0.5 8.0 ± 0.3 Table 1 Characteristic of soil samples collected in the Podlasie Province (agricultural type of soil: L—light, M—medium, H— heavy, O—organic) Environ Sci Pollut Res (2016) 23:25093–25104 25096 with ultrasounds for 7 min at power of 15 W was chosen for simultaneous extraction of Cd, Pb and Ni into an acid-soluble fraction (FI). The recoveries of metals released from soil sam- ples under optimized working conditions are higher than those obtained by Canepari et al. (2005) and comparable to results obtained by Kazi et al. (2006) and Pérez-Cid et al. (1998). for 10 min at 15 W. Optimization of the UASE procedure Even some differences between light, medium and heavy soils have been observed; the recoveries of all metals were in the range of 81–113 %. In order to confirm the accuracy of results obtained by means of the above procedures, they were applied to the analysis of certified reference material BCR-701 (lake sediment). As can be seen in Table 3, the results obtained for all metals by conventional procedure are generally in good agreement with certified values (recoveries in the range 92– Environ Sci Pollut Res (2016) 23:25093–25104 25097 a) Fraction I b) Fraction II 0 20 40 60 80 100 120 140 160 Recovery, % Power of ultrasonic probe, W Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H 0 20 40 60 80 100 120 140 160 10 13 15 19 22 26 1 3 5 7 10 12 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H 0 20 40 60 80 100 120 140 160 Recovery, % Power of ultrasonic probe, W Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M 0 20 40 60 80 100 120 140 160 10 13 15 19 22 26 1 3 5 7 10 12 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H c) fraction III 0 20 40 60 80 100 120 140 160 3 6 8 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H Ni-H Fig. Optimization of the UASE procedure 1 Recovery of Cd, Pb and Ni in fractions extracted from soil in dependence of the power of ultrasonic probe (fixed sonication time 5 min) and the sonication time (fixed sonication power 15 W): a fraction I, b fraction II, c fraction III; (L—light soil, M—medium soil, H—heavy soil) a) Fraction I 0 20 40 60 80 100 120 140 160 Recovery, % Power of ultrasonic probe, W Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H 0 20 40 60 80 100 120 140 160 10 13 15 19 22 26 1 3 5 7 10 12 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H a) Fraction I 0 20 40 60 80 100 120 140 160 Recovery, % Power of ultrasonic probe, W Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H 10 13 15 19 22 26 0 20 40 60 80 100 120 140 160 1 3 5 7 10 12 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H Recovery, % b) Fraction II 0 20 40 60 80 100 Recovery, Power of ultrasonic probe, W 0 20 40 60 80 100 10 13 15 19 22 26 1 3 5 7 10 12 Recovery, Sonication time, min 0 20 40 60 80 100 120 140 160 Recovery, % Power of ultrasonic probe, W Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M 0 20 40 60 80 100 120 140 160 10 13 15 19 22 26 1 3 5 7 10 12 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H Ni-H b) b) Fraction II 0 20 40 60 80 100 120 140 160 Recovery, % Power of ultrasonic probe, W Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M 10 13 15 19 22 26 Ni-H 0 20 40 60 80 100 120 140 160 1 3 5 7 10 12 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H Recovery, % c) fraction III 0 20 40 60 80 100 120 140 160 3 6 8 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H Fig. Optimization of the UASE procedure 1 Recovery of Cd, Pb and Ni in fractions extracted from soil in dependence of the power of ultrasonic probe (fixed sonication time 5 min) and the sonication time (fixed sonication power 15 W): a fraction I, b fraction II, c fraction III; (L—light soil, M—medium soil, H—heavy soil) Table 2 Recovery of Cd, Pb and Ni in fraction of soil extracted according to the developed ultrasound-assisted method in comparison to the conventional modified BCR method Soil sample Fraction Recovery ± SD, % (n = 3) Cd Pb Ni L—light soil (pH 4.3) F I 89.1 ± 7.7 81.2 ± 12.9 91.3 ± 6.3 F II 91.3 ± 3.9 89.5 ± 3.1 91.3 ± 3.9 F III 99.8 ± 5.0 87.7 ± 9.3 86.6 ± 3.0 M—medium soil (pH 7.1) F I 103.8 ± 7.5 112.4 ± 9.9 92.6 ± 9.9 F II 95.2 ± 3.4 92.8 ± 3.2 97.4 ± 2.9 F III 100.9 ± 15.8 113.4 ± 15.2 101.5 ± 6.8 H—heavy soil (pH 4.3) F I 87.6 ± 8.8 98.3 ± 2.7 99.0 ± 5.5 F II 88.0 ± 4.2 104.6 ± 2.0 102.2 ± 7.7 F III 84.6 ± 9.0 96.6 ± 10.4 100.6 ± 2.7 Table 2 Recovery of Cd, Pb and Ni in fraction of soil extracted according to the developed ultrasound-assisted method in comparison to the conventional modified BCR method Environ Sci Pollut Res (2016) 23:25093–25104 25098 Environ Sci Pollut Res (2016) 23: Table 3 Comparison of the results for CRM BCR 701 by the conventional sequential extraction (CSE) method and ultrasound-assisted sequential extraction (UASE) method Content ± U, mg kg−1 (k = 2) Recovery, % Content ± U, mg kg−1 (k = 2) Recovery, % Certified value CSE method USAE method Fraction I Cd 7.34 ± 0.35 7.40 ± 1.70 100.8 6.81 ± 0.83 92.8 Pb 3.18 ± 0.21 3.61 ± 0.36 113.5 3.3 ± 0.36 103.8 Ni 15.4 ± 0.9 15.1 ± 1.6 98.0 12.6 ± 2.1 81.8 Cu 49.3 ± 1.7 53.0 ± 5.2 107.5 40.2 ± 5.0 81.6 Fraction II Cd 3.77 ± 0.28 4.10 ± 0.82 108.7 3.75 ± 0.42 99.5 Pb 126 ± 3 128 ± 13 101.6 129 ± 8 102.4 Ni 26.6 ± 1.3 27.6 ± 3.4 103.7 24.2 ± 2.8 91.0 Cu 124 ± 3 132.5 ± 10.2 106.8 117.2 ± 8.6 94.5 Fraction III Cd 0.27 ± 0.06 0.30 ± 0.08 111.1 0.29 ± 0.07 107.4 Pb 9.3 ± 2.0 10.2 ± 2.5 109.7 9.8 ± 2.3 105.4 Ni 15.3 ± 0.9 14.1 ± 2.0 92.2 14.1 ± 1.7 92.2 Cu 55.2 ± 4.0 61.5 ± 8.8 111.4 56.1 ± 7.8 101.6 Sum of fractions I+II+III Cd 11.38 11.81 103.7 10.85 95.3 Pb 138.48 142.21 102.7 142.1 102.6 Ni 57.3 57.1 99.7 50.9 88.8 Cu 228.5 247 108.1 213.5 93.4 113 %), while those obtained by accelerated procedure are slightly lower (especially for Ni in FI) (recoveries in the range 82–105 %). Optimization of the UASE procedure 1 Recovery of Cd, Pb and Ni in fractions extracted from soil in dependence of the power of ultrasonic probe (fixed sonication time 5 min) and the sonication time (fixed sonication power 15 W): a fraction I, b fraction II, c fraction III; (L—light soil, M—medium soil, H—heavy soil) c) fraction III 0 20 40 60 80 100 120 140 160 3 6 8 Recovery, % Sonication time, min Cd-L Cd-M Cd-H Pb-L Pb-M Pb-H Ni-L Ni-M Ni-H Fig. Optimization of the UASE procedure These discrepancies are probably an effect of different particle sizes and type of analysed material (sediment vs. soil) during procedure optimization. Nevertheless, concen- trations of all metal fractions in BCR-701 determined by UASE procedure are within the results reported in the litera- ture and compiled by Sutherland (2010). Good agreement of the results with certified values was also obtained for overall metal recoveries in fractions FI-FIII (93–103 %), except for Ni extraction (89 %), that was also reported by Pérez et al. (2008) and Ipolyi et al. (2002). These studies indicate a great potential of UASE procedure for fast monitoring of mobile metal frac- tions in soil. However, the re-adsorption phenomenon sug- gests that metal distribution has to be cautiously interpreted, principally that of Pb and Ni. Even though the above de- scribed procedure provided accurate results, it requires valida- tion before any further application. For the evaluation of the linearity of calibration graphs, the standards of Cd, Pb and Ni in the extraction solutions were prepared. The linearity of the calibration graph was considered acceptable when the correlation factor was higher than 0.995. The sensitivity of measurements of Cd and Pb (expressed as a slope of calibration graph) was dependent on the type of ex- traction solution, being the highest for fraction II (in diluted HNO3 after evaporation of NH2OH HCl). This phenomenon affected the linear range of calibration graphs of both metals. Such effect was not observed for measurements of Ni (Table 4). The selectivity of the method was evaluated by the comparison of calibration graphs obtained by external cal- ibration procedure (reagent-matched standard solutions) and standard addition method (extract of soil spiked with increas- ing amounts of analyte). As the slopes of calibration graphs obtained by these techniques were the same in the range of analytical error, the external calibration procedure was used for quantification of metal fractions in soil by the ETAAS technique. The LOD was calculated according to the follow- ing equation: LOD = blank + 3SDblank, where the extraction solution was used as the blank sample. The LOQ was calcu- lated as LOQ = blank + 6SDblank. In order to assess these parameters for soil samples, the volume of extraction solution and the mass of soil sample were used for calculations. Validation of the UASE procedure The precision of measurements of analyte absorbance in extraction solutions, defined as a degree of agreement be- tween a set of results, was assessed by six independent Environ Sci Pollut Res (2016) 23:25093–25104 25099 Table 4 Validation parameters for the ultrasound-assisted extraction method for determination of Cd, Pb and Ni fractions in soil by the ETAAS technique Table 4 Validation parameters for the ultrasound-assisted extraction method for determination of Cd, Pb and Ni fractions in soil by the ETAAS technique Validation parameter Soil fraction: extraction solution F I: 0.11 mol L−1 CH3COOH F II: 0.5 mol L−1 NH2OH HCla F III: 1 mol L−1 CH3COONH4 Cd Pb Ni Cd Pb Ni Cd Pb Ni Linear range of calibration graph, ng mL−1 0.3–10 0.85–25 3–120 0.1–4 1.3–20 4–100 0.1–10 2.3–20 5–120 Slope of calibration graph 0.0333 0.0135 0.0031 0.0911 0.0164 0.0033 0.0475 0.0147 0.0032 Regression coefficient, r 0.9997 0.9996 0.9996 0.9994 0.9996 0.9996 0.9975 0.9982 0.9992 Limit of detection for extraction solution, ng mL−1 0.06 0.52 2.0 0.06 0.62 3.1 0.02 1.53 3.5 Limit of quantification for extraction solution, ng mL−1 0.29 0.85 2.8 0.12 1.31 3.9 0.06 2.31 5.1 Precision of absorbance measurements for soil extract as RSD, % (n = 6) 2.2 2.0 2.6 2.8 3.3 2.9 2.2 3.7 2.5 Limit of detection for soil fraction, ng g−1 2.5 20 81 2.4 25 122 1.0 76 175 Limit of quantification for soil fraction, ng g−1 11.5 34 140 4.9 52 195 3.0 92 255 Repeatability of metal determination in BCR 701 fraction as RSD, %, (n = 6) 4.4 5.6 12.5 5.3 2.2 6.6 3.4 3.8 2.8 Trueness of the procedureb Found content ± U, k = 2, mg kg−1 6.81 ± 0.83 3.30 ± 0.36 12.6 ± 2.1 3.75 ± 0.42 128.6 ± 8.2 24.2 ± 2.8 0.290 ± 0.068 9.8 ± 2.3 14.1 ± 1.7 Bias, % −7.2 3.8 −18.2 −5.3 2.1 −9.0 7.4 −3.2 −7.8 a After evaporation and dilution in 0.1 mol L−1 HNO3 b As compared to certified value of BCR 701 Table 4 Validation parameters for the ultrasound-assisted extraction method for determination of Cd, Pb and Ni fractions in soil by the ETAAS technique e 4 Validation parameters for the ultrasound-assisted extraction method for determination of Cd, Pb and Ni fraction i fraction as RSD, %, (n = 6) ueness of the procedureb und content ± U k = 2 6.81 ± 0.83 3.30 ± 0.36 12.6 ± 2.1 3.75 ± 0.42 128.6 ± 8.2 24.2 ± 2.8 0.290 ± 0.068 9.8 ± 2.3 14.1 ± 1.7 fraction as RSD, %, (n = 6) ueness of the procedureb und content ± U, k = 2, mg kg−1 6.81 ± 0.83 3.30 ± 0.36 12.6 ± 2.1 3.75 ± 0.42 128.6 ± 8.2 24.2 ± 2.8 0.290 ± 0.068 9.8 ± 2.3 14.1 ± 1.7 as, % −7.2 3.8 −18.2 −5.3 2.1 −9.0 7.4 −3.2 −7.8 6.81 ± 0.83 3.30 ± 0.36 12.6 ± 2.1 3.75 ± 0.42 128.6 ± 8.2 24.2 ± 2.8 0.290 ± 0.068 9.8 ± 2.3 14.1 ± 1.7 below 7.5 %. Validation of the UASE procedure The validation of the developed UASE procedure for fraction- ation of Cd, Pb and Ni in soil was performed according to the international guidelines ISO/IEC 17025 (2005). Various ana- lytical parameters, such as linearity, limit of detection (LOD) and limit of quantification (LOQ), precision, selectivity and trueness, were estimated. Validation of the UASE procedure uc cMe ð Þ cMe ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ u ms ð Þ ms 2 þ u Ve ð Þ Ve 2 þ u cal ð Þ ð Þ2 þ u R ð Þ R 2 þ u fð Þ f 2 þ u repeat:extr: ð Þ2 s a) b) c) d) 22.8 10.1 10.8 9.8 20.1 10.2 12.2 7.7 28.3 24.4 14.1 14.3 0 5 10 15 20 25 30 Cd Pb Ni Cu Expannded uncertainty, % FI FII FIII 12.2 10.8 16.5 12.5 11.2 5.3 11.6 7.3 23.6 23.6 12.2 13.9 0 5 10 15 20 25 Cd Pb Ni Cu Expanded uncertainty, % FI FII FIII 0% 20% 40% 60% 80% 100% Cd-FI Cd-FII Cd-FIII Pb-FI Pb-FII Pb-FIII Ni-FI Ni-FII Ni-FIII Cu-I Cu-II Cu-III % in combined uncertainty u(cal)/cal u(repeat.) u(R)/R 0% 20% 40% 60% 80% 100% Cd-FI Cd-FII Cd-FIII Pb-FI Pb-FIIPb-FIII Ni-FI Ni-FII Ni-FIII Cu-I Cu-II Cu-III % in combined uncertainty u(cal)/cal u(repeat.) u(R)/R a) b) c) d) 22.8 10.1 10.8 9.8 20.1 10.2 12.2 7.7 28.3 24.4 14.1 14.3 0 5 10 15 20 25 30 Cd Pb Ni Cu Expannded uncertainty, % FI FII FIII 12.2 10.8 16.5 12.5 11.2 5.3 11.6 7.3 23.6 23.6 12.2 13.9 0 5 10 15 20 25 Cd Pb Ni Cu Expanded uncertainty, % FI FII FIII 0% 20% 40% 60% 80% 100% Cd-FI Cd-FII Cd-FIII Pb-FI Pb-FII Pb-FIII Ni-FI Ni-FII Ni-FIII Cu-I Cu-II Cu-III % in combined uncertainty u(cal)/cal u(repeat.) u(R)/R 0% 20% 40% 60% 80% 100% Cd-FI Cd-FII Cd-FIII Pb-FI Pb-FIIPb-FIII Ni-FI Ni-FII Ni-FIII Cu-I Cu-II Cu-III % in combined uncertainty u(cal)/cal u(repeat.) u(R)/R Fig. 2 Uncertainty budget: Expanded uncertainty (in %, k = 2) of content of metals in fraction I, II and III determined (a) after ultrasound-assisted extraction of soil and (c) after conventional modified BCR procedure. Validation of the UASE procedure All results are within the range of data for BCR- 701 compiled from 33 literature data sets (Sutherland 2010). The validation parameters of the developed UASE method are summarized in Table 3. measurements of the same sample. It was expressed as the relative standard deviation (RSD) and gave values below 2.9 %. The repeatability of extraction of metals from soil sam- ples and BCR-701 by the UASE method was evaluated on the basis of six independent extractions of the same sample under the same condition in a short period of time. It was expressed as RSD and gave values in the range 2–13 % for soil and 2– 7 % for BCR-701. Better repeatability obtained for certified reference material (CRM) results from better homogeneity of this material. The limits of detection and quantification of metals in soil fractions are even 40–50 times higher than those obtained for pure extraction solutions. Evaluation of expanded uncertainty of Cd, Pb and Ni content in soil fractions by the developed UASE procedure was performed in accordance with the Guide to the Expression of Uncertainty in Measurement (2008) using a modelling approach, similarly to the scheme presented in Leśniewska et al. (2016). For that purpose, possible sources of uncertainty of the measurement procedure were identified and individual standard uncertainties of these components were estimated. The combined standard uncertainty of results was calculated according to the law of an uncertainty propa- gation; next, the uncertainty budget was estimated. For calcu- lation of the content of metals in soil fractions, the following model equation was used: The trueness of the developed procedure of sequential ex- traction of Cd, Pb and Ni, defined as a closeness of the mean value of obtained results to the true value, was evaluated by analysis of BCR-701. The content of Cd, Pb and Ni in all fractions was compared with the certified values and the bias of the UASE method was evaluated. The bias of the method was calculated as a difference between the mean value of the obtained results and the reference value. Validation of the UASE procedure The highest bias of the method was obtained for fractionation of Ni (from −18.2 % in FI to −7.8 % in FIII), but for other metals, was cMe ¼ As−a b ⋅Ve⋅f ms⋅R cMe ¼ As−a b ⋅Ve⋅f ms⋅R ms⋅R 25100 Environ Sci Pollut Res (2016) 23:25093–25104 The parameters included in the model equation were identi- fied as components significantly contributing to the measure- ment result. On the basis of their individual standard uncer- tainties and the law of propagation of uncertainty, the combined standard uncertainty of analyte content in fractions of soil uc(cMe) was evaluated according to the following equation: where cMe denotes the metal content in fraction of soil (mg kg−1), As is the absorbance of analyte in given soil extract, a is the intercept of the calibration graph, b is the slope of the calibration graph, Ve is the volume of given soil extract (L), f is the dilution factor of extract, ms is the mass of soil (g) and R is the recovery of analyte in fraction of BCR-701. where cMe denotes the metal content in fraction of soil (mg kg−1), As is the absorbance of analyte in given soil extract, a is the intercept of the calibration graph, b is the slope of the calibration graph, Ve is the volume of given soil extract (L), f is the dilution factor of extract, ms is the mass of soil (g) and R is the recovery of analyte in fraction of BCR-701. Validation of the UASE procedure The percentage contribution of uncertainty of component in a combined uncertainty of content of metals in fraction of soil determined (b) after ultrasound-assisted extraction of soil and (d) after the conventional modified BCR procedure; (u(ms)/ms, u(Ve)/Ve, u(cal)/cal, u(R)/R, u( f )/f, and u(repeat.extr.) denote relative uncertainty of mass of soil, volume of extract, calibration, recovery, dilution factor and repeatability of extraction process; u(ms)/ms and u(Ve)/Ve were <0.1 %; u( f )/f < 0.5 %) a) 12.2 10.8 16.5 12.5 11.2 5.3 11.6 7.3 23.6 23.6 12.2 13.9 0 5 10 15 20 25 Cd Pb Ni Cu Expanded uncertainty, % FI FII FIII Fig. 2 Uncertainty budget: Expanded uncertainty (in %, k = 2) of content of metals in fraction I, II and III determined (a) after ultrasound-assisted extraction of soil and (c) after conventional modified BCR procedure. The percentage contribution of uncertainty of component in a combined uncertainty of content of metals in fraction of soil determined (b) after ultrasound-assisted extraction of soil and (d) after the conventional modified BCR procedure; (u(ms)/ms, u(Ve)/Ve, u(cal)/cal, u(R)/R, u( f )/f, and u(repeat.extr.) denote relative uncertainty of mass of soil, volume of extract, calibration, recovery, dilution factor and repeatability of extraction process; u(ms)/ms and u(Ve)/Ve were <0.1 %; u( f )/f < 0.5 %) b) b) 0% 20% 40% 60% 80% 100% Cd-FI Cd-FII Cd-FIII Pb-FI Pb-FIIPb-FIII Ni-FI Ni-FII Ni-FIII Cu-I Cu-II Cu-III % in combined uncertainty u(cal)/cal u(repeat.) u(R)/R c) 22.8 10.1 10.8 9.8 20.1 10.2 12.2 7.7 28.3 24.4 14.1 14.3 0 5 10 15 20 25 30 Cd Pb Ni Cu Expannded uncertainty, % FI FII FIII c) Cu d) 0% 20% 40% 60% 80% 100% Cd-FI Cd-FII Cd-FIII Pb-FI Pb-FII Pb-FIII Ni-FI Ni-FII Ni-FIII Cu-I Cu-II Cu-III % in combined uncertainty u(cal)/cal u(repeat.) u(R)/R d) Environ Sci Pollut Res (2016) 23:25093–25104 25101 Fig. 2 and Table 3. In order to study capability of the developed UASE procedure, the uncertainties of results of fractionation of Cu in soil obtained in our earlier study (Leśniewska et al. 2014) have been also calculated (Fig. 2 and Table 3). The highest expanded uncertainty of results was observed for content of metals in FIII (12–24 %), while for FI and FII, was in the range 5–16 % (Fig. 2a) that is similar to the pattern represented for BCR-701 and resulted from the complexity of the sequential extraction procedure. Validation of the UASE procedure However, for CSE procedure, the 0.00 0.05 0.10 0.15 0.20 0.25 0.30 L1 (1.6) L2 (0.24) M1 (2.2) M2 (1.6) M3 (0.7) M4 (7.9) M5 (0.4) O1 (2.1) O2 (1.6) O3 (1.6) Content of Cd, mg kg-1 Sample of soil Cd-FI Cd-FII Cd-FIII 1.2 2.2 2.2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 L1 (5.6) L2 (3.0) M1 (7.6) M2 (9.6) M3 (7.0) M4 (8.7) M5 (9.8) O1 (8.1) O2 (12.1) O3 (8.0) Content of Ni, mg kg-1 Sample of soil Ni-FI Ni-FII Ni-FIII 3.4 5.0 4.2 0.0 0.4 0.8 1.2 1.6 2.0 L1 (11.9) L2 (9.8) M1 (8.5) M2 (7.3) M3 (9.3) M4 (8.8) M5 (7.8) O1 (18.8) O2 (28.6) O3 (22.5) Content of Pb, mg kg-1 Sample of soil Pb-FI Pb-FII Pb-FIII 3.0 3.4 3.9 3.8 2.3 2.2 1.8 8.2 6.2 9.4 0.00 0.05 0.10 0.15 0.20 0.25 0.30 L1 (1.6) L2 (0.24) M1 (2.2) M2 (1.6) M3 (0.7) M4 (7.9) M5 (0.4) O1 (2.1) O2 (1.6) O3 (1.6) Content of Cd, mg kg-1 Sample of soil Cd-FI Cd-FII Cd-FIII 1.2 2.2 2.2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 L1 (5.6) L2 (3.0) M1 (7.6) M2 (9.6) M3 (7.0) M4 (8.7) M5 (9.8) O1 (8.1) O2 (12.1) O3 (8.0) Content of Ni, mg kg-1 Sample of soil Ni-FI Ni-FII Ni-FIII 3.4 5.0 4.2 0.0 0.4 0.8 1.2 1.6 2.0 L1 (11.9) L2 (9.8) M1 (8.5) M2 (7.3) M3 (9.3) M4 (8.8) M5 (7.8) O1 (18.8) O2 (28.6) O3 (22.5) Content of Pb, mg kg-1 Sample of soil Pb-FI Pb-FII Pb-FIII 3.0 3.4 3.9 3.8 2.3 2.2 1.8 8.2 6.2 9.4 Fig. 3 Content of Cd, Pb and Ni in fractions of soil collected from the Podlasie Province after ultrasound-assisted extraction with expanded uncertainty (k = 2) (soil category: L—light, M—me- dium, O—organic; in brackets, the pseudo-total content of metal in mg kg−1) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 L1 (1.6) L2 (0.24) M1 (2.2) M2 (1.6) M3 (0.7) M4 (7.9) M5 (0.4) O1 (2.1) O2 (1.6) O3 (1.6) Content of Cd, mg kg-1 Sample of soil Cd-FI Cd-FII Cd-FIII 1.2 2.2 2.2 Fig. Validation of the UASE procedure A distribution of expanded uncertainty of con- tent of metals in soil fractions obtained by the CSE meth- od was analogous. Again, the highest values of expanded uncertainty were obtained for metal content in fraction III (14.1–28.3 %). However, for CSE procedure, the where u(ms), u(Ve), u(cal), u(R), u( f ), and u(repeat.extr.) denote standard uncertainties of mass of soil, volume of extract, cal- ibration, recovery, dilution factor and repeatability of extrac- tion process, respectively. where u(ms), u(Ve), u(cal), u(R), u( f ), and u(repeat.extr.) denote standard uncertainties of mass of soil, volume of extract, cal- ibration, recovery, dilution factor and repeatability of extrac- tion process, respectively. To obtain an expanded uncertainty (U) of the result at the 95 % confidence level, the combined standard uncertainty of analyte content in fractions was multiplied by the coverage factor k of 2. The expanded uncertainties of content of metals in fractions FI, FII and FIII obtained for the same samples using the modified BCR method with conventional sequential extraction (CSE) were calculated accordingly. The values of relative uncertainty of each component in combined uncertainty and expanded uncertainty of con- tent of metals in fractions FI, FII and FIII are presented in FIII are presented in (14.1–28.3 %). However, for CSE procedure, the 0.00 0.05 0.10 0.15 0.20 0.25 0.30 L1 (1.6) L2 (0.24) M1 (2.2) M2 (1.6) M3 (0.7) M4 (7.9) M5 (0.4) O1 (2.1) O2 (1.6) O3 (1.6) Content of Cd, mg kg-1 Sample of soil Cd-FI Cd-FII Cd-FIII 1.2 2.2 2.2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 L1 (5.6) L2 (3.0) M1 (7.6) M2 (9.6) M3 (7.0) M4 (8.7) M5 (9.8) O1 (8.1) O2 (12.1) O3 (8.0) Content of Ni, mg kg-1 Sample of soil Ni-FI Ni-FII Ni-FIII 3.4 5.0 4.2 0.0 0.4 0.8 1.2 1.6 2.0 L1 (11.9) L2 (9.8) M1 (8.5) M2 (7.3) M3 (9.3) M4 (8.8) M5 (7.8) O1 (18.8) O2 (28.6) O3 (22.5) Content of Pb, mg kg-1 Sample of soil Pb-FI Pb-FII Pb-FIII 3.0 3.4 3.9 3.8 2.3 2.2 1.8 8.2 6.2 9.4 II are presented in (14.1–28.3 %). Validation of the UASE procedure 3 Content of Cd, Pb and Ni in fractions of soil collected from the Podlasie Province after ultrasound-assisted extraction with expanded uncertainty (k = 2) (soil category: L—light, M—me- dium, O—organic; in brackets, the pseudo-total content of metal in mg kg−1) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 L1 (1.6) L2 (0.24) M1 (2.2) M2 (1.6) M3 (0.7) M4 (7.9) M5 (0.4) O1 (2.1) O2 (1.6) O3 (1.6) Content of Cd, mg kg-1 Sample of soil Cd-FI Cd-FII Cd-FIII 1.2 2.2 2.2 0.0 0.4 0.8 1.2 1.6 2.0 L1 (11.9) L2 (9.8) M1 (8.5) M2 (7.3) M3 (9.3) M4 (8.8) M5 (7.8) O1 (18.8) O2 (28.6) O3 (22.5) Content of Pb, mg kg-1 Sample of soil Pb-FI Pb-FII Pb-FIII 3.0 3.4 3.9 3.8 2.3 2.2 1.8 8.2 6.2 9.4 Content of Pb, mg kg-1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 L1 (5.6) L2 (3.0) M1 (7.6) M2 (9.6) M3 (7.0) M4 (8.7) M5 (9.8) O1 (8.1) O2 (12.1) O3 (8.0) Content of Ni, mg kg-1 Sample of soil Ni-FI Ni-FII Ni-FIII 3.4 5.0 4.2 Environ Sci Pollut Res (2016) 23:25093–25104 25102 expanded uncertainty of Pb, Ni and Cu content in FI was lower, probably due to higher recoveries of these metals in FI. fractions of mineral soil is similar, while for organic soil, its highest content was determined in FIII. The distribution of metals among fractions in soils of the Podlasie Province is not discussed in detail as these issues are beyond the scope of this work and due to variety of properties of analysed soil (Table 1). However, it is worth to point out that the results of fractionation of four metals in soil can be achieved within 2 h (taking into account 87 min for extraction of metals from soil and 30 min for their determination by AAS), indicating us- ability of the procedure for fast screening of mobile metal fractions. Finally, the uncertainty budget was constructed in order to indicate critical control points of the developed UASE and CSE methods. The percentage contribution of uncertainty of each com- ponent in combined uncertainty was calculated (as, e.g. [u(ms)/ ms]2/[uc(cMe)/cMe]2). As can be seen in Fig. Validation of the UASE procedure 2b, d, the uncertainty of metal content in all fractions is strongly influenced by their recoveries (38–92 % for UASE and 33–83 % for CSE), calibra- tion (9–48 % for UASE and 3–49 % for CSE) and repeatability of extraction step (1–38 % for UASE and 5–43 % for CSE). Uncertainties of the mass of sample, the volume of extract and the dilution factor were neglected in combined uncertainty of metal content in all cases as their values were below 0.5 %. The largest shares in uncertainties of recovery of analytes derive from the uncertainties of certified values of CRM (Table 3). However, low recoveries of Cd, Ni and Cu in FI obtained by using the UASE method influenced uncertainties of their content. The sec- ond important source of uncertainty was the concentration of analytes in the extract of soil that was calculated from the calibra- tion graph. Among the three sources of uncertainty in the calibra- tion step, a slope of the calibration graph and repeatability of measurements of analyte absorbance had significant effect on the determined concentration of analytes. Preparation of standard solutions for calibration by dilution of stock standard solutions introduced the lowest uncertainty in that step. Repeatability of the extraction step significantly affected the combined uncertainty of metal content in all fractions of soil (Fig. 2b, d). Such effect was strongly observed when the CSE procedure that was applied as contribution of u(repeat.) in the combined uncertainty was in the range 16–43 % almost for metals in all fractions, whereas for developed UASE, its share was mostly below 15 %. Conclusions In order to facilitate the method for determination of Cd, Pb and Ni fractions in soil, a fast ultrasound-assisted sequential extraction (UASE) procedure based on modified BCR proto- col was proposed. Application of ultrasounds allowed to shorten the total time of procedure from 51 h to 87 min (27 min of sonication of soil and 1 h heating at 85 °C). During method validation, it was found that external cali- bration technique using sample-matched solutions is appropri- ate for quantification of elements in soil extracts. The trueness of the developed method assessed by analysis of BCR-701 was acceptable, varying for different metals and fractions. The extended uncertainty of content of metal fractions in soil was in the range of 5.3–23.6 %, being the highest for fraction III. The values of extended uncertainty obtained for the devel- oped UASE procedure were generally lower than for the CSE modified BCR procedure. The analysis of uncertainty budget indicated that the main share in uncertainty of results derived from the evaluation of the recovery of metals in CRM and the quantification of metals in soil extracts (including the calibra- tion step). 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The pseudo-total content of metals de- termined in all analysed samples did not exceed its permissi- ble limit for agricultural soil in Poland, which was set at 4 mg kg−1 for Cd, 100 mg kg−1 for Pb and 100 mg kg−1 for Ni (Ordinance of the Minister of Environment, Poland 2002). Therefore, the analysed soil samples were considered as un- polluted. The content of metals in organic soils was generally higher than in mineral soils. Distribution of metals among soil fraction was different for various elements, what is consistent with literature data. The highest content of Cd was determined in the reducible fraction (FII) and lower in fraction III and fraction I. In case of Pb, the highest content was observed in fraction III, while the lowest in fraction I. The Ni content in In our work, ultrasonic treatment caused lower amounts of Pb and Ni to be released in the first fraction of soil. These results are probably an effect of re-adsorption of metals on the surface of soil during the extraction step, which is enhanced when ultrasounds are applied. Apart of a better penetration of solvent into the solid sample to extract the metal, the ultrasounds cause the activation of adsorptive sites of soil particles. However, the results obtained for fractionation of elements in BCR-701 were in the range of results published by others (Sutherland 2010). The metal distribution in soil has to be cautiously interpreted, but the procedure is very suitable for fast screening of mobile metal fractions in soil, so it can be recommended for eco- toxicological studies and environmental risk assessment. Environ Sci Pollut Res (2016) 23:25093–25104 25103 Acknowledgments This work was supported by the Polish Ministry of Science and Higher Education—research Project N N310 375539. Acknowledgments This work was supported by the Polish Ministry of Science and Higher Education—research Project N N310 375539. Application of the UASE method ISO 11466:1995 Soil quality—extraction of trace elements soluble in aqua regia ISO 11466:1995 Soil quality—extraction of trace elements soluble in aqua regia ISO/IEC 17025:2005 General requirements for the competence of testing and calibration laboratories Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Kazi TG, Jamali MK, Siddiqui A, Kazi GH, Arain MB, Afridi HI (2006) An ultrasonic assisted extraction method to release heavy metals from untreated sewage sludge samples. 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Anal Chem 51: 844–851 Sutherland RA (2010) BCR-701: a review of 10-years of sequential ex- traction analyses. Anal Chim Acta 680:10–20 Environ Sci Pollut Res (2016) 23:25093–25104 Sutherland RA (2010) BCR-701: a review of 10-years of sequential ex- traction analyses. Anal Chim Acta 680:10–20 Tessier A, Campbell PGC, Bisson M (1979) Sequential extraction proce- dure for the speciation of particulate trace metals. Anal Chem 51: 844–851 References J Environ Monit 4:950–955 Ipolyi I, Brunori C, Cremisini C, Fodor P, Macaluso L, Morabito R (2002) Evaluation of performance of time-saving extraction devices in the BCR three-step sequential extraction procedure. J Environ Monit 4:541–548 Rusnak R, Halasz G, Horvath M, Remeteiova D (2010) Preliminary results on the intensification of the BCR sequential extraction with sonication for sediments, soils, and gravitation sediment samples. 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https://openalex.org/W2055048897	https://europepmc.org/articles/pmc4419512?pdf=render	English	null	Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus	Retrovirology	2,015	cc-by	11,157	* Correspondence: tinoush@umd.edu; wlodawer@nih.gov; okeefeba@mail.nih.gov 1Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA 2Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Full list of author information is available at the end of the article Moulaei et al. Retrovirology (2015) 12:6 DOI 10.1186/s12977-014-0127-3 Open Access Abstract Background: The lectin griffithsin (GRFT) is a potent antiviral agent capable of prevention and treatment of infections caused by a number of enveloped viruses and is currently under development as an anti-HIV microbicide. In addition to its broad antiviral activity, GRFT is stable at high temperature and at a broad pH range, displays little toxicity and immunogenicity, and is amenable to large-scale manufacturing. Native GRFT is a domain-swapped homodimer that binds to viral envelope glycoproteins and has displayed mid-picomolar activity in cell-based anti-HIV assays. Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM. Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity. Results: Here we present data on engineered tandem repeats of mGRFT (mGRFT tandemers) with antiviral activity at concentrations as low as one picomolar in whole-cell anti-HIV assays. mGRFT tandemers were analyzed thermodynamically, both individually and in complex with HIV-1 gp120. We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions. This establishes that, although the intra-virion crosslinking of HIV envelope glycoproteins is likely integral to their activity, the antiviral activity of these lectins is not due to virus aggregation caused by inter-virion crosslinking. Conclusions: The engineered tandemer constructs of mGRFT may provide novel and powerful agents for prevention of infection by HIV and other enveloped viruses. Keywords: Lectins, HIV, Griffithsin Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus Tinoush Moulaei1,2,3, Kabamba B Alexandre1, Shilpa R Shenoy1,4, Joel R Meyerson5, Lauren RH Krumpe1,4, Brian Constantine1, Jennifer Wilson1, Robert W Buckheit Jr.6, James B McMahon1, Sriram Subramaniam5, Alexander Wlodawer2 and Barry R O’Keefe1* * Correspondence: tinoush@umd.edu; wlodawer@nih.gov; okeefeba@mail.nih.gov 1Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA 2Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA Full list of author information is available at the end of the article © 2015 Moulaei et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background structural features through conformational occlusion and oligomerization [2,3], sequence hyper-variability, es- pecially within loops that mask conserved epitopes [4], and extensive posttranslational glycosylation [5]. The surface glycoproteins of enveloped viruses act as an- chors for docking and fusion with the target cell’s mem- brane [1]. These glycoproteins are the most prominent features of the viral surface that can be recognized within the host cellular background and are targeted for antibody neutralization. Consequently, viruses have evolved a number of strategies for shielding the spike structures formed by their surface glycoproteins. These strategies include restriction of access to conserved N-linked carbohydrates compose approximately 50% of the molecular weight of HIV gp120 [6]. This glycan armor hides the underlying protein structures on one face of gp120, whereas the epitopes on the other face of gp120 are masked by the quaternary structure of the tri- meric spike [7]. The effectiveness of glycans in this de- fensive mechanism may in part hinge on the generally weak interactions between proteins and carbohydrates [8]. The glycan modifications of HIV Env are also essen- tial for its folding and function [9,10]. Evolution of mu- tations in the position and frequency of N-linked glycans Page 2 of 14 Moulaei et al. Retrovirology (2015) 12:6 a number of mono- and disaccharides [26-28]. In its na- tive form GRFT is a rigid domain-swapped homodimer. We also engineered several monomeric forms of GRFT (mGRFT) (Figure 1B) and determined their structures, including the structure of a complex with nonamannoside, an analogue of Man9 that forms a common glycosylation pattern found on HIV surface glycoproteins [29]. The anti-HIV activity of mGRFT was approximately 1000-fold lower than that of GRFT, although both the monomeric and dimeric forms of this lectin have very similar oligosaccharide-binding affinities. Therefore, we suggested that the presence of two binding domains in native, di- meric GRFT was important for its activity by allowing the cross-linking of gp120-associated oligosaccharides. Such a mechanism could also lead to the agglutination of HIV vi- rions [30]. We hypothesized that providing more flexibility and varying the distance between the two domains might improve antiviral properties of the derivatives of GRFT. Here we describe the design and characterization of four new lectins constructed from two, three, and four tandem repeats of mGRFT (mGRFT tandemers), which allow for variation in the distance between the carbohydrate binding domains and provide significant freedom to their orienta- tion angle. Background These novel mGRFT tandemers displayed po- tent anti-HIV activity down to a level of 1.0 pM and were able to do so without engendering viral aggregation. on the HIV spike is a contributing factor to viral escape during the course of infection and appears to occur within limits affected by immune evasion, efficient pro- cessing, and access to receptor binding sites [11]. Logic- ally, mu that would reduce the glycosylation levels of HIV Env could affect viral processing and maturation, leading to attenuated infectivity, as well as further ex- posing the virus to the immune system. Therefore, tar- geting this viral glycan shield may be an effective way of combating HIV infection. g Lectins are small proteins that have evolved to bind carbohydrates with high affinity and specificity. A num- ber of lectins have been shown to display potent antiviral activity [12]. Currently, the most potent reported anti- HIV lectin is griffithsin (GRFT), an obligate domain- swapped dimer in which each domain has jacalin-like fold (Figure 1A) [13]. GRFT has anti-HIV EC50 of ~50 pM in cell-based assays [14] and exhibits synergistic ef- fects against HIV in combination with other antiviral agents [15-17]. Additionally, GRFT has been reported to exhibit antiviral activity against the coronavirus respon- sible for SARS [18,19], the hepatitis C virus [20], the Japanese encephalitis virus [21], and the murine herpes simplex virus type 2 [22]. There are a number of charac- teristics that make GRFT an attractive candidate for devel- opment as an antiviral therapeutic. GRFT is thermostable, can survive in a wide range of conditions including those found in the macaque vaginal environment, and exhibits little or no toxicity and immunogenicity [23]. Subcutane- ous administration of GRFT in rodent models has also shown that this lectin accumulates to therapeutically ef- fective levels in serum and plasma with minimal toxicity [24]. Finally, low-cost, large-scale production of GRFT in genetically modified tobacco plants has been demon- strated [25]. Single cycle pseudo-HIV-1 virus neutralization assay Single cycle pseudo-HIV-1 virus neutralization assay All mGRFT tandemers as well as mGRFT and native di- meric GRFT were tested for their activity against a spectrum of HIV-1 strains from different subtypes in a Tzm-bl single cycle neutralization assay system. As expected, the activity of all GRFT molecules tested was reduced when compared to the multiple cycle HIV-1 induced cytopathicity assay. The results, as shown in Table 2, indicate that the 3mGRFT and 4mGRFT tande- mers were, on average, more potent than native dimeric GRFT, whereas the 2mGRFT and 2mGRFT3 tandemers were not significantly better than native dimeric GRFT. Strikingly, two viral strains, subtype B CAAN5342.2 and subtype C CAP206.8 were shown to be resistant to native dimeric GRFT but retained sensitivity to the 3mGRFT and 4mGRFT tandemers, with the 3mGRFT and 4mGRFT tandemers at least 10-fold more active than native dimeric GRFT. A similar result was also seen with the subtype A virus Q168.a2 which was also ~10 Cloning, expression, and purification of mGRFT and GRFT tandemers The GRFT monomer 1GS-S ([30], PDB ID 3LL2, Figure 1B) was chosen as the repeating unit in the design of mGRFT tandemers, because the L2S mutation at its N terminus rendered this monomer more susceptible to proteolytic cleavage of its N-terminal affinity tag. All further references to mGRFT in this work are to 1GS-S. Schematic models of mGRFT tandemers constructed We have previously determined the structures of unli- ganded, native GRFT (Figure 1A) and its complexes with Figure 1 Structure and binding site orientation of dimeric GRFT and mutations used to generate monomeric GRFT. Dimeric GRFT (A) is a domain-swapped dimer with two identical carbohydrate-binding domains (circled in blue) separated by 50 Å and at a relative angle of ~160° from each other. Obligate monomeric GRFT (B) was generated by the addition of Gly-Ser residues in the hinge region of wild-type GRFT. The enhanced flexibility of the hinge region resulted in the collapse of the swapped domain to form an obligate monomer, mGRFT. Figure 1 Structure and binding site orientation of dimeric GRFT and mutations used to generate monomeric GRFT. Dimeric GRFT (A) is a domain-swapped dimer with two identical carbohydrate-binding domains (circled in blue) separated by 50 Å and at a relative angle of ~160° from each other. Obligate monomeric GRFT (B) was generated by the addition of Gly-Ser residues in the hinge region of wild-type GRFT. The enhanced flexibility of the hinge region resulted in the collapse of the swapped domain to form an obligate monomer, mGRFT. Page 3 of 14 Moulaei et al. Retrovirology (2015) 12:6 based on the structure of mGRFT [30] are depicted in Figure 2. The linker between each mGRFT domain was chosen to be flexible and unstructured and consisted of single and triple repeats of Gly-Thr-Gly. However, the flexibility of the linker rendered all attempts at crystal- lizing the tandemers unsuccessful. Purification of tande- mers was performed largely as described previously [30]. The relevant information for GRFT, mGRFT, and the tandemers is listed in Table 1. The relative thermal stability of the mGRFT tandemers was evaluated by dif- ferential scanning calorimetry (Additional file 1: Table S1). It was found that the mGRFT tandemers have simi- lar melting temperatures to mGRFT, indicating that the lectin domains were properly folded. based on the structure of mGRFT [30] are depicted in Figure 2. Cloning, expression, and purification of mGRFT and GRFT tandemers The linker between each mGRFT domain was chosen to be flexible and unstructured and consisted of single and triple repeats of Gly-Thr-Gly. However, the flexibility of the linker rendered all attempts at crystal- lizing the tandemers unsuccessful. Purification of tande- mers was performed largely as described previously [30]. The relevant information for GRFT, mGRFT, and the tandemers is listed in Table 1. The relative thermal stability of the mGRFT tandemers was evaluated by dif- ferential scanning calorimetry (Additional file 1: Table S1). It was found that the mGRFT tandemers have simi- lar melting temperatures to mGRFT, indicating that the lectin domains were properly folded. enhancement of antiviral activity was observed for constructs with additional mGRFT domains, since the anti-HIV activity of 4mGRFT (EC50 = 1.2 pM) was similar to 3mGRFT. The similar anti-HIV activity of 2mGRFT and 2mGRFT3 indicated that the longer interdomain linker did not affect the potency of the tandemers. None of the mGRFT tandemers displayed any toxicity to the CEM-SS cells at the highest tested doses (see Additional file 2: Figure S1). Anti-HIV cytopathicity assay y y y The mGRFT tandemers, as well as mGRFT and GRFT, were tested simultaneously in a whole-cell anti-HIV assay that measures HIV-1RF-induced cytopathicity in the T-lymphoblastic cell line CEM-SS. This assay also tests for compound-induced cytotoxicity to uninfected CEM-SS cells. The results (Table 1) showed that, as re- ported previously, mGRFT was significantly weaker than GRFT, with an EC50 value of 119.3 nM. For the mGRFT tandemers, 2mGRFT (EC50 = 2.7 pM) and 2mGRFT3 (EC50 = 2.6 pM) were 5-fold more active than GRFT (EC50 = 13.8 pM). The antiviral activity displayed by the 3mGRFT tandemer was enhanced by another 5 fold, with an EC50 = 1.0 pM. However, no further Figure 2 Theoretical structures of GRFT-tandemers. Griffithsin tandemers 2mGRFT, 2mGRFT3, 3mGRFT, and 4mGRFT are shown as models of monomeric units attached by flexible linker regions. The N and C termini (red and black, respectively) in a single mGRFT domain are approximately 10 Å apart, causing the individual domains in the tandemers to branch out. Each Gly-Thr-Gly linker (blue) is also approximately 10 Å long in its extended conformation. Figure 2 Theoretical structures of GRFT-tandemers. Griffithsin tandemers 2mGRFT, 2mGRFT3, 3mGRFT, and 4mGRFT are shown as models of monomeric units attached by flexible linker regions. The N and C termini (red and black, respectively) in a single mGRFT domain are approximately 10 Å apart, causing the individual domains in the tandemers to branch out. Each Gly-Thr-Gly linker (blue) is also approximately 10 Å long in its extended conformation. Page 4 of 14 Moulaei et al. Retrovirology (2015) 12:6 Table 1 Physical characteristics an anti-HIV activity of griffithsin tandemers Name Number of domains Linker Molecular Weight (kDa) Anti-HIV EC50 1 (pM) Terminal binding surface distance (Å) GRFT 2 none 25.6 13.8 ± 0.3 50 mGRFT (1GS-S [30]) 1 none 12.8 119,300 ± 19,300 n.a. 2mGRFT 2 Gly-Thr-Gly 25.8 2.7 ± 2.7 70 2mGRFT3 2 (Gly-Thr-Gly)3 26.2 2.6 ± 5.3 90 3mGRFT 3 Gly-Thr-Gly 38.8 1.0 ± 2.9 90 4mGRFT 4 Gly-Thr-Gly 51.8 1.2 ± 0.3 100 1Effective concentration at which 50% of CEM-SS cells are protected from the cytopathic effects of HIV-1RF infection. Table 1 Physical characteristics an anti-HIV activity of griffithsin tandemers times more sensitive to the 3mGRFT and 4mGRFT tandemers. y Isothermal titration calorimetry was used to characterize the thermodynamics of binding of the mGRFT tande- mers to the HIV envelope glycoprotein gp120, as shown previously with GRFT [14]. Anti-HIV cytopathicity assay Anti-HIV activity was visualized using the single-cycle Tzm-bl assay system with activity reported as the effective concentration that inhibited infection by 50% (EC50). All experiments were done in triplicate with the average value reported. Numbers in red indicate fold-difference in EC50 values between wild-type and mutant gp120 bearing pseudoviruses. Figure 3 Effect of the addition of glycans at positions 234 N and 295 N on HIV-1 sensitivity to GRFT and mGRFT-tandemers. Pseudoviruses from either (A) HIV-1 CAAN5342.A2 or (B) HIV-1 CAP206.8 bearing either wild-type gp120 (black bars) or re-engineered mutant gp120 restoring the glycosylation sites at position 234 and 295 (white bars) were tested for their sensitivity to native GRFT or mGRFT tandemers. Anti-HIV activity was visualized using the single-cycle Tzm-bl assay system with activity reported as the effective concentration that inhibited infection by 50% (EC50). All experiments were done in triplicate with the average value reported. Numbers in red indicate fold-difference in EC50 values between wild-type and mutant gp120 bearing pseudoviruses. 2mGRFT and 3mGRFT binding events were the most exothermic, the 2mGRFT3 construct produced the best overall binding profile among the mGRFT tandemers, showing very little entropic cost for binding (TΔS = − 35 kcal/mol). The enthalpy/entropy compensation (ΔH/ ΔS) ratio for 2mGRFT3 was 1.33, compared to 1.16 and 1.19 for 2mGRFT and 3mGRFT, respectively (Table 3). As with any tethered constructs, the crosslinking inter- actions with gp120 and formation of water filled aggre- gates would be entropically disfavored, primarily due to the decreased entropy of the “caged” water molecules within the crosslinked complexes [31]. It was clear from the enthalpy-entropy compensation that the mGRFT tandemers either did not form large aggregates with gp120, or if they did, the crosslinked complexes were soluble and flexible enough to prevent any great degree of trapping of the solvent water molecules. The gp120 ti- trated solutions of the mGRFT tandemers were far less cloudy than the post-titration solution of GRFT, suggest- ing that little or no crosslinked aggregation had taken place. In particular, solution of the 2mGRFT3 complex with gp120 was essentially clear. oligosaccharides of gp120. Anti-HIV cytopathicity assay Since 4mGRFT exhibited no better activity than 3mGRFT in anti-HIV assays, it was not investigated further. The gp120 binding of 2mGRFT, 2mGRFT3, and 3mGRFT was fully characterized and the thermodynamic parameters of the interactions are detailed in Table 3. Binding of gp120 to the mGRFT tan- demers was revealed to be exothermic in nature. The exothermic heats of interaction (negative ΔH values; Table 2) were likely due to polar and electrostatic interac- tions between mGRFT domains and the high-mannose To evaluate the role of specific oligosaccharides in the sensitivity of subtype B CAAN5342.2 and subtype C CAP206.8 to wild type dimeric GRFT compared to the 3mGRFT and 4mGRFT tandemers we undertook to restore two specific oligosaccharide attachment sites on gp120 into these Env constructs by mutating specific residues at positions 234 N and 295 N. As can be seen in Figure 3, insertion of these two oligosac- charide attachment sites restored sensitivity to native dimeric GRFT (as well as the 2mGRFT and 2mGRFT3 tademers) but did not significantly affect the activity of the 3mGRt and 4mGRFT tandemers. Table 2 Anti-HIV-1 activity of mGRFT tandemers, mGRFT and native dimeric GRFT in a single cycle Tzm-bl pseudovirus assay system Envelope IC50 (nM) 2MG 2MG3 3MG 4MG GRFT Subtype B PVO.4 0.301±0.0818 0.558±0.105 0.270±0.0344 0.185±0.0151 0.0370±0.0173 QH0692.42 0.202±0.0842 0.513±0.0882 0.223±0.0834 0.170±0.0119 0.0473±0.0152 JR-FL 2.09±3.28 5.77±2.16 0.325±0.0702 0.287±0.0476 0.922±0.354 CAAN5342.A2 5.27±3.28 16.9±1.41 0.326±0.0834 0.232±0.0167 7.34±0.927 Median 1.20 3.16 0.298 0.0208 0.485 Subtype C Du156.12 0.0182±0.00133 0.0616±0.00335 0.0794±0.0252 0.0887±0.0185 0.0324±0.00204 Du179.14 0.158±0.0962 0.144±0.0438 0.124±0.0262 0.0934±0.0291 0.606±0.139 COT6.15 1.46±0.452 2.54±1.01 0.412±0.0588 0.273±0.0396 0.734±0.326 DU151.2 0.694±0.144 1.70±0.0306 0.308±0.0169 0.300±0.0285 1.49±0.225 CAP206.8 0.178±0.0750 0.696±0.0141 0.181±0.0546 0.164±0.0586 2.14±1.26 Median 0.178 0.696 0.181 0.164 0.734 Subtype A Q23.7 7.26±0.225 7.16±0.512 0.432±0.101 0.276±0.0710 1.19±0.535 Q168.a2 1.55±0.723 5.28±1.48 0.331±0.0398 0.273±0.0167 3.80±1.03 Median 4.41 6.22 0.396 0.274 2.5 ivity of mGRFT tandemers, mGRFT and native dimeric GRFT in a single cycle Tzm-bl pseudovirus Table 2 Anti-HIV-1 activity of mGRFT tandemers, mGRFT and native dimer assay system Page 5 of 14 Moulaei et al. Retrovirology (2015) 12:6 Figure 3 Effect of the addition of glycans at positions 234 N and 295 N on HIV-1 sensitivity to GRFT and mGRFT-tandemers. Pseudoviruses from either (A) HIV-1 CAAN5342.A2 or (B) HIV-1 CAP206.8 bearing either wild-type gp120 (black bars) or re-engineered mutant gp120 restoring the glycosylation sites at position 234 and 295 (white bars) were tested for their sensitivity to native GRFT or mGRFT tandemers. Anti-HIV cytopathicity assay As reported previously for the binding between GRFT and high-mannose oligosacharides [30], an enthalpy-entropy compensation was apparent, in- dicating that the favorable binding contacts between GRFT and gp120 had overcome the unfavorable conform- ational penalties of the bound partners and the unfavor- able entropy of water (negative TΔS values; Table 3) at the interface and around the bound complex, resulting in energetically-favored binding with gp120 (negative ΔG values; Table 3). The observed tight, nanomolar binding affinities were close to the accuracy limits of the calorimetric instru- ment but well within the experimental error and the cal- culated Kd values (Table 3), showed that the mGRFT tandemers bound gp120 with slightly stronger affinity than GRFT. Particularly in the case of the 2mGRFT3 and 3mGRFT constructs, a 3 nM Kd value was calcu- lated for both tandemers. Deconstruction of the gp120 binding affinities into their respective enthalpic and en- tropic contributions provided further insight into the na- ture of the binding between the mGRFT tandemers and gp120. From an examination of the enthalpy of binding (ΔH) it was clear that the mGRFT tandemers mediated a higher number of binding contacts with gp120 than did GRFT (Table 3). Dynamic light scattering One of the common attributes of many lectins is their capacity to agglutinate pathogens [32]. Although anti- viral lectins such as GRFT have been shown not to The enthalpy of binding was, however, not the sole de- terminant of an optimal binding interaction. Although Table 3 Thermodynamic parameters of griffithsin binding to HIV-1 gp120 Table 3 Thermodynamic parameters of griffithsin binding to HIV-1 gp120 Affinity (μM) ΔH (kcal/mol) ΔG TΔS ΔH/TΔS GRFT 0.008 ± 0.004 – 30.4 + 0.3 – 11.2 ± 0.3 – 19.2 ± 0.3 1.58 mGRFT 0.112 ± 0.050 – 35.5 + 0.6 – 9.5 ± 0.7 – 26.0 ± 0.7 1.36 2mGRFT 0.005 ± 0.002 – 69.6 + 0.5 – 11.6 ± 0.6 – 59.9 ± 0.5 1.16 2mGRFT3 0.003 ± 0.001 – 47.2 + 0.6 – 11.8 ± 0.2 – 35.4 ± 0.6 1.33 3mGRFT 0.003 ± 0.001 – 74.4 + 0.4 – 11.8 ± 0.2 – 62.7 ± 0.4 1.19 Moulaei et al. Retrovirology (2015) 12:6 Page 6 of 14 agglutinate human cells [14], it has been suggested that these lectins, which contain multiple binding domains, aggregate viruses [30]. To determine if this was true and to evaluate the role that the enhanced flexibility in bind- ing domains may play in this phenomenon, we used dynamic light scattering to measure the aggregation of HIV-1BAL virions after treatment with mGRFT, GRFT, or one of the four GRFT tandemers. As shown in Figure 4, GRFT did aggregate HIV-1BAL virions to a significant ex- tent when compared to untreated virions. As expected, mGRFT, with only a single binding domain, did not ag- gregate virions but, interestingly, also none of the GRFT tandemers, including 4mGRFT with its four binding do- mains, caused virions to aggregate. The overall spatial distribution and shape of the vi- rions in the presence of mGRFT appeared similar to that of the negative control (Figure 5D). However, differences in spike size and structural profile were evident (Figure 5E and F, red arrows). Viruses exposed to mGRFT displayed an additional density on the spike that was irregular in both shape and the degree of enlargement. The effect of GRFT on HIV-1 differed dramatically from that of mGRFT. Virions treated with GRFT formed large aggre- gates having lateral dimensions on the micrometer scale (Figure 5G). These aggregates appear in the micrograph as large electron-dense swathes. Cryo-electron microscopy y py The interactions of GRFT, mGRFT, and the tandemers with purified HIV-1BaL virions were evaluated using cryo-electron microscopy. The control experiments with HIV-1Bal in the absence of any lectins showed a uniform distribution of virions in the imaging field, with virions having approximately 100 nm diameter and spherical shape (Figure 5A). Ten-nanometer gold fiducials appear as electron dense spots in the micrographs. When virions were imaged at high magnification with cryo-electron tomography, individual envelope glycoprotein spikes were visible (Figures 5B, C, red arrows). The spikes were of the expected height of ~ 120 Å as measured from the mem- brane, with a structural profile consistent with previous electron tomographic studies [33-38]. Dynamic light scattering Individual virions could be identified at the periphery of the aggregates, and though they seem to maintain their membrane integrity, their shapes were highly distorted. Tomography revealed dense masses of protein at the interfaces between virions (Figures 5H and I), which consistently co-localized to the interface between neighboring virions. The mGRFT tande- mers displayed mutually similar effects on HIV-1 virions and envelope glycoproteins (Figures 5J, K, I, and M, re- spectively), but differed significantly from virions treated with GRFT. Tomographic imaging showed that virions treated with the mGRFT tandemers remained monodis- perse, similar to the results with mGRFT and dissimilar from GRFT. Discussion Retrovirology (2015) 12:6 (See figure on previous page.) Figure 5 Electron micrographs of the HIV-1 virions interacting with different constructs of GRFT-based lectins. (A) Projection of untreated HIV-1 virions. Virions are observed as ~ 100 nm circles in the imaging field which includes 10-nanometer-sized protein-A gold fiducials which appear as dark spots. (B, C) Slices through tomograms of untreated HIV-1 virions with red arrows indicating individual Env proteins. (D) Projection of HIV-1 virions that were incubated with mGRFT prior to vitrification. (E, F) Slices through tomograms of HIV-1 virions treated with mGRFT. Representative Env glycoprotein spikes are indicated by red arrows (E). A patch of glycoprotein spikes is indicated by a red arc (F). (G) Projection of HIV-1 virions that were incubated with native GRFT prior to vitrification. (H, I) Slices through tomograms of virions at the periphery of the aggregates in virions treated with native GRFT. (J–M) Slices through tomograms collected from vitreous preparations of HIV-1 virions with GRFT tandemer constructs, 2mGRFT (J), 2mGRFT3 (K), 3mGRFT (I), and 4mGRFT (M). Scale bars are 500 nm in panels (A, D, and G) and 50 nm in the remaining panels. GRFT on its anti-HIV activity. By allowing for more flexibility in the linker region of the mGRFT tandemer structures we enhanced the ability of the second mGRFT bining site to sample the local oligosaccharide environment on individual virions. The larger mGRFT tandemers (2mGRFT3, 3mGRFT and 4mGRFT) also in- creased the distance between the individual mGRFT binding domains, thus increasing the potential scope of GRFT-oligosaccharide interactions to distances greater than 50 Å. GRFT on its anti-HIV activity. By allowing for more flexibility in the linker region of the mGRFT tandemer structures we enhanced the ability of the second mGRFT bining site to sample the local oligosaccharide environment on individual virions. The larger mGRFT tandemers (2mGRFT3, 3mGRFT and 4mGRFT) also in- creased the distance between the individual mGRFT binding domains, thus increasing the potential scope of GRFT-oligosaccharide interactions to distances greater than 50 Å. native, dimeric GRFT in the same assay system (Table 1). We also tested the mGRFT tandemers in a TzmBl single cycle pseudovirus assay system against several different strains of HIV-1 to assess their broader activity com- pared to both mGRFT and native GRFT. The results showed that the 3mGRFT and 4mGRFT tandemers had better overall anti-HIV activity than wild-type GRFT with significant strain variance in EC50 values (Table 2). Discussion The data also showed that the 3mGRFT and 4mGRFT tandemers retained activity against strains of virus which displayed a level of resistance to native dimeric GRFT. All of the designed mGRFT tandemers were readily expressed in E. coli and purified to homogeneity. The mGRFT tandemers are distinct from other recently pub- lished GRFT tandemer constructs [42] in that they were created using obligate monomeric forms of GRFT [30], thereby preventing any potential for domain swapping by the expressed mGRFT tandemers. The maintenance of the mGRFT form of individual lectin domains in the mGRFT tandemers was confirmed by differential scan- ning calorimetry which showed that mGRFT tandemers melted at temperatures corresponding to mGRFT and not native, dimeric GRFT (Additional file 1: Table S1). We evaluated the data to determine whether or not in- creasing the length of inter-domain linkers would affect anti-HIV activity. The maximum theoretical distance be- tween the terminal carbohydrate-binding surfaces of each mGRFT tandemer was calculated based on basic models derived from the X-ray crystal structure of mGRFT (Table 1, Figure 2). In native GRFT the distance between the centers of the carbohydrate-binding regions on each domain was ~50 Å (Figure 1A). Increasing the linear length of the mGRFT tandemers from ~70 Å (2mGRFT) to ~90 Å (2mGRFT3) resulted in a nearly identical or slightly worse anti-HIV activity overall, sug- gesting that this increase in potential distance between mGRFT domains was not a determining factor in anti- HIV potency. Increasing the number of mGRFT do- mains did enhance antiviral activity. However, this effect was limited to three mGRFT domains as evidenced from the nearly identical anti-HIV activities of 3mGRFT and 4mGRFT (Tables 1 and 2). The limit on the number of mGRFT domains was possibly due to geometric con- straints that could limit access to glycan ligands by the fourth mGRFT domain of 4mGRFT and/or saturation of local viral oligosaccharides with mGRFT domains. Previous calorimetric evaluations of the binding of high-mannose oligosaccharides by GRFT [14,18,30] and our present data on the binding of the mGRFT tande- mers to gp120 showed an overall similar mode of inter- action, primarily involving favorable polar-electrostatic contacts. Clear differences among the antiviral proteins were evident, however, when the bindings were parsed into individual thermodynamic contributions (Table 3). Discussion The functional unit of native GRFT is an obligate domain- swapped homodimer [39], with two carbohydrate-binding surfaces located at the opposite ends of the dimeric mol- ecule. The first 15 residues of each monomer form a strand-loop-strand structure that completes the β-prism-II fold of the opposite monomer. Each β-prism harbors three carbohydrate-binding sites on one of the terminal facets of the prism. The centers of the carbohydrate-binding sites form an equilateral triangle with 15 Å sides. The domains are oriented at a ~160° angle from each other, with the centers of the triangular carbohydrate-binding surfaces positioned 50 Å apart (Figure 1A). The orientation of the domains is fixed, since the dimer is formed by domain swapping involving two β strands, resulting in a rigid mol- ecule [26-28] The three carbohydrate-binding sites on the surface of each domain act as a selective mechanism for specifically targeting high-mannose, branched carbohydrate environments typically found on viral envelope glycopro- teins [40]. Viral glycoproteins present a preponderance of high mannose oligosaccharides when compared to human cellular glycoproteins, which have a larger proportion of sialylated oligosaccharides. Alignments of glycans identified on gp120 in vitro [6,41] with N-linked sequon variations of gp120 identified in silico suggests that high-mannose modi- fications dominate the conserved regions of gp120 [11]. Figure 4 Results of dynamic light scattering experiments on HIV-1BAL virions treated with GRFT, mGRFT, or the mGRFT tandemers. Dynamic light scattering traces are shown for HIV-1BAL viruses without lectin, with mGRFT, with GRFT, or with one of the tandemers. Negative controls with buffers or purified lectin did not measurably scatter light. Figure 4 Results of dynamic light scattering experiments on HIV-1BAL virions treated with GRFT, mGRFT, or the mGRFT tandemers. Dynamic light scattering traces are shown for HIV-1BAL viruses without lectin, with mGRFT, with GRFT, or with one of the tandemers. Negative controls with buffers or purified lectin did not measurably scatter light. The mGRFT tandemers described here were designed to evaluate the role of the fixed spatial orientation of Page 7 of 14 Page 7 of 14 Moulaei et al. Retrovirology (2015) 12:6 nd on next page.) igure 5 (See legend on next page.) Figure 5 (See legend on next page.) (See legend on next page.) page.) n next page.) Figure 5 (See legend on next page.) Page 8 of 14 Moulaei et al. Discussion Given the molecular dimensions of the mGRFT tandemers (<100 Å across), and the fact that neighboring spikes on the virus are spaced apart by significantly greater and variable distances (each trimeric Env itself is ~ 150 Å across), it follows that the multiple sites on the mGRFT tandemers are occupied by carbohydrates that are displayed within the same Env trimer, with a high likelihood of crosslinking across protomers within the trimer. Cryo-electron microscopic studies have shown that CD4-induced opening of the trimeric Env spike is necessary for exposure of gp41 that initiates the first step in the fusion of viral and target cell membranes [34,36,46]. The crosslinking of gp120 protomers may essentially block the opening of the trimeric spike and prevent exposure of gp41 and its fusogenic components required for viral entry, thus providing a likely mechanism for the potent function of GRFT and mGRFT tandemers as antiviral agents. The inability of the mGRFT tandemers to cause viral aggregation likely stems from the conformational flexi- bility of the linkers between the mGRFT domains. In na- tive GRFT the two domains are rigidly held across from each other. When one domain of GRFT binds to an Env spike, the second domain would be oriented away at a ~160° angle from that spike and is likely more available for binding the glycans on other Env spikes as well as the glycans on the same spike. In the case of the mGRFT tandemers, after binding of the first mGRFT domain to an Env spike, the flexible linkers would allow the remaining mGRFT domains to more readily sample the local environment and bind to the nearest available carbohydrate ligand. The local concentration of ligands available for subsequent mGRFT domains is potentially higher on the same spike than in the overall solution. Therefore, the mGRFT tandemers, owing to the flexibil- ity of their linkers, are more likely to bind glycans on the same virion, whereas native GRFT, due to its con- formational rigidity, would more favor inter-virion cross- linking. These observations have implications for understand- ing the mechanism whereby the mGRFT tandemers neutralize HIV. The antiviral activity of GRFT, mGRFT, and the mGRFT tandemers stems from their selective binding to high mannose oligosaccharides on Env spikes. Natural mutations that removed glycosylation at posi- tions Asn234 and Asn295 have been reported to impart resistance to GRFT [43]. In addition, Huang et al. Discussion The data suggested that steric hindrance and/or lack of flexibility in GRFT appeared to hamper its domains from making full contact with gp120, whereas the flexible linkers in the GRFT tandemer structures allowed their mGRFT domains to sample more extensively the gp120 surface glycans (comparing ΔH values in Table 3). The anti-HIV activity of mGRFT tandemers does not stem from aggregation of virions, as evidenced by the absence of virion agglutination in dynamic light scatter- ing experiments (Figure 4). In contrast, native GRFT binding to HIV-1 resulted in extensive aggregation of vi- rions (Figure 4). The dimeric structure of native GRFT enhanced its anti-HIV activity by orders of magnitude but also led to viral aggregation. While this aggregation is a possible contributing factor to the antiviral activity of native GRFT, the ability to cause aggregation is clearly The mGRFT constructed in our previous work dis- played a large reduction in its anti-HIV activity in the CEM-SS HIV-1-induced cytotoxicity assay system. It is important to note that, whereas mGRFT was far less ac- tive than GRFT, its anti-HIV activity was still in the nanomolar range [30]. The mGRFT tandemers created for this study all showed anti-HIV activity significantly better than mGRFT and five- to ten-fold better than Moulaei et al. Retrovirology (2015) 12:6 Page 9 of 14 range of oligosaccharides on gp120 than the smaller mGRFT tandemers or the conformationally-restricted wild-type GRFT. not the determining factor for the anti-HIV activity of the mGRFT tandemers. This was evident from both the electron microscopy and light scattering results for HIV treated with the four tandemers. All tandemers were equally or more active than GRFT but none of the mGRFT tandemers caused aggregation of HIV virions. Mapping the location of the Asn234, Asn295, and Asn448 glycans on trimeric spike structures (Figure 6) suggests that glycans decorating these asparagines are located on the lateral edges of HIV spikes. The ar- rangement of the griffithsin monomers in the mGRFT tandemers is such that they are able to bind carbohy- drate moieties on the same virion, but not in a way that bridges separate virions, as demonstrated by the tomographic and light scattering experiments. Discussion showed that deglycosylation of gp120 at Asn295 or Asn448 also resulted in resistance to GRFT [44]. The lo- cation of these oligosaccharide attachment sites on gp120 are shown in Figure 6. To investigate the role of specific oligosaccharides in the sensitivity to GRFT tan- demers, we utilized genetically-engineered pseudotype viruses including either HIV-1 clade B CAAN5342 gp120 (Figure 3A) or clade C CAP206.8 gp120 (Figure 3B) sequences. We tested both strains in either the presence or absence of oligosaccharide attachment sites at positions 234 N and 295 N of the gp120 protein. Figure 3 displays the results of our assessment of the activity of either wild-type GRFT, mGRFT, or mGRFT tandemers against these viruses. The 3mGRFT and 4mGRFT tandemers retain significantly more activity against wild-type GRFT- resistant viruses than the 2mGRFT, 2mGRFT3, or wild-type GRFT. We speculate that this increased ac- tivity against resistant viruses is due to the ability of these larger mGRFT tandemers to sample a broader Methods a TEV protease cleavage site [30] was used as a template to amplify a second 1GS-S cassette using standard PCR protocols and PfuUltra® Hotstart PCR Master Mix (Strata- gene). The amplified cassette contained only the 1GS-S gene terminated with a stop codon and flanked by two XhoI restriction endonuclease sites. The amplified 1GS-S cassette was inserted at the XhoI site downstream of the first 1GS-S cassette in the original expression vector used as a template. The correct orientation of the second cas- sette was verified by sequencing. Site-directed mutagenesis was used to mutate the DNA sequence between the two 1GS-S cassettes to 5’-ggtaccgcgggctagcatatgtcgaccggt-3’, removing the stop codon at the 3’-terminus of the first 1GS-S cassette and introducing a new multiple-cloning site (MCS) with flanking KpnI and AgeI sites (p420TG). In order to create an expression plasmid for 2mGRFT (p2mGRFT), site-directed mutagenesis was used to alter Cloning, expression and purification of GRFT, mGRFT and mGRFT tandemers All restriction enzymes were purchased from New England Biolabs. All chemicals were obtained from American Bioanalytical, unless otherwise stated. All primers were pur- chased from Integrated DNA Technologies. Protein purifi- cation was performed as described previously for the monomeric forms of GRFT [32], except that buffer A con- tained 50 mM maltose. Conclusion The mGRFT tandemers display picomolar anti-HIV activity and, unlike native GRFT, do not aggregate viral particles. mGRFT tandemers also retain potency against viruses resistant to native GRFT. The data presented here demonstrate the potential utility of mGRFT tandemers for the microbicide development, as well as highlight the complex spatial and orientation requirements for viral neutralization by carbohydrate- binding agents such as griffithsin. The 3mGRFTand 4mGRFT tandemers display some advantages over na- tive GRFT (e.g. activity against GRFT-resistant viruses) but the addition of flexible linkers makes these con- structs more susceptible to proteolytic degradation and less thermally stable. The advantages or disadvan- tages of the absence of viral aggregation by mGRFT tandemers, though perhaps responsible for their mod- erately improved in vitro activity, is of undetermined value in an in vivo setting. Further studies on the full physiological and immunological consequences of mGRFT tandemer administration will be necessary to fully evaluate the potential of these engineered lectins for microbicide development. Page 10 of 14 Moulaei et al. Retrovirology (2015) 12:6 Figure 6 Location of GRFT binding-associated oligosaccharides (coupled to Asn234, Asn295, and Asn448) in the context of HIV trimeric spike. Disaccharide modification of Asn234 (blue), Asn295 (red) and Asn448 (yellow) are displayed on a gp120 trimer. X-ray crystal structure of the partially glycosylated HIV-1HXBc2 gp120 (PDB 1GC1 [45]) was superimposed on unliganded gp120 electron tomography structure (PDB 3DNN [34]). Figure 6 Location of GRFT binding-associated oligosaccharides (coupled to Asn234, Asn295, and Asn448) in the context of HIV trimeric spike. Disaccharide modification of Asn234 (blue), Asn295 (red) and Asn448 (yellow) are displayed on a gp120 trimer. X-ray crystal structure of the partially glycosylated HIV-1HXBc2 gp120 (PDB 1GC1 [45]) was superimposed on unliganded gp120 electron tomography structure (PDB 3DNN [34]). Construction of plasmid p420TG and p2mGRFT For the construction of the mGRFT tandemers we chose a construct (1GS-S) that, as reported previously [30], pro- duced obligate monomeric GRFT. An expression vector containing the gene for 1GS-S (see Ref. [30] for nomencla- ture of the different constructs of mGRFT) downstream of Page 11 of 14 Moulaei et al. Retrovirology (2015) 12:6 the MCS in p420TG to 5’-ggtacaggt-3’. The resulting vec- tor contained a single ORF expressing two 1GS-S domains preceded by a TEV protease cleavage site and linked by a GlyThrGly linker. then dialyzed against pure deionized water, or against 20 mM Tris (pH 8.0), 100 mM NaCl. All purification pro- cedures were performed at room temperature. As with the native GRFT, the GRFT tandemers were all stable to pH ranges from 1–8 and to both heat (up to 50°C) and or- ganic solvents (i.e. alcohols, acetonitrile). Differential scanning calorimetry Primers 5’- ggaccggtgccgtactgttcatagtagatgtccaggctatc-3’ and 5’-ggggtaccggcagctcgacccatcgcaag-3’ were used with p2mGRFT as the template in a standard PCR reaction. Two amplicons corresponding to single and double 1GS-S cassettes were separated and purified from an agarose gel. Each amplicon contained a KpnI site followed by a glycine codon (ggc) at the 5’ terminus and the same glycine codon followed by a AgeI site at the 3’ terminus. Differential scanning calorimetry (DSC) experiments were carried out with a Microcal VP-DSC microcalorim- eter (Microcal, Northampton, MA). The concentrations of all tandemer proteins were determined by amino acid analysis, and a 60 μM concentrated sample of a tande- mer protein was evaluated per experiment. By routine protocol, buffer (50 mM Tris, 60 mM NaCl) was intro- duced to both the reference and sample cells and the calorimeter was allowed to ramp through one cycle of a heat-cool cycle (10°C to 90°C) at a heating/cooling rate of 60°C/hr. During the down scan at 25°C, the buffer so- lution from the sample cell was quickly and efficiently replaced with a degassed tandemer protein sample. The entire system was re-pressurized to approximately 30 psi of positive pressure to prevent evaporation at higher temperatures, and the experiment was allowed to con- tinue. A total of 6 alternating up-down scans (10°C to 90°C) were performed to measure possible reversibility of folding/unfolding of the tandemers. According to manufacturing protocol, Origin DSC Analysis software was used to correct for buffer effects and to carry out the integration of the unfolding transitions of the tande- mers. The baseline corrected thermograms were fitted to a two-state melting model and the calorimetric transi- tion enthalpy (ΔHunf) was obtained from the area under the excess heat capacity peak, the midpoint of the transi- tion calculated as the melting temperature (Tm). The single and double 1GS-S cassettes were ligated in between the KpnI and AgeI sites in p420TG to yield p3mGRFT and p4mGRFT, respectively. Site-directed mutagenesis was used to alter the GlyThrGly linker in p2mGRFT to a GlyThrGlyGlyThrGlyGlyThrGly linker, yielding p2mGRFT3 for expression of 2mGRFT3. The complete DNA and amino acid sequences of the GRFT tandemers are included in Additional file 3. Purification protocol for tandemers All tandemers used in the experiments outlined in this paper were purified generally as described previously for the monomeric forms of GRFT [30], except that the cell lysis buffer contained 50 mM maltose to improve recov- ery. Briefly, cells were lysed in 50 mM Tris (pH 8.0), 200 mM NaCl, 50 mM maltose and 5% v/v BugBuster 10x Protein Extraction Reagent (Merck), with continu- ous stirring at room temperature overnight. The lysate was then purified by either (i) heating at 50°C for 10 mi- nutes, (ii) adjusting the buffer to 15% v/v ethanol, or (iii) adjusting the buffer to 10% v/v isopropanol. The lysate was centrifuged at 17,000 rpm in a SS-34 rotor for 30 min. The supernatant was loaded onto a Ni-NTA Superflow column (QIAGEN), equilibrated with five column volumes of buffer A (20 mM Tris [pH 8.0], 200 mM NaCl). The column was washed with buffer A and eluted with buffer A containing 250 mM imidazole. TEV protease was added in 1:100 molar ratio of protease to eluted protein and the sample was incubated at room temperature for 1 hr. The TEV-digested sample was then passed over the same Ni-NTA Superflow column equili- brated with five column volumes of buffer A. The flow- through was collected. For further purification the pooled fractions were loaded onto an amylose column (New England Biolabs) equilibrated with five column volumes of buffer A. The column was washed with buffer A and eluted with buffer A additionally containing 50 mM maltose. The pooled fractions containing tandemers were HIV-induced cytotoxicity bioassay A 2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazo- lium-5-carboxanilide inner salt (XTT)-tetrazolium-based assay was used to determine the anti-HIV activity of mGRFT, GRFT, and mGRFT tandemers against HIV-1RF challenged T-lymphoblastic CEM-SS cells, as described previously [47]. XTT was graciously supplied by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute. CEM-SS cells were maintained in RPMI 1640 media without phenol red and supplemented with 5% fetal bovine serum (BioWhittaker), 2 mM L-glutamine (BioWhittaker), and 50 μg/ml gentamicin (BioWhittaker) (complete medium). Exponentially growing cells were washed and resuspended in complete medium, and a 50 μl ali- quot containing 5 × 103 cells was added to individual wells of a 96-well round-bottom microtiter plate containing Moulaei et al. Retrovirology (2015) 12:6 Page 12 of 14 Page 12 of 14 serial dilutions of GRFT, mGRFT or the mGRFT tande- mers (2mGRFT, 2mGRFT3, 3mGRFT, 4mGRFT) in a vol- ume of 100 μl of medium. Stock supernatants of HIV-1RF were diluted in complete medium to yield sufficient cyto- pathicity (80–90% cell kill in 6 days), and a 50 μl aliquot was added to appropriate wells. Plates were incubated for 6 days at 37°C and then stained for cellular viability using XTT. All experiments were performed in triplicate. To as- sess activity EC50 values were determined by calculating the point at which the dose–response sigmoid curve of increas- ing cell-survival crossed the point at which 50% of the cells survived. Compound-induced direct cytotoxicity to CEM- SS cells was tested simultaneously by adding identical con- centrations of test compounds to wells containing only CEM-SS cells and no virus. Cell viability in these wells was measured at the same time point and by the same method- ology as employed for wells used to evaluate the protection against-HIV-1-induced cytopathicity. injections. The titrations were all done in a rapidly stir- ring solution (300 rpm) held at a constant temperature of 30°C. The heats of binding were recorded as the ex- cess power compensation required for maintaining the same temperature during the course of the titration. Baseline experiments of tandemer titration into buffer were done to calculate heats of dilution and this value was subtracted from the experimental heats of binding. The resulting isotherms were fitted using Origin 5.0 nonlinear least-squares program according to manufac- turer’s protocol, and the values for the enthalpy of bind- ing (ΔH) and the dissociation constant were obtained. Cryo-electron microscopy d Cryo-electron microscopy Imaging experiments used purified suspensions of HIV- 1BaL virions with estimated concentration of ~ 1011 vi- rions/mL (AIDS and Cancer Virus Program, Leidos Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA). Prior to use, viruses were inactivated with Aldrithiol-2 which preserves viral entry capacity and antigenic integrity at levels similar to those of untreated virus. Sample mixtures were prepared by adding 10 nm protein-A gold colloid (Cell Microscopy Center, Utrecht University, 3584 CH Utrecht, The Netherlands) to virus suspension, followed by addition of one of six griffithsin constructs and incubation at 4°C for 30 min. All GRFT constructs were added to the Dynamic light scattering Dynamic light scattering Whole HIV-1 viruses (AIDS and Cancer Virus Program, Leidos Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA) at stock concen- tration (~ 1011 virions/mL) were diluted 1,000-fold in di- lution buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4), and 1 mL of this diluted virus volume was transferred to a plastic cuvette. Autocorrelation mea- surements were carried out at 25°C using a DynaPro NanoStar instrument (Wyatt Technology, Santa Barbara, CA 93117, USA) that provided particle size distributions, the peak of which was taken to be the mean particle size. For dynamic light scattering (DLS) experiments, the vi- ruses were pre-mixed with lectin at the same concentra- tions used in cryo-electron microscopy experiments. This ensured a valid comparison between dynamic light scattering experiments, and the imaging experiments in which there was no sample dilution before mixing. Dy- namic light scattering measurements were carried out with the assistance from Dr. Grzegorz Piszczek (National Institutes of Health, National Heart, Lung and Blood In- stitute, Biophysics Facility, Bethesda, MD 20814, USA). Single-cycle Tzm-bl HIV-1 pseudovirus assay Th l l d The single-cycle pseudovirus assay was performed as previously reported using virus strains as described in Alexandre et al. [43]. Briefly, HIV-1 psudoviruses were generated using pSG3ΔEnv plasmids, which were co- transfected with various Env from different virus strains into 293 T cells using the Fugene transfection reagent (Roche Applied Sciences, Indianapolis, IN). The single cycle pseudovirus neutralization assay was performed in a manner similar to that previously re- ported by Montefiori [48]. Briefly, various dilutions of each compound (GRFT, mGRFT, or mGRFT tandemers) were added to 100 μL of DMEM media augmented with 10% FBS in individual wells of a 96-well plate. To each test well 200 TCID50 of pseudovirus in 50 μL of media was added an incubated for 1 hr at 37°C. Then, 100 μL of TZM-bl cells at a concentration of 1×105 cells/mL was added and cultured at 37°C for an additional 48 hrs. Viral infections were visualized by measuring the activity of firefly luciferase. Inhibitory concentrations (IC50) were calculated as the concentration resulting in a 50% reduc- tion of relative light units (RLU) compared to virus con- trols. All measurements were performed in triplicate. HIV-induced cytotoxicity bioassay From the dissociation constant, a value for the free en- ergy of binding (ΔG) was extrapolated (ΔG = −RTlnKa), and from this value, the entropy of binding (ΔS) was lastly calculated (ΔG = ΔH –TΔS). Acknowledgements h k l R 11. Zhang M, Gaschen B, Blay W, Foley B, Haigwood N, Kuiken C, et al. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology. 2004;14:1229–46. 11. Zhang M, Gaschen B, Blay W, Foley B, Haigwood N, Kuiken C, et al. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology. 2004;14:1229–46. We thank Nicole LaRonde (University of Maryland) and Maria Miller (NCI-Frederick) for helpful discussions regarding experimental design. We thank Steven Fellini, Susan Chacko, and colleagues for support with our use of the Biowulf Linux cluster (National Institutes of Health, Bethesda, MD 20814, USA, http:// biowulf.nih.gov); Jeffrey D. Lifson and Julian W. Bess for providing purified, AT-2 treated HIV-1 virions (AIDS and Cancer Virus Program, Leidos Inc., Frederick Na- tional Laboratory for Cancer Research, Frederick, MD 21702, USA); David Schauder (now of Medical College of Wisconsin) for assistance with cryo-electron micros- copy and analysis; and Grzegorz Piszczek for assistance with the dynamic light scattering experiments (National Institutes of Health, National Heart, Lung and Blood Institute, Biophysics Facility, Bethesda, MD 20814, USA). This work was supported in part by funds from the NIH Intramural AIDS Targeted Antiviral Program and Center for Cancer Research at the National Cancer Institute (to B.O, A.W. and S.S.), and an NIH Intramural AIDS Research Fellowship (to J.R.M.). This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN26120080001E, and supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 12. Balzarini J. Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses? Antivir Chem Chemother. 2007;18:1–11. 12. Balzarini J. Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses? Antivir Chem Chemother. 2007;18:1–11. 13. Bourne Y, Astoul CH, Zamboni V, Peumans WJ, Menu-Bouaouiche L, Van Damme EJ, et al. Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose. Biochem J. 2002;364:173–80. 14. Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA. 6Imquest BioSciences, Frederick, MD 21704, USA. virion suspension at equimolar concentrations with re- spect to the griffithsin monomer. Two microliters of sample mixture were applied to plasma cleaned carbon- coated 200-mesh grids (Quantifoil Micro Tools, 07745 Jena, Germany) and immediately blotted and plunge fro- zen using a Mark III Vitrobot (FEI Company, Hillsboro, Oregon 97124, USA) maintained at 25°C and 100% humid- ity. Data was collected on samples maintained at −193°C using a Tecnai G2 Polara transmission electron microscopy (FEI Company, Hillsboro, Oregon 97124, USA) operated at 200 kV and equipped with an energy-filter and 2 K x 2 K post-energy filter CCD camera (Gatan Incorporated, Pleas- anton, CA 94588, USA). Projections were acquired with a 10–20 e−/Å2 dose at 4.5 kX magnification with −70 μm underfocus. Tilt series spanned an angular range of +/−65° with 2° tilt increments and were acquired at −2.5 μm underfocus with a per-tilt dose of 1 – 2 e−/Å2. Tilt series were aligned using RAPTOR as implemented in IMOD [49,50], and tomograms were reconstructed using R- weighted back projection as implemented in IMOD. virion suspension at equimolar concentrations with re- spect to the griffithsin monomer. Two microliters of sample mixture were applied to plasma cleaned carbon- coated 200-mesh grids (Quantifoil Micro Tools, 07745 Jena, Germany) and immediately blotted and plunge fro- zen using a Mark III Vitrobot (FEI Company, Hillsboro, Oregon 97124, USA) maintained at 25°C and 100% humid- ity. Data was collected on samples maintained at −193°C using a Tecnai G2 Polara transmission electron microscopy (FEI Company, Hillsboro, Oregon 97124, USA) operated at 200 kV and equipped with an energy-filter and 2 K x 2 K post-energy filter CCD camera (Gatan Incorporated, Pleas- anton, CA 94588, USA). Projections were acquired with a 10–20 e−/Å2 dose at 4.5 kX magnification with −70 μm underfocus. Tilt series spanned an angular range of +/−65° with 2° tilt increments and were acquired at −2.5 μm underfocus with a per-tilt dose of 1 – 2 e−/Å2. Tilt series were aligned using RAPTOR as implemented in IMOD [49,50], and tomograms were reconstructed using R- weighted back projection as implemented in IMOD. Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA. 6Imquest BioSciences, Frederick, MD 21704, USA. 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https://openalex.org/W3121089048	https://europepmc.org/articles/pmc7788801?pdf=render	English	null	Circ_0000396 inhibits rheumatoid arthritis synovial fibroblast growth and inflammatory response via miR-203/HBP1 axis	Journal of Biological Research - Thessaloniki	2,021	cc-by	7,725	Abstract Background: Circ_0000396 was found to be down-regulated in the rheumatoid arthritis (RA) patients and had a high diagnostic value. However, the function and mechanisms underlying circ_0000396 in RA progression remain unclear. Methods: The expression of circ_0000396, microRNA (miR)-203 and HMG-box transcription factor 1 (HBP1) was detected using qRT-PCR and western blot. The proliferative and apoptotic capabilities of rheumatoid arthritis synovial fibroblasts (RASFs) were measured by colony formation, CCK-8, flow cytometry and western blot assays, respectively. The levels of interleukins (IL)-6, IL-1β, IL-8 and tumor necrosis factor-α (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The target correlations between miR-203 and circ_0000396 or HBP1 were validated using pull-down and dual-luciferase reporter assay. Results: Circ_0000396 was decreased in RA synovial tissues and RASFs, and overexpression of circ_0000396 sup- pressed cell proliferation, induced cell apoptosis and reduced the release of inflammatory cytokine IL-6, IL-1β, IL-8 and TNF-α in RASFs, while circ_0000396 deletion functioned oppositely. MiR-203 was confirmed to be a target of circ_0000396, and miR-203 reversed the protective effects of circ_0000396 on the dysfunction and inflammation of RASFs. HBP1 was a target of miR-203, and silencing miR-203 inhibited RASFs malignant changes by regulating HBP1. In addition, circ_0000396 could regulate HBP1 by sponging miR-203, and HBP1 decrease attenuated the effects of circ_0000396 on RASF growth and inflammation. Results: Circ_0000396 was decreased in RA synovial tissues and RASFs, and overexpression of circ_0000396 sup- pressed cell proliferation, induced cell apoptosis and reduced the release of inflammatory cytokine IL-6, IL-1β, IL-8 and TNF-α in RASFs, while circ_0000396 deletion functioned oppositely. MiR-203 was confirmed to be a target of circ_0000396, and miR-203 reversed the protective effects of circ_0000396 on the dysfunction and inflammation of RASFs. HBP1 was a target of miR-203, and silencing miR-203 inhibited RASFs malignant changes by regulating HBP1. In addition, circ_0000396 could regulate HBP1 by sponging miR-203, and HBP1 decrease attenuated the effects of circ_0000396 on RASF growth and inflammation. Conclusion: Circ_0000396 inhibited the growth and inflammation in RASFs by regulating miR-203/HBP1 axis, provid- ing a potential therapeutic target for RA. Keywords: circ_0000396, Rheumatoid arthritis, Synovial fibroblasts, miR-203; HBP1 Keywords: circ_0000396, Rheumatoid arthritis, Synovial fibroblasts, miR-203; HBP1 effects on the pathogenesis of RA [3, 4]. It is reported that RASFs have similar characteristics to tumor cells, such as tumor-like proliferation, migration, invasion, and resistance to apoptosis [5, 6]. © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Laifang Wang, Qing Zhao, Na Wang, Yanjie Ding, Lingli Kong and Jing Wang Laifang Wang, Qing Zhao, Na Wang, Yanjie Ding, Lingli Kong and Jing Wang Abstract Besides, over-secretion of cytokines in RA is another key feature of RASFs, which activates the immune responses, and in turn, stimulate RASFs, ultimately leading to joint destruction [7]. Thus, a better knowledge of RASFs is necessary for improving the therapy of RA. Open Access Open Access Background Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic disorder that is characterized by hypertrophy, hyperplasia, and angiogenesis of synovial tissues [1, 2]. The pathogenesis of RA is complex; increasing evidence suggests that synovial fibroblasts of RA (RASFs), also known as fibroblast-like synoviocytes, perform important Correspondence: sigtbs@163.com Department of Rheumatism and Immunology, Huaihe Hospital of Henan University, No. 115 Ximen Street, Kaifeng 475000, Henan, China Correspondence: sigtbs@163.com Department of Rheumatism and Immunology, Huaihe Hospital of Henan University, No. 115 Ximen Street, Kaifeng 475000, Henan, China Circular RNAs (circRNAs) are a category of endog- enous RNAs with conserved closed continuous loops Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 https://doi.org/10.1186/s40709-020-00131-4 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 https://doi.org/10.1186/s40709-020-00131-4 Journal of Biological Research-Thessaloniki Page 2 of 11 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 RNA isolation and qRT‑PCR l A d b Total RNA was prepared by using TRIzol reagent (Invitro- gen, Carlsbad, CA, USA) following the standard procedure. Then extracts were interacted with Rnase R (Epicentre, Madison, WI, USA), and the yield and purity were meas- ured by a NanoDrop 2000 instrument (Thermo Fisher Scientific, Rochester, NY, USA). Subsequently, first strand cDNA was generated using a PrimeScript RT Reagent Kit (Takara, Dalian, China), and then qRT-PCR was con- ducted with SYBR Green methods (Takara, Dalian, China) on the ABI7500 system (Applied Biosystems, Foster City, CA, USA). The relative fold changes in expression were assessed using 2−ΔΔCt method and normalized by GADPH and U6. The experiment was performed in triplicate, and the average was taken. The primers were presented as fol- lows: circ_0000396: F 5΄- GCTCACACCAATCCCCTG -3΄, R 5΄-GCCTGGCACACAGTAGACAC-3΄; GADPH: F 5΄-GATATTGTTGCCATCAATGAC-3΄, R 5΄-TTGATT TTGGAGGGATCTCG-3΄; miR-203 F 5΄-TGCTCTAGA GGCGTCTAAGGCGTCCG-3΄, R 5΄- CCCAAGCTT CACCTCCCAGCAGCACTTG-3΄; HBP1: F 5΄- TGA AGGCTGTGATAATGAGGAAGAT-3΄, R 5΄- CATAGA AAGGGTGGTCCAGCTTA-3΄; U6: F 5΄-CTCGCTTCG GCAGCACA-3΄, R 5΄-ACGCTTCACGAATTTGCGT-3΄. This study mainly focused on identifying the expres- sion patterns of circ_0000396 in RA synovial tissues and RASFs, exploring the roles and the underlying mecha- nism of circ_0000396 in RASF biological behavior and inflammation. Cell proliferation analysis For colony formation assay, transfected RASFs were seeded into a 12-well plate at a density of 1000 cells per well and then cultured in a CO2 incubator at 37 °C for 2 weeks. Subsequently, cells were washed with phos- phate-buffered saline (PBS), and then fixed with 4% para- formaldehyde for 15 min and stained with 0.5% crystal violet for 20 min. The colonies were photographed and imaged using a microscope reader (Bio-Rad, Hercules, CA, USA). The experiments were replicated at least three time. Clinical samples Samples of RA synovial tissues were collected from 31 RA patients who received knee joint replacement surgery at Huaihe Hospital of Henan University. All RA patients were diagnosed in line with the American College of Rheumatology classification. For non-RA control, the synovial tissue samples were collected from 25 patients who underwent emergency trauma amputation. None of the healthy controls had RA or osteoarthritis. All samples were stored in liquid nitrogen for further analysis. Cell transfectionh structures, and widely exist in the eukaryotic transcrip- tome [8]. Recent studies have found that circRNAs are the potential regulators in a variety of diseases, includ- ing cancers, cardiovascular and autoimmune diseases, by regulating numerous biological events in cells [9–11]. Additionally, the involvement of circRNAs in the pro- gression of RA also was identified [12]. For example, Luo et al. [13] proved that hsa_circ_0044235 in peripheral blood was the potential biomarker for the diagnosis of RA patients. Li et al. [14] found circFADS2 knockdown protected chondrocytes against extracellular matrix (ECM) degradation, inflammatory cytokines release, and apoptosis by modulating miR-498/mTOR pathway. Li et al. [15] demonstrated hsa_circ_0001859 up-regulated ATF2 through miR-204/211 to stimulate inflammation in SW982 cells. Thus, circRNAs may be promising can- didates for the treatment of RA. Circ_0000396 is a novel identified circRNA; it has been found that it was down- regulated in the RA patients versus the healthy group, and showed a higher receiver operating characteristic (ROC) area under the curve (AUC), while circ_0000396 might be a potential diagnostic biomarker for RA patients [16]. However, the roles of circ_0000396 in RASF dys- function remain unclear. The mimic or inhibitor targeting miR-203 (miR-203 or in- miR-203), and the corresponding negative control (miR-NC and in-miR-NC) were purchased from RIBOBIO (Guang- zhou, China). The small interfering RNA (siRNA) targeting circ_0000396 covalent closed junction (si-circ_0000396), siRNA targeting HMG-box transcription factor 1 (HBP1) (si-HBP1), siRNA negative control (si-NC), empty vec- tor (pcDNA), pcDNA-circ_0000396 overexpression vector (circ_0000396), pcDNA-HBP1 overexpression vector (HBP1) were designed by Genepharma (Shanghai, China). Cell trans- fection was carried out with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Enzyme linked immunosorbent assay (ELISA) Cytokine concentrations from the harvested supernatants of RASFs after appropriate treatment were measured by using commercial interleukins IL-6, IL-1β and IL-8 and tumor necrosis factor-α (TNF-α) ELISA kits (R&D Sys- tems, Minneapolis, USA) according to the instructions of the manufacturer. Each sample was prepared in triplicate, and the experiment was repeated three times. The expression of circ_0000396 in RA synovial tissues and RASFsi To investigate the expression profile of circ_0000396 in RA, qRT-PCR analysis was performed. The results showed that circ_0000396 expression was significantly decreased in synovial tissues with RA relative to these in non-RA controls (Fig. 1a). Besides, it was also found that circ_0000396 expression was lower in RASFs compared with that in NSFs (Fig. 1b). These results indicated that circ_0000396 expression might be associated with the progression of RA. Statistical analysis Data was manifested as mean ± standard deviation (SD) and analyzed using GraphPad Prism 7 software (Graph- Pad, San Diego, CA, USA). The differences in different groups were analyzed by Student’s t test, Mann–Whitney U-test or one-way analysis of variance (ANOVA) accord- ing to the data normality. p value less than 0.05 exhibited a statistical significance. Western blot l Total proteins were prepared using RIPA buffer, and then qualified by the bicinchoninic acid (BCA) method. Sub- sequently, the extracted proteins were separated by 10% SDS-PAGE, and afterwards shifted to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Then immunoblotting was performed with the antibody against HBP1 (1:5000, ab83402, Abcam, Cambridge, MA, USA), Ki67 (1:5000, ab16667, Abcam), cleaved PARP (1:3000, ab32064, Abcam), cleaved caspase3 (1:3000, ab2302, Abcam), and β-actin (1:1000, Cat#4967, Cell Signaling Technology, Boston, MA, USA), followed by interaction with HRP-conjugated secondary antibody and visualized by electrochemiluminescence. Each sam- ple was prepared in triplicate, and the same experiment was repeated three times. Pull‑down assay l b l d Biotin-labeled miR-203 (Bio-miR-203), Bio-miR-203 MUT, and Bio-miR-NC generated by GenePharma were transfected into RASFs for 48 h. Then RASFs were lysed, and cell lysates were incubated with Dynabead M-280 streptavidin beads (Invitrogen, Carlsbad, CA, USA) over- night. After elution, the bead-bound was purified using TRIzol and analyzed using qRT-PCR. Cell apoptosis assay After transfection, a total of 1 × 105 RASFs from each group were washed with PBS. Then, cells were collected and resuspended in binding buffer, followed by incuba- tion with annexin V-FITC (10 μL) and PI (10 μL) in the dark (BD Biosciences, San Jose, CA, USA). Finally, apop- totic cells were analyzed by a flow cytometer with Flow J software (FACSCanto™ II, BD Biosciences, San Jose, CA, USA). In each independent experiment, three parallel wells were made, and the procedures were carried out in triplicate. Cell culture RA samples and healthy control samples were cut into small pieces. After washing with Hank’s solution, all pieces were digested with trypsin solution. Subsequently, RASFs and normal synovial cells (NSFs) were separated using the fluorescence activated cell sorting (FACS) tech- nique based on the expression of CD68 (a macrophage marker) and CD90 (a fibroblast marker) (Additional file 1: Fig. S1). All collected cells were grown in DMEM (Gibco, Carlsbad, CA, USA) harboring 10% FBS and incubated with 5% CO2 at 37 °C. Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Page 3 of 11 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 UTR containing the potential binding sites of miR-203 were cloned into the pmiR-RB-Report luciferase vector (Promega, Shanghai, China) to generate the wild-type pmiR-RB-Report-circ_0000396/HBP1 3΄ UTR plasmids (circ_0000396 WT or HBP1 3΄ UTR WT) or mutated type pmiR-RB-Report-circ_0000396/HBP1 3΄ UTR plasmids (circ_0000396 MUT or HBP1 3΄ UTR MUT), respectively. Subsequently, RASFs were co-transfected with these constructed luciferase plasmids and miR-203 mimic or miR-NC using Lipofectamine™ 3000 (Invitro- gen, Carlsbad, CA, USA) for 48 h. Finally, the luciferase activity was detected by a dual luciferase assay kit (Pro- mega, Shanghai, China) and normalized with Ranilla luciferase activity. UTR containing the potential binding sites of miR-203 were cloned into the pmiR-RB-Report luciferase vector (Promega, Shanghai, China) to generate the wild-type pmiR-RB-Report-circ_0000396/HBP1 3΄ UTR plasmids (circ_0000396 WT or HBP1 3΄ UTR WT) or mutated type pmiR-RB-Report-circ_0000396/HBP1 3΄ UTR plasmids (circ_0000396 MUT or HBP1 3΄ UTR MUT), respectively. Subsequently, RASFs were co-transfected with these constructed luciferase plasmids and miR-203 mimic or miR-NC using Lipofectamine™ 3000 (Invitro- gen, Carlsbad, CA, USA) for 48 h. Finally, the luciferase activity was detected by a dual luciferase assay kit (Pro- mega, Shanghai, China) and normalized with Ranilla luciferase activity. For CCK-8 assay, RASFs were seeded into 96-well plates with 5000 cells per well overnight following trans- fection, and then incubated with 10 μL CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) for 2 h. The absorbance of each well at 450 nm was determined by a microplate reader in the indicated time. Each sample was prepared in quadruplicate, and the entire experiment was repeated three times. Dual‑luciferase reporter assayh To identify the potential role of circ_0000396 in the pro- gression of RA, circ_0000396 was up-regulated or down- regulated in RASFs by transfecting with circ_0000396 or si-circ_0000396. As expected, circ_0000396 pcDNA notable increased the expression of circ_0000396, while The circinteractome (https://circinteractome.nia.nih. gov/) and DIANA TOOLS databases (http://diana.imis. athena-innovation.gr/DianaTools/index.php) were used to predict potential binding sites. The wild-type (WT) or mutant (MUT) circ_0000396 and HBP1 3΄ The circinteractome (https://circinteractome.nia.nih. gov/) and DIANA TOOLS databases (http://diana.imis. athena-innovation.gr/DianaTools/index.php) were h used to predict potential binding sites. The wild-type (WT) or mutant (MUT) circ_0000396 and HBP1 3΄ Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Wang et al. J of Biol Res-Thessaloniki Page 4 of 11 Fig. 1 The expression of circ_0000396 in RA synovial tissues and RASFs. a Expression of circ_0000396 in RA synovial tissues and healthy control tissues was measured by qRT-PCR. b qRT-PCR analysis of circ_0000396 expression in RASFs and NSFs was performed. p < 0.05, p < 0.01, p < 0.001, *p < 0.0001 (Student’s t test) Fig. 1 The expression of circ_0000396 in RA synovial tissues and RASFs. a Expression of circ_0000396 in RA synovial tissues and healthy control tissues was measured by qRT-PCR. b qRT-PCR analysis of circ_0000396 expression in RASFs and NSFs was performed. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 (Student’s t test) Fig. 2 Effects of circ_0000396 on the growth and inflammation of RASFs. RASFs were transfected with pcNDA, circ_0000396, si-NC, or si-circ_0000396. After transfection, a circ_0000396 expression was detected by qRT-PCR (Student’s t test); b, c the proliferation of RASFs was analyzed using colony formation assay and CCK-8 assay, respectively (Mann–Whitney U-test); d the number of apoptotic RASFs was examined by flow cytometry (Student’s t test); e western bolt analysis of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was performed (Student’s t test); f–I the level of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was measured using ELISA assay (Student’s t test). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Fig. 2 Effects of circ_0000396 on the growth and inflammation of RASFs. RASFs were transfected with pcNDA, circ_0000396, si-NC, or si-circ_0000396. Dual‑luciferase reporter assayh After transfection, a circ_0000396 expression was detected by qRT-PCR (Student’s t test); b, c the proliferation of RASFs was analyzed using colony formation assay and CCK-8 assay, respectively (Mann–Whitney U-test); d the number of apoptotic RASFs was examined by flow cytometry (Student’s t test); e western bolt analysis of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was performed (Student’s t test); f–I the level of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was measured using ELISA assay (Student’s t test). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Page 5 of 11 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Fig. 3 MiR-203 is identified as a direct target of circ_0000396. a The putative binding sits between circ_0000396 and miR-203 were predicted. b–d The interaction between circ_0000396 and miR-203 was confirmed by the dual-luciferase reporter assay (Mann–Whitney U-test) and pull-down assay in RASFs (ANOVA). e, f Expression of miR-203 in RA synovial tissues and healthy control tissues as well as in RASFs and NSFs was detected by qRT-PCR (Student’s t test). g qRT-PCR analysis of miR-203 expression in RASFs transfected with pcNDA, circ_0000396, si-NC, or si-circ_0000396 was conducted (Student’s t test). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 results displayed that, by contrast with the negative con- trol, the secretion levels of IL-6, IL-1β, IL-8, and TNF-α were suppressed by overexpressed circ_0000396, but was elevated by the depletion of circ_0000396 (Fig. 2f–i). Taken together, circ_0000396 inhibited cell proliferation and inflammatory response, but induced cell apoptosis in RASFs. MiR‑203 is identified as a direct target of circ_0000396 It has been reported that circRNAs can function as sponges of microRNAs (miRNAs) to regulate gene expression [17]. Thus, to study the mechanism underlying the action of circ_0000396 on RASF dysfunction, the direct miRNA tar- gets of circ_0000396 were predicted by searching circinter- actome database, and the binding sites between miR-203 and circ_0000396 were presented (Fig. 3a). Afterwards, a dual luciferase reporter assay was performed and a decline of luciferase activity in RASFs co-transfected with circ_0000396 WT plasmids and miR-203 was observed, but there was no obvious impact on circ_0000396 MUT plasmids compared with the control group after up-regu- lation of miR-203 expression in cells (Fig. 3b). MiR‑203 is identified as a direct target of circ_0000396 Fig. 3 MiR-203 is identified as a direct target of circ_0000396. a Fig. 3 MiR-203 is identified as a direct target of circ_0000396. a The putative binding sits between circ_0000396 and miR-203 were predicted. b–d The interaction between circ_0000396 and miR-203 was confirmed by the dual-luciferase reporter assay (Mann–Whitney U-test) and pull-down assay in RASFs (ANOVA). e, f Expression of miR-203 in RA synovial tissues and healthy control tissues as well as in RASFs and NSFs was detected by qRT-PCR (Student’s t test). g qRT-PCR analysis of miR-203 expression in RASFs transfected with pcNDA, circ_0000396, si-NC, or si-circ_0000396 was conducted (Student’s t test). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Fig. 3 MiR-203 is identified as a direct target of circ_0000396. a The putative binding sits between circ_0000396 and miR-203 were predicted. b–d The interaction between circ_0000396 and miR-203 was confirmed by the dual-luciferase reporter assay (Mann–Whitney U-test) and pull-down assay in RASFs (ANOVA). e, f Expression of miR-203 in RA synovial tissues and healthy control tissues as well as in RASFs and NSFs was detected by qRT-PCR (Student’s t test). g qRT-PCR analysis of miR-203 expression in RASFs transfected with pcNDA, circ_0000396, si-NC, or si-circ_0000396 was conducted (Student’s t test). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 si-circ_0000396 transfection effectively decreased its expression in RASFs, and transfection of negative con- trol had no effect on circ_0000396 expression (Fig. 2a). After that, results of colony formation and CCK-8 assay showed circ_0000396 up-regulation significantly inhibited colonies formation and the proliferation of RASFs, while circ_0000396 down-regulation promoted colonies formation and RASF proliferation (Fig. 2b, c). Inversely, the number of apoptotic RASFs was increased by circ_0000396 overexpression and was decreased by circ_0000396 knockdown (Fig. 2d). Moreover, western blot analysis displayed that circ_0000396 up-regula- tion decreased Ki67 level and increased cleaved PARP and cleaved caspase3 expression levels in RASFs, while circ_0000396 down-regulation showed inverse effects (Fig. 2e), further indicating circ_0000396 suppressed cell proliferation and induced apoptosis in RASFs. Mean- while, the expression of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α was detected using ELISA, and Dual‑luciferase reporter assayh Meanwhile, we also found that the inhibition of miR-203 enhanced the luciferase activity of the circ_0000396 WT reporter vector but not mutant reporter vector in RASFs (Fig. 3c). Additionally, the data of pull-down analysis exhibited a significant enrichment of circ_0000396 in Bio-miR-203 group relative to Bio-miR-203 MUT or Bio-miR-NC group (Fig. 3d). These data confirmed that circ_0000396 directly bound to miR-203 in RASFs. After that, the expression of miR-203 was analyzed; qRT-PCR analysis showed miR-203 was up-regulated in RA synovial tissues and RASFs relative to the normal controls (Fig. 3e, f), and miR-203 was overex- pressed in RASFs when circ_0000396 was down-regulated, but was decreased when circ_0000396 was up-regulated (Fig. 3g). Therefore, circ_0000396 negatively regulated miR- 203 expression in RASFs. Circ_0000396 affects the growth and inflammation of RASFs by binding to miR‑203 Moreover, circ_0000396 was also discovered to suppress HBP1 expression at mRNA and protein levels, but this inhibition could be abated by overexpressed miR-203 in RASFs (Fig. 5h, i). In sum- mary, HBP1 was a target of miR-203, and circ_0000396 could directly regulate HBP1 expression by binding to miR-203 in RASFs. apoptosis (Fig. 4d, e) in RASFs. In addition, ELISA analysis showed the massive release of inflammatory cytokines IL-6, IL-1β, IL-8, and TNF-α induced by circ_0000396 in RASFs was also attenuated by miR-203 up-regulation (Fig. 4f–i). In all, circ_0000396 regulated RA progression by interacting with miR-203. Circ_0000396 affects the growth and inflammation of RASFs by binding to miR‑203 We further explored whether miR-203 involved in the protective functions of circ_0000396 on RASFs malignant changes and inflammation. First, RASFs were transfected with pcDNA, circ_0000396, circ_0000396 + miR-NC, circ_0000396 + miR-203. After transfection, we found that the expression of miR-203 was inhibited by circ_0000396 overexpression, which was rescued by miR-203 mimic (Fig. 4a), indicating the successful transfection. Afterwards, rescue assay was conducted, and results displayed miR-203 overexpres- sion reversed circ_0000396 restoration-mediated inhi- bition of proliferation (Fig. 4b, c, e), and promotion of Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Page 6 of 11 Fig. 4 Circ_0000396 affects the growth and inflammation of RASFs by binding to miR-203. RASFs were transfected with pcDNA, circ_0000396, circ_0000396 + miR-NC, circ_0000396 + miR-203. After transfection, a the expression of miR-203 was measured by qRT-PCR; b, c RASF proliferation was analyzed by colony formation assay and CCK-8 assay, respectively; d the apoptosis of RASFs was measured by flow cytometry; e the expression of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was detected using western blot; f–i The level of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was detected by ELISA assay. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 (ANOVA) Fig. 4 Circ_0000396 affects the growth and inflammation of RASFs by binding to miR-203. RASFs were transfected with pcDNA, circ_0000396, circ_0000396 + miR-NC, circ_0000396 + miR-203. After transfection, a the expression of miR-203 was measured by qRT-PCR; b, c RASF proliferation was analyzed by colony formation assay and CCK-8 assay, respectively; d the apoptosis of RASFs was measured by flow cytometry; e the expression of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was detected using western blot; f–i The level of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was detected by ELISA assay. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 (ANOVA) WT vectors and miR-203 (Fig. 5b), and the increase of luciferase activity in RASFs co-transfected with HBP1 3΄UTR WT vectors and in-miR-203 (Fig. 5c) confirmed the direct interaction between HBP1 and miR-203. After that, qRT-PCR analysis showed HBP1 expression was both decreased in RA synovial tissues and RASFs (Fig. 5d, e), and miR-203 inhibited the expression of HBP1 in RASFs (Fig. 5f, g). Circ_0000396 can directly regulate miR‑203 targeted gene HBP1 in RASFs Through searching the online program DIANA TOOLS, HBP1 was found to have the potential binding sites in miR-203 (Fig. 5a). Immediately, the reduction of lucif- erase activity in RASFs co-transfected with HBP1 3΄UTR Page 7 of 11 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Fig. 5 Circ_0000396 can directly regulate miR-203 targeted gene HBP1 in RASFs. a The putative binding sits between HBP1 3΄UTR and miR-203 were listed. b, c The interaction between circ_0000396 and miR-203 was confirmed by the dual-luciferase reporter assay in RASFs (Mann–Whitney U-test). d, e The protein expression of HBP1 in RA synovial tissues and healthy control tissues, as well as in RASFs and NSFs was detected by western blot (Student’s t test). f, g qRT-PCR or western blot analysis of HBP1 expression in RASFs transfected with miR-NC, or miR-203 was conducted (Student’s t test). h, i Levels of HBP1 in RASFs transfected pcDNA, circ_0000396, circ_0000396 + miR-NC, circ_0000396 + miR-203 were detected by qRT-PCR or western blot (ANOVA). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 (Fig. 6a, b), suggesting the successful transfection. Sub- sequently, we found miR-203 inhibition mediated proliferation arrest (Fig. 6c, d, f), apoptosis elevation (Fig. 6e, f), and release suppression of inflammatory cytokines (Fig. 6g–j) in RASFs; however, these func- tions could be reversed by HBP1 silencing (Fig. 6b–j). Altogether, miR-203 inhibition suppressed the growth and inflammation of RASFs by regulating HBP1. (Fig. 6a, b), suggesting the successful transfection. Sub- sequently, we found miR-203 inhibition mediated proliferation arrest (Fig. 6c, d, f), apoptosis elevation (Fig. 6e, f), and release suppression of inflammatory cytokines (Fig. 6g–j) in RASFs; however, these func- tions could be reversed by HBP1 silencing (Fig. 6b–j). Altogether, miR-203 inhibition suppressed the growth and inflammation of RASFs by regulating HBP1. Circ_0000396 affects the growth and inflammation of RASFs through modulating HBP1 Given that circ_0000396 could regulate HBP1 expression via miR-203, whether circ_0000396 performed protective effects on RASFs by regulating HBP1 was investigated. RASFs were transfected with pcDNA, circ_0000396, circ_0000396 + si-NC, or circ_0000396 + si-HBP1, then we found si-HBP1 attenuated circ_0000396-mediated up-regulation of HBP1 expression in RASFs (Fig. 7a, b). Subsequently, rescue assay was conducted, results exhib- ited that knockdown of HBP1 overturned circ_0000396- induced inhibition on malignant phenotypes and inflammation, evidenced by the increase of RASF pro- liferation (Fig. 7c, d, f) and decrease of apoptotic RASFs (Fig. 7e, f), as well as the release of IL-6, IL-1β, IL-8, and TNF-α in RASFs (Fig. 7g–j). Thus, we conformed that circ_0000396 regulated RA progression by modulating HBP1. Discussion Growing studies have indicated that circRNAs are impor- tant regulators in complex human pathologies, and circR- NAs are found to play a vital role in the immune system, and are greatly connected with the onset and progression of autoimmune diseases, including RA [18]. In this study, we also found circ_0000396 was decreased in RA syno- vial tissues and RASFs, which was consistent with pre- vious study [16]. Then, functional experiments showed circ_0000396 overexpression inhibited cell proliferation and induced cell apoptosis in RASFs, while circ_0000396 decrease showed opposite effects. Besides, we also found the secretion of IL-6, IL-1β, IL-8, and TNF-α inflam- matory cytokines was suppressed by overexpressed circ_0000396, thus suppressing inflammation in RASFs. Fig. 5 Circ_0000396 can directly regulate miR-203 targeted gene HBP1 in RASFs. a The putative binding sits between HBP1 3΄UTR and miR-203 were listed. b, c The interaction between circ_0000396 and miR-203 was confirmed by the dual-luciferase reporter assay in RASFs (Mann–Whitney U-test). d, e The protein expression of HBP1 in RA synovial tissues and healthy control tissues, as well as in RASFs and NSFs was detected by western blot (Student’s t test). f, g qRT-PCR or western blot analysis of HBP1 expression in RASFs transfected with miR-NC, or miR-203 was conducted (Student’s t test). h, i Levels of HBP1 in RASFs transfected pcDNA, circ_0000396, circ_0000396 + miR-NC, circ_0000396 + miR-203 were detected by qRT-PCR or western blot (ANOVA). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 l RASF stimulation is a pivotal factor in the transform of affected synovium to healthy synovium, thus induc- ing the expansion of arthritis and distant joints destruc- tion [19, 20]. It has been shown RA is closely associated with the abnormal hyperplasia of RASFs [21]. RASFs can produce plentiful cytokines and matrix metalloprotein- ases (MMPs) that, in turn, stimulate the migration and invasion of themselves [19]. In addition, proliferation HBP1 silence reverses the function of miR‑203 on RASF growth and inflammation The effects of miR-203 on RASFs were investigated. RASFs were transfected with in-miR-NC, in-miR-203, in-miR-203 + si-NC, or in-miR-203 + si-HBP1. Then, results showed that HBP1 expression was promoted by miR-203 inhibition, which was rescued by HBP1 knockdown in RASFs at mRNA and protein levels HBP1 silence reverses the function of miR‑203 on RASF growth and inflammation HBP1 silence reverses the function of miR‑203 on RASF growth and inflammation The effects of miR-203 on RASFs were investigated. RASFs were transfected with in-miR-NC, in-miR-203, in-miR-203 + si-NC, or in-miR-203 + si-HBP1. Then, results showed that HBP1 expression was promoted by miR-203 inhibition, which was rescued by HBP1 knockdown in RASFs at mRNA and protein levels Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Page 8 of 11 Fig. 6 HBP1 silence reverses the function of miR-203 on RASF growth and inflammation. RASFs were transfected with in-miR-NC, in-miR-203, in-miR-203 + si-NC, or in-miR-203 + si-HBP1. a, b Transfection efficiency was analyzed by using qRT-PCR or western blot. c, d The proliferation of RASFs was determined by colony formation assay and CCK-8 assay, respectively. e Flow cytometry was applied to detect the apoptosis of RASFs. f Western bolt analysis of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was carried out. g–j The level of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was detected by ELISA assay. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 (ANOVA) Fig. 6 HBP1 silence reverses the function of miR-203 on RASF growth and inflammation. RASFs were transfected with in-miR-NC, in-miR-203, in-miR-203 + si-NC, or in-miR-203 + si-HBP1. a, b Transfection efficiency was analyzed by using qRT-PCR or western blot. c, d The proliferation of RASFs was determined by colony formation assay and CCK-8 assay, respectively. e Flow cytometry was applied to detect the apoptosis of RASFs. f Western bolt analysis of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was carried out. g–j The level of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was detected by ELISA assay. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 (ANOVA) may be a promising therapeutic target to prevent RA progression. elevation combined with insufficient apoptosis may also induce the spread of RASFs, giving rise to the bone destruction [22]. Importantly, RASF proliferation also results in the immunoreaction, and ultimately contrib- utes to the joint damage [23]. RA patients always highly express inflammatory cytokines, such as IL-6, IL-1β, IL-8 and TNF-α, which activate STAT3 either directly or indi- rectly, and in turn boosts the expression of these inflam- matory cytokines in an autocrine manner in RASFs. HBP1 silence reverses the function of miR‑203 on RASF growth and inflammation Besides, STAT3 activation also stimulates the expression of receptor activator of nuclear factor kappa B ligand (RANKL), which is necessary for osteoclastogenesis; all these changes further promoted chronic inflamma- tion and joint destruction [24, 25]. Thus, circ_0000396 It is well believed that circRNAs act as miRNA sponges to regulate gene expression [17]. MiRNAs are small non- protein coding RNAs, and have been classified to play a multifunctional role in the pathogenesis of RA. For example, miR-20a inhibited proliferation and induced apoptosis of RASFs by down-regulating the expression of STAT3 [26]. MiR-338-5p contributed to the promotion of proliferation, invasiveness and the release of pro-inflam- matory cytokines in RASFs by mediating the inhibition of SPRY1 [27]. MiR-650 interacted with AKT2 to suppress the biological behaviors of RASFs to impede the progres- sion of RA [28]. In our study, we confirmed circ_0000396 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Page 9 of 11 Fig. 7 Circ_0000396 affects the growth and inflammation of RASFs through modulating HBP1. RASFs were transfected with pcDNA, circ_0000396, circ_0000396 + si-NC, or circ_0000396 + si-HBP1. a, b Transfection efficiency was analyzed by using qRT-PCR or western blot. c, d The proliferation of RASFs was determined using colony formation assay and CCK-8 assay. e The apoptosis rate of RASFs was detected using flow cytometry. f The expression of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was detected using western blot. g–j Levels of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was detected by ELISA assay. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 (ANOVA) Fig. 7 Circ_0000396 affects the growth and inflammation of RASFs through modulating HBP1. RASFs were transfected with pcDNA, circ_0000396, circ_0000396 + si-NC, or circ_0000396 + si-HBP1. a, b Transfection efficiency was analyzed by using qRT-PCR or western blot. c, d The proliferation of RASFs was determined using colony formation assay and CCK-8 assay. e The apoptosis rate of RASFs was detected using flow cytometry. f The expression of Ki67, cleaved caspase3 and cleaved PARP expression in RASFs was detected using western blot. g–j Levels of inflammatory cytokines IL-6, IL-1β, IL-8 and TNF-α in RASFs was detected by ELISA assay. p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001 (ANOVA) directly targeted miR-203. MiR-203 was increased in RA synovial tissues and RASFs, and inhibition of miR- 203 suppressed cell growth and inflammation in RASFs. References 1. González-Chica DA, Vanlint S, Hoon E, Stocks N. Epidemiology of arthritis, chronic back pain, gout, osteoporosis, spondyloarthropathies and rheu- matoid arthritis among 1.5 million patients in Australian general practice: NPS MedicineWise MedicineInsight dataset. BMC Musculoskelet Disord. 2018;19:20. Competing interests h h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 14 August 2020 Accepted: 21 December 2020 Received: 14 August 2020 Accepted: 21 December 2020 Received: 14 August 2020 Accepted: 21 December 2020 Funding g This study was supported by grant from the Henan Province Medical Science and Technology Project Joint Development Project (no. 2018020329) and Henan Province Traditional Chinese Medicine Scientific Research Project (no. 2017ZY3044). Consent for publication Not applicable. expression through binding to miR-203, and knockdown of HBP1 reversed the protective effects of circ_0000396 on RASF dysfunction. In all, these results suggest a promising therapeutic target for the molecular therapy of RA patients. However, the function of circ_0000396 in healthy cell lines should be detected before the applica- tion of circ_0000396 to clinical use, in order to ensure the safety and efficiency. Additionally, the delivery methods of RNA remain the challenges for clinical application. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Fig. 8 Schematic model showing the role of circ_0000396 in the growth and inflammation of RASFs. Circ_0000396 could suppress RASF growth and inflammation by up-regulating HBP1 through absorbing miR-203 Fig. 8 Schematic model showing the role of circ_0000396 in the growth and inflammation of RASFs. Circ_0000396 could suppress RASF growth and inflammation by up-regulating HBP1 through absorbing miR-203 Ethics approval and consent to participate The study was approved by the Ethics Committee of the Huaihe Hospital of Henan University and was carried out according to the guidelines of Declara- tion of Helsinki. All participants had provided written informed consent before the study. Authors’ contributions QZ designed and supervised the study. LW conducted the experiments and drafted the manuscript. NW conducted the experiments and supervised the study. YD collected and analyzed the data. LK and JW contributed the methodology and analyzed the data. All authors read and approved the final manuscript. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s40709-020-00131-4. The online version contains supplementary material available at https://doi. org/10.1186/s40709-020-00131-4. Additional file 1: Fig. S1. Identification of RASFs and NSFs by flow cytom- etry. The purity of the third generation of RASFs (A) and NSFs (B) of the primary culture reached 98.0% and 99.0%, respectively. Additional file 1: Fig. S1. Identification of RASFs and NSFs by flow cytom- etry. The purity of the third generation of RASFs (A) and NSFs (B) of the primary culture reached 98.0% and 99.0%, respectively. HBP1 silence reverses the function of miR‑203 on RASF growth and inflammation Besides, miR-203 overexpression reversed the protective effects of circ_0000396 on RASFs dysfunction. of p53-dependent apoptosis, to reinforce the stability of p53 by repressing p53 ubiquitination induced by Mdm2 [30]. Chen et al. [31] revealed that HBP1 decreased the expression of macrophage migration inhibitory fac- tor (MIF), an important pro-inflammatory chemokine, to hinder the release of inflammatory cytokines TNF-α and IL-6. Consistently in the current study, HBP1 was decreased in RA synovial tissues and RASFs, and HBP1 knockdown attenuated miR-203 inhibition-mediated dysfunction and inflammation in RASFs. In addition, we also observed circ_0000396 indirectly regulated HBP1 f We also confirmed miR-203 directly bound to HBP1. HBP1 is a ubiquitous transcription factor, and plays significant roles in senescence, apoptosis, differentia- tion termination, and tumor suppression in various cell types [29]. HBP1 can accelerate cell death through up- regulating the expression of p21, an imperative mediator Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 Wang et al. J of Biol Res-Thessaloniki Page 10 of 11 Fig. 8 Schematic model showing the role of circ_0000396 in the growth and inflammation of RASFs. Circ_0000396 could suppress RASF growth and inflammation by up-regulating HBP1 through absorbing miR-203 Supplementary Information ; 2. Gibofsky A. Epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis: a synopsis. Am J Manag Care. 2014;20:S128–35. 3. Liu L, Zuo Y, Xu Y, Zhang Z, Li Y, Pang J. MiR-613 inhibits proliferation and invasion and induces apoptosis of rheumatoid arthritis synovial fibro- blasts by direct down-regulation of DKK1. Cell Mol Biol Lett. 2019;24:8. 4. Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T. Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology. 2006;45:669–75. 5. Ye Y, Gao X, Yang N. LncRNA ZFAS1 promotes cell migration and invasion of fibroblast-like synoviocytes by suppression of miR-27a in rheumatoid arthritis. Hum Cell. 2018;31:14–21. 6. Shang C-h, Zhang Q-q, Zhou J-h. Oridonin inhibits cell proliferation and induces apoptosis in rheumatoid arthritis fibroblast-like synovio- cytes. Inflammation. 2016;39:873–80. 7. Noss EH, Brenner MB. The role and therapeutic implications of fibroblast-like synoviocytes in inflammation and cartilage erosion in rheumatoid arthritis. Immunol Rev. 2008;223:252–70. Conclusion 2. Gibofsky A. Epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis: a synopsis. Am J Manag Care. 2014;20:S128–35. In conclusion, our findings demonstrated circ_0000396 could suppress RASF growth and inflammation by up- regulating HBP1 through miR-203 (Fig. 8), which sug- gested a novel idea into understanding the pathogenesis of RA process. Nevertheless, there are still some limita- tions. The data presented are based on a limited num- ber of cells in vitro, in vivo assay and a larger cohort of the disease analysis are necessary for the verification of these conclusions in the future. Moreover, the potent, long-lasting, and safe immune responses of circ_0000396 in animal models should be investigated before clinical application. 3. Liu L, Zuo Y, Xu Y, Zhang Z, Li Y, Pang J. MiR-613 inhibits proliferation and invasion and induces apoptosis of rheumatoid arthritis synovial fibro- blasts by direct down-regulation of DKK1. Cell Mol Biol Lett. 2019;24:8. 4. Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T. Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology. 2006;45:669–75. y y 5. Ye Y, Gao X, Yang N. LncRNA ZFAS1 promotes cell migration and invasion of fibroblast-like synoviocytes by suppression of miR-27a in rheumatoid arthritis. Hum Cell. 2018;31:14–21. 6. Shang C-h, Zhang Q-q, Zhou J-h. Oridonin inhibits cell proliferation and induces apoptosis in rheumatoid arthritis fibroblast-like synovio- cytes. Inflammation. 2016;39:873–80. l 7. Noss EH, Brenner MB. The role and therapeutic implications of fibroblast-like synoviocytes in inflammation and cartilage erosion in rheumatoid arthritis. Immunol Rev. 2008;223:252–70. Page 11 of 11 Wang et al. J of Biol Res-Thessaloniki (2021) 28:1 8. Qu S, Yang X, Li X, Wang J, Gao Y, Shang R, et al. Circular RNA: a new star of noncoding RNAs. Cancer Lett. 2015;365:141–8. 22. Pope RM. Apoptosis as a therapeutic tool in rheumatoid arthritis. Nat Rev Immunol. 2002;2:527–35. 22. Pope RM. Apoptosis as a therapeutic tool in rheumatoid arthritis. Nat Rev Immunol. 2002;2:527–35. 23. Kaneko S, Kondo Y, Yokosawa M. Sumida T [Rheumatoid arthritis and cytokines]. Nihon Rinsho. 2016;74:913–8. 9. Altesha MA, Ni T, Khan A, Liu K, Zheng X. Circular RNA in cardiovascular disease. J Cell Physiol. 2019;234:5588–600. 10. Du J, Zhang G, Qiu H, Yu H, Yuan W. The novel circular RNA circ- CAMK2A enhances lung adenocarcinoma metastasis by regulating the miR-615-5p/fibronectin 1 pathway. Cell Mol Biol Lett. 2019;24:72. 24. Mori T, Miyamoto T, Yoshida H, Asakawa M, Kawasumi M, Kobayashi T, et al. Conclusion Mol Cell Biol. 2013;33:887–903. 29. Pan K, Chen Y, Roth M, Wang W, Wang S, Yee AS, et al. HBP1-mediated transcriptional regulation of DNA methyltransferase 1 and its impact on cell senescence. Mol Cell Biol. 2013;33:887–903. 16. Yang X, Li J, Wu Y, Ni B, Zhang B. Aberrant dysregulated circular RNAs in the peripheral blood mononuclear cells of patients with rheumatoid arthritis revealed by RNA sequencing: novel diagnostic markers for RA. Scand J Clin Lab Invest. 2019;79:551–9. 30. Ren B, Liu J, Wu K, Zhang J, Lv Y, Wang S, et al. TNF-alpha-elicited miR-29b potentiates resistance to apoptosis in peripheral blood monocytes from patients with rheumatoid arthritis. Apoptosis. 2019;24:892–904. 31. Chen H, Li X, Liu S, Gu L, Zhou X. MircroRNA-19a promotes vascular inflammation and foam cell formation by targeting HBP-1 in atherogen- esis. Sci Rep. 2017;7:12089. 17. Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard CK, et al. Natural RNA circles function as efficient microRNA sponges. Nature. 2013;495:384–8. 18. Zhou Z, Sun B, Huang S, Zhao L. Roles of circular RNAs in immune regula- tion and autoimmune diseases. Cell Death Dis. 2019;10:503. Conclusion IL-1β and TNFα-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis. Int Immunol. 2011;23:701–12. i y 11. Li LJ, Zhu ZW, Zhao W, Tao SS, Li BZ, Xu SZ, et al. Circular RNA expres- sion profile and potential function of hsa_circ_0045272 in systemic lupus erythematosus. Immunology. 2018;155:137–49. 25. Yoshida GJ, Azuma A, Miura Y, Orimo A. Activated fibroblast program orchestrates tumor initiation and progression; molecular mechanisms and the associated therapeutic strategies. Int J Mol Sci. 2019;20:2256. y y 12. Zheng F, Yu X, Huang J, Dai Y. Circular RNA expression profiles of peripheral blood mononuclear cells in rheumatoid arthritis patients, based on microarray chip technology. Mol Med Rep. 2017;16:8029–36. 26. Wei XJ, Li XW, Lu JL, Long ZX, Liang JQ, Wei SB, et al. MiR-20a regulates fibroblast-like synoviocyte proliferation and apoptosis in rheumatoid arthritis. Eur Rev Med Pharmacol Sci. 2020;24:7578. y y 13. Luo Q, Zhang L, Li X, Fu B, Deng Z, Qing C, et al. Identification of circu- lar RNAs hsa_circ_0044235 in peripheral blood as novel biomarkers for rheumatoid arthritis. Clin Exp Immunol. 2018;194:118–24. 27. Yang Y, Wang Y, Liang Q, Yao L, Gu S, Bai X. MiR-338-5p promotes inflam- matory response of fibroblast-like synoviocytes in rheumatoid arthritis via targeting SPRY1. J Cell Biochem. 2017;118:2295–301. 14. Li G, Tan W, Fang Y, Wu X, Zhou W, Zhang C, et al. circFADS2 protects LPS-treated chondrocytes from apoptosis acting as an interceptor of miR-498/mTOR cross-talking. Aging (Albany NY). 2019;11:3348–61. 14. Li G, Tan W, Fang Y, Wu X, Zhou W, Zhang C, et al. circFADS2 protects LPS-treated chondrocytes from apoptosis acting as an interceptor of miR-498/mTOR cross-talking. Aging (Albany NY). 2019;11:3348–61. 28. Xu X, Chen H, Zhang Q, Xu J, Shi Q, Wang M. MiR-650 inhibits prolifera- tion, migration and invasion of rheumatoid arthritis synovial fibroblasts by targeting AKT2. Biomed Pharmacother. 2017;88:535–41. 28. Xu X, Chen H, Zhang Q, Xu J, Shi Q, Wang M. MiR-650 inhibits prolifera- tion, migration and invasion of rheumatoid arthritis synovial fibroblasts by targeting AKT2. Biomed Pharmacother. 2017;88:535–41. 15. Li B, Li N, Zhang L, Li K, Xie Y, Xue M, et al. Hsa_circ_0001859 Regulates ATF2 Expression by Functioning as an MiR-204/211 Sponge in Human Rheumatoid Arthritis. J Immunol Res. 2018;2018:9412387. 29. Pan K, Chen Y, Roth M, Wang W, Wang S, Yee AS, et al. HBP1-mediated transcriptional regulation of DNA methyltransferase 1 and its impact on cell senescence. Publisher’s Note 19. Xing R, Jin Y, Sun L, Yang L, Li C, Li Z, et al. Interleukin-21 induces migra- tion and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis. Clin Exp Immunol. 2016;184:147–58. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 20. Juarez M, Filer A, Buckley CD. Fibroblasts as therapeutic targets in rheu- matoid arthritis and cancer. Swiss Med Wkly. 2012;142:w13529. 21. Baltimore D, Boldin MP, O’Connell RM, Rao DS, Taganov KD. MicroRNAs: new regulators of immune cell development and function. 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https://openalex.org/W2152359555	https://europepmc.org/articles/pmc544568?pdf=render	English	null	Small area mapping of prostate cancer incidence in New York State (USA) using fully Bayesian hierarchical modelling.	International journal of health geographics	2,004	cc-by	7,517	Small area mapping of prostate cancer incidence in New York State (USA) using fully Bayesian hierarchical modelling l h 1 2 Address: 1New York State Cancer Registry, New York State Department of Health, Albany, NY, USA and 2Department of Environmental Health and Toxicology, School of Public Health, University at Albany, Albany, NY, USA Address: 1New York State Cancer Registry, New York State Department of Health, Albany, NY, USA and 2Departmen Toxicology, School of Public Health, University at Albany, Albany, NY, USA Email: Glen D Johnson* - gdj01@health.state.ny.us * Corresponding author * Corresponding author Received: 01 September 2004 Accepted: 08 December 2004 Received: 01 September 2004 Accepted: 08 December 2004 Published: 08 December 2004 International Journal of Health Geographics 2004, 3:29 doi:10.1186/1476-072X-3-29 International Journal of Health Geographics 2004, 3:29 doi:10.1186/1476-072X-3-29 This article is available from: http://www.ij-healthgeographics.com/content/3/1/29 s article is available from: http://www.ij-healthgeographics.com © 2004 Johnson; licensee BioMed Central Ltd. J ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. his is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecom hich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Health Geographics BioMed Central Open Access Page 1 of 12 (page number not for citation purposes) Abstract Background: As part of a long-term initiative to improve cancer surveillance in New York State, small area maps of relative risk, expressed as standardized incidence ratios (SIRs), were produced for the most common cancers. This includes prostate cancer, the focus of this paper, since it is the most common non-dermatologic malignancy diagnosed among men and the second leading cause of cancer deaths for men in the United States. ZIP codes were chosen as mapping units for several reasons, including the need to balance between protecting personal privacy and public demand for fine geographic resolution. Since the population size varies greatly among such small mapping units, hierarchical Bayes spatial modelling was applied in this paper to produce a map of smoothed SIRs. It is further demonstrated how other characteristics of the large sample from the stationary posterior distribution of SIRs can be mapped to investigate various aspects of the statewide spatial pattern of prostate cancer incidence. Results: Thematic mapping of the median and 95 percentile range of SIRs provided, respectively, a map of spatially smoothed values and the uncertainty associated with these smoothed values. Maps were also produced to identify ZIP codes expressing a 95% probability, in the Bayesian paradigm, of being less than or greater than the null value of 1. Conclusion: The model behaved as expected since areas that were statistically elevated coincided with areas identified by the spatial scan statistic, plus the relative uncertainty increased as a ZIP code's population decreased, with an exaggerated effect for low population ZIP codes on the edge of the state border. The overall smoothed pattern, along with identified high and low areas, may reflect difference across the state with respect to socio-demographics and risk factors; however, this is confounded by potential differences in screening and diagnostic follow-up. Nevertheless, the Bayes modelling approach is shown to provide not only smoothed results, but also considerable other information from a large empirical distribution of outcomes associated with each mapping unit. understanding spatial or spatial-temporal patterns that may help to identify discrepancies in disease burden Background understanding spatial or spatial-temporal patterns that may help to identify discrepancies in disease burden g Geographic surveillance of chronic disease is central to Page 1 of 12 (page number not for citation purposes) Page 1 of 12 (page number not for citation purposes) http://www.ij-healthgeographics.com/content/3/1/29 g p y g All stage prostate cancer Incidence by ZIP Code in New York State, 1994–1998. ZIP code-level ratios of observed incidence to age- and race-adjusted expected incidence, along with significant spatial scan statistic circles that are non-overlap- ping within specified ranges of standardized incidence ratios, based on reference [1]. Select cities and regions overlaid for reference. http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 crepancies in mortality rates do exist [4]. Furthermore, several treatment options appear to be associated with excellent long-term disease-specific survival for otherwise healthy men with localized disease [5]. among different regions or communities. As part of ongo- ing efforts in New York State to understand spatial pat- terns of cancer and to help implement cancer prevention and control programs, small area maps of cancer relative risk, expressed as standardized incidence ratios (SIRs), have been produced and shared with the public [1] for the most common anatomical cancer sites. Results for prostate cancer (all stages combined) are reproduced in Figure 1, where ZIP code-level standardized incidence ratios (SIRs) are presented along with results from analyzing these data with the spatial scan statistic [6]. The circles in Figure 1 represent statistically elevated regions based on Poisson likelihood ratios comparing rates inside the circle to those outside the circle. Details of how the scan statistic results were reduced to the circles presented in Figure 1 are found in Boscoe et al [7]. Prostate cancer, the focus of this paper, was included because it is the most common non-dermatologic malig- nancy diagnosed among men and the second leading cause of cancer deaths for men in the United States (US) [2]. Although mortality from this disease in the US has statistically significantly decreased at a rate of 2.6% per year from 1990 to 2000 [3], unexplained geographic dis- All stage p Figure 1 All stage prostate cancer Incidence by ZIP Code in New York State, 1994 1998 Figure 1 All stage prostate cancer Incidence by ZIP Code in New York State, 1994–1998. ZIP code-level ratios of observed incidence to age- and race-adjusted expected incidence, along with significant spatial scan statistic circles that are non-overlap- ping within specified ranges of standardized incidence ratios, based on reference [1]. Select cities and regions overlaid for reference g p y g All stage prostate cancer Incidence by ZIP Code in New York State, 1994–1998. ZIP code-level ratios of observed incidence to age- and race-adjusted expected incidence, along with significant spatial scan statistic circles that are non-overlap- ping within specified ranges of standardized incidence ratios, based on reference [1]. Select cities and regions overlaid for reference. Page 2 of 12 (page number not for citation purposes) http://www.ij-healthgeographics.com/content/3/1/29 http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 porates both a heterogeneous and spatially structured random effect [18], thus allowing the most flexibility in model development. Several reviews consistently support the fully Bayesian approach over empirical Bayes model- ling [16,17,19]. It is well recognized that the stability of population-based statistics like the SIRs in Figure 1 can vary greatly among small geographic areas due to varying population size. Different methods of smoothing have been developed to address this issue, where all are based on the phenome- non that observations close together in space are more likely to share similar properties than those that are far apart [8]. While this positive spatial autocorrelation may be problematic for statistical methods that require inde- pendent observations, it can also be embraced to help smooth noisy maps by borrowing strength from neigh- bors for those mapping units with small populations. Of the different approaches to spatial smoothing, only a few appear to have gained acceptance in spatial epidemiology. Non-parametric approaches include spatial filtering [9,10] and the head-banging algorithm [11], both of which are basically variations on a moving window ker- nel-type smoother. Since there are no closed form analytical solutions for parameter estimates of a fully Bayesian model, nor likeli- hood profiles to maximize, Markov Chain Monte Carlo (MCMC) methods are used to generate large samples from the posterior distributions of all stochastic nodes of the hierarchical model, given the likelihood describing the original data distribution and all appropriate prior and hyperprior distributions of the likelihood parameters [16]. All stage p Figure 1 Estimation and inference in the fully Bayesian para- digm are based upon these large sample approximations of the posterior distributions. In what follows, the fully Bayesian approach is applied to simulating large samples from the posterior distribution of prostate cancer relative risk in each of 1412 ZIP codes in New York State. Various aspects of these distributions are then mapped to reveal information on the geographic patterns of prostate cancer. The parametric approach of generalized linear modelling [12] treats the observed response, y, as a random variable that has arisen from a probability distribution with expec- tation θ. This expectation is modeled, via an appropriate link g(·), as a linear function g(θ) = α + x'β + ε, for a com- mon value α, explanatory covariates x'β and a random effect ε that captures unexplained variation. If the random effect is associated with exchangeable spatial heterogene- ity, estimates are smoothed towards a global mean, whereas if the random effect is associated with local spa- tial autocorrelation, estimates are smoothed towards a local neighborhood mean, which is typically more mean- ingful in geographic epidemiology. There are different approaches to modelling local spatial dependence, and section 6.3 of Cressie [13] presents several arguments in favor of the conditional autoregressive (CAR) model orig- inally conceived by Besag [14]. Page 3 of 12 (page number not for citation purposes) Results The model defined by equations 1–5 was applied to sim- ulate a sample of 1000 independent observations from the stationary posterior distribution of standardized inci- dence ratios for each ZIP code. Summary statistics and graphical analysis of these empirical distributions indi- cated that they arose from generally symmetric posterior distributions. Since the sample mean, median and mode were very similar for each ZIP code, the median was cho- sen to represent central tendency and is mapped in Figure 2. This "smoothed" map of SIRs provides a picture of spa- tial pattern inherent in the raw data mapped in Figure 1. Uncertainty associated with these estimates of relative risk is mapped in Figure 3 as the 95 percentile range (97.5th – 2.5th percentile) of the 1000 values sampled from the pos- terior distribution of SIRs for each ZIP code. Estimation of model parameters can proceed by maxi- mum likelihood [13]; however, the hierarchical nature of generalized linear models lends itself well to Bayesian analysis whereby linear terms in the model are assigned prior distributions that, in turn, have "hyperprior" param- eters. Earlier applications employed empirical Bayes methods [15], where hyperparameters are estimated directly from the data. This approach is limited because it assigns a point estimate to the hyperparameter without allowing for variability that may be associated with it, and this variability can be large [16,17]. The posterior distributions can also be used to identify ZIP codes where a specified mass of the distribution of rel- ative risk is greater or less than the null value. For exam- ple, Figure 4 shows ZIP codes where 95% of the simulated SIRs exceed the value of one. In the Bayesian paradigm, those ZIP codes highlighted in Figure 4 have a 95% prob- ability of higher than expected risk. Likewise, Figure 5 highlights ZIP codes expressing a 95% probability of lower than expected risk. Fully Bayesian modelling assigns hyperprior distributions to these hyperparameters, so that every parameter of the hierarchical model is allowed to vary over a prior distribu- tion and no single point estimate is used to represent an unknown parameter value. Furthermore, the fully Baye- sian approach allows the convolution model that incor- Page 3 of 12 (page number not for citation purposes) International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 Bayesian smoothed prostate cancer incidence Figure 2 Bayesian smoothed prostate cancer incidence. Results Median of the posterior distribution of ZIP code-level standardized inc ence ratios. Thematic categories based on natural breaks method, with slight adjustment. Bayesian smoothed prostate cancer incidence Figure 2 Bayesian smoothed prostate cancer incidence. Median of the posterior distribution of ZIP code-level standardized inci- dence ratios. Thematic categories based on natural breaks method, with slight adjustment. y p g Bayesian smoothed prostate cancer incidence. Median of the posterior distribution of ZIP code-level standardized inci- dence ratios. Thematic categories based on natural breaks method, with slight adjustment. Page 4 of 12 (page number not for citation purposes) Discussion Methodology Most applications to date use the first order binary weight- ing scheme where wij = 1 if a mapping unit j shares a com- mon border with unit i, and wij = 0 otherwise. This weighting scheme actually has its roots in image analysis, for which this type of modelling was developed [14], and it makes sense when the spatial units are equal size and shape pixels and the response variable has a constant var- iance for each pixel. However, this is not the case when smoothing disease maps where the mapping units are of irregular size and shape, and stability of the response var- iable estimates varies with changing population size. This problem is especially relevant for mapping units like ZIP codes. The Poisson model applied in this paper is a particular application of hierarchical Bayes spatial generalized linear models for the exponential family of likelihoods [20]. Particular models are specified by the likelihood that is assumed to give rise to the observations, the structure of the prior, and the hyperprior distributions of variance components, which are typically vague to allow learning from the data. An aspect of these models that will influ- ence outcomes is the neighborhood weights, as in Equa- tion (4); however, defining these weights remains an open area of research. Page 4 of 12 (page number not for citation purposes) Page 4 of 12 (page number not for citation purposes) International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 Uncertainty of Bayesian Smoothed prostate cancer incidence Figure 3 Uncertainty of Bayesian Smoothed prostate cancer incidence. ZIP code-level 95th percentile range of posterior distri- bution of standardized incidence ratios. Thematic categories based on Natural Breaks Method. Uncertainty of Bayesian Smoothed prostate cancer incidence Figure 3 Uncertainty of Bayesian Smoothed prostate cancer incidence. ZIP code-level 95th percentile range of posterior distri- bution of standardized incidence ratios. Thematic categories based on Natural Breaks Method. A proper approach may be to define weights as a decay function of geographic distance between population- weighted centroids of the mapping units. This function may be obtained by fitting a model correlogram to resid- uals that are obtained from a model that does not include a random effect to account for spatial autocorrelation. Cressie and Chan [21] used the empirical variogram of the response variable to determine the range of spatial auto- correlation. Discussion Methodology For neighborhood distances within this range, weights were defined as a function of Euclidean distance. Meanwhile, Griffith [22] provides some "rules of thumb" for defining geographic weights, but they are very general. Ferrandiz, et al [23] used a weight of ninj/dij for neighboring mapping units separated by geographic dis- tance dij and of population sizes ni and nj. These authors applied such a weight to prostate cancer mortality map- ping; however, this gravity-type weighting may be better suited for infectious disease, not chronic disease. If a decay function is fit from the data, the varying stability of disease rates among the mapping units presents a chal- lenge. Since the Bayesian model is designed to adjust for varying stability, perhaps a hierarchical model like the one applied in this paper can be extended so that the Page 5 of 12 (page number not for citation purposes) Page 5 of 12 (page number not for citation purposes) International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 ZIP codes with a 95% probability of relative risk exceeding 1 Figure 4 ZIP codes with a 95% probability of relative risk exceeding 1. The lower 5th percentile of the posterior distribution of standardized incidence ratios exceeds or equals 1. ZIP codes with a 95% probability of relative risk exceeding 1 Figure 4 ZIP codes with a 95% probability of relative risk exceeding 1. The lower 5th percentile of the posterior distribution of standardized incidence ratios exceeds or equals 1. ZIP codes with a 95% probability of relative risk exceeding 1 Figure 4 ZIP codes with a 95% probability of relative risk exceeding 1. The lower 5th percentile of the posterior distribution of standardized incidence ratios exceeds or equals 1. weights in Equation (4) are defined as a decay function whose unknown parameters can be assigned "hyperprior" distributions. spatial pattern that is inherent in the data of Figure 1. It is demonstrated how hierarchical Bayes spatial modelling has the appealing feature of providing a whole distribution of possible outcomes that can be used for not only smoothing, but also to explore other aspects of spa- tial pattern. Application to prostate cancer mapping Maps like in Figure 1 present a compromise between the need to protect personal privacy and public demand for fine geographic resolution. Such small mapping units are necessary for discerning among communities that can vary drastically across a region with respect to possible risk factors and both population density and demographics. However, this comes with the cost of unstable risk esti- mates for many mapping units that have small popula- tions. Smoothing is therefore applied to help visualize Viewing Figures 1 through 3 indicate that the Bayesian model is behaving as expected since the smoothed esti- mates are increasingly dependent on the prior model as uncertainty increases due to decreasing population, whereas for ZIP codes with large populations, like in New York City, the smoothed estimates are similar to the raw SIRs. Page 6 of 12 (page number not for citation purposes) International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 ZIP codes with a 95% probability of relative risk being less than 1 Figure 5 ZIP codes with a 95% probability of relative risk being less than 1. The upper 95th percentile of the posterior distribu ion of standardized incidence ratios is less than or equal to 1. ZIP codes with a 95% probability of relative risk being less than 1 Figure 5 ZIP codes with a 95% probability of relative risk being less than 1. The upper 95th percentile of the posterior distribu- tion of standardized incidence ratios is less than or equal to 1. influenced by many factors, including differences between regions of the state in terms of racial, ethnic and socio-demographic composition. Yet it is well recognized that interpreting any possible relationships with risk factors is confounded by differences in screening and diagnostic practices across the state. Prostate-specific anti- gen (PSA) testing remains high among US males over 40 [25] and there is evidence of a steady increase in testing rates in New York State during the years corresponding to the data analyzed in this paper [26]. Along these lines, we note that a relatively large proportion of the four most populated counties of New York City reveal a high proba- bility of less than expected incidence (see Figure 5 inset). Page 7 of 12 (page number not for citation purposes) http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 extreme values associated with small populations may simply reflect random noise. population in these four counties, as indicated by much higher proportions of people who are foreign-born and/ or do not speak English at home [27], which may translate to lower screening rates. Other methods like the spatial scan statistic can be used in conjunction with Bayes smoothing to strengthen overall spatial analysis. Statistically significant scan statistic cir- cles like those in Figure 1 can vary in size, potentially encompassing many ZIP codes, so are not restricted to only pre-defined neighborhoods like the conditional autoregressive model used by the Bayes smoothing algo- rithm. In this regard, the spatial scan statistic is similar to smoothing by spatial filtering with variable-radius circles [10]. General regions of statistically elevated relative risk may be identified by the scan statistic, with supporting evidence from Bayesian posterior distributions to help identify the mapping units that contribute strongly to a scan statistic circle. Indeed, each spatial scan statistic circle reported in Figure 1 contains at least one elevated ZIP code identified in Figure 4 by the Bayesian model. Although the patterns seen in Figures 2, 4 and 5 may be partially explained by geographic variations in PSA testing and diagnostic follow up, such variation is not actually known, therefore we cannot adjust for this potential con- founder. In the neighboring state of Connecticut, geo- graphic variation of invasive prostate cancer incidence was large and revealed some consistency before and after the introduction of PSA testing, while the pattern was com- pletely different and variation was much smaller during the years of PSA introduction [28]. These authors suggest that such a space-time pattern reflects the impact of intro- ducing PSA testing, although this cannot be confirmed in the absence of data on geographic differences in PSA use. Some areas appear elevated that are in popular vacation spots, such as the eastern forks of Long Island and the "north country" of New York State including the Thou- sand Islands area along the Saint Lawrence River (near Watertown in Figure 1) and the Adirondack region. This may possibly be due to a seasonal residence effect whereby vacation areas tend to have artificially inflated chronic disease rates [29]. http://www.ij-healthgeographics.com/content/3/1/29 This occurs when seasonal resi- dents provide a health care provider with a local address of a vacation home, while their primary residence is where they are counted by the decennial US census. Conse- quently, if their residence at time of diagnoses is in the vacation area, this record inflates the SIR numerator for that area, while they are counted in the denominator for the area of their primary residence as captured by the cen- sus. This effect is enhanced since the population spending extended periods in vacation areas tends to be over age 55, which is the age cohort at highest risk of chronic disease. Boscoe and Mclaughlin [29] have presented evidence of increased overall cancer rates in areas with seasonally res- ident populations in New York State, especially the Thou- sand Islands area. This uncertainty is reflected in Figure 3 where the width of the posterior distribution of SIRs for these areas is relatively large. There is ample flexibility for exploratory analysis by vary- ing the display parameters for results from these two methods. For example, the results in Figure 4 can be either more generalized or further focused by identifying ZIP codes where, say, 90% or 99%, respectively, of the poste- rior probability mass exceeds the null value of one. At the same time, we can display the set of non-overlapping scan statistic circles that correspond to lower levels of relative risk than are shown in Figure 1, thus capturing more geo- graphic area. In fact, when this is done for scan statistic cir- cles corresponding to 15–49% relative risk (not shown), all of the elevated ZIP codes in Figure 4 are spatially asso- ciated with significant scan statistic circles. There is extensive literature on hierarchical Bayes spatial modelling for disease mapping; however, most papers are theoretical in nature and use illustrative examples, often with the same data sets. One exception was recently pub- lished by Short et al [31], who applied Bayes modelling to produce maps of cancer control variables. Specifically, they smoothed maps of different outcomes (mortality, incidence, staging and screening) for each of breast, color- ectal and lung cancer in Minnesota (USA) counties. Can- cer control maps were created for each cancer site by obtaining a weighted sum of each smoothed outcome, and an overall cancer control map was obtained by a weighted sum of the individual cancer control map val- ues. Application to prostate cancer mapping This may be partially explained by the large immigrant We also see that many edge ZIP codes in less populated areas tend to have greater uncertainty relative to their non- edge neighbors because there are fewer neighbors to bor- row strength from. For the heterogeneous Poisson model applied in this paper, Lawson et al [24] suggest treating edge mapping units as a guard and not as part of the actual study area. However, presenting the width of Bayesian posterior distributions provides a way to retain the edge units while also showing the relative uncertainty associ- ated with their smoothed values. The smoothed pattern in Figure 2 is highlighted for areas of high and low incidence in Figures 4 and 5, respectively. Geographic patterns seen in these maps are potentially Page 7 of 12 (page number not for citation purposes) Page 7 of 12 (page number not for citation purposes) Methods D Level 2, link to a linear function: log(θi) = α + ui + vi (2) Level 2, link to a linear function: log(θi) = α + ui + vi (2) (2) Observed and expected values of prostate cancer inci- dence used to calculate SIRs for 1412 New York State ZIP codes [1] were obtained for the years 1994 to 1998 from the New York State Cancer Registry (NYSCR). The expected values used in the SIR denominator are based on indirect standardization using the age-by-race distribution in each ZIP code and the statewide age- and race-specific incidence rates as a reference. Age and race distributions correspond to the year 1997, as estimated by the Claritas Corporation™ based on prior census values. ZIP code boundaries were delineated by the GDT Corporation™ in 1999. Level 3, assign prior distributions: α ~ N(0,0.0001), not- ing that 0.0001 is the precision, thus defining a vague prior, u v v v i u i i v i i ~N and N ( , ) [ \| ] ~ ( , ) ( ) 0 3 2 2 σ σ δ ( ) 3 where and v w v w w i ij j j ij j v v ij j i i i i = = ∈ ∈ ∈ ∑ ∑ ∑ δ δ δ σ σ 2 2 4 ( ) 4 ( ) ZIP Code delivery areas are prone to change over time [32], particularly in rapidly growing parts of the country. According to the NYSCR [personal communication], a review of all of the issues of the Postal Bulletin, where these changes are documented, from 1990 to the present revealed that New York has had stable ZIP Code delivery areas. Approximately 50 small, rural post offices were closed, 3 new post offices were added, and none were rea- ligned. ZIP codes were combined in instances where serv- ice delivery area changed between 1990 and 1999 or for confidentiality reasons where necessary [33]. for a neighborhood of geographic units δi with respect to unit i and wij is a weight defining the relationship between geographic unit i and its neighbor j. The weight is defined simply as wij = 1 if ZIP codes i and j are adjacent (share a common border) and wij = 0 otherwise. Modelling i h for shape parameters a and c, and inverse scale parameters b and d. Letting the geographic domain (New York State) be sub- divided into i = 1, ..., n distinct mapping units (n = 1412 ZIP codes for our application), the number of cases within each unit, Yi, conditional on location i, is defined as a Poisson random variable with expectation Eiθi, where Ei equals the age- and race-adjusted expected number of prostate cancer cases, and θi equals the area-specific rela- tive risk. Given an observed response yi, note that the max- imum likelihood estimate of relative risk is = yi / Ei, the standardized incidence ratio (SIR). ˆθi This is the convolution model originally proposed by Besag, York and Mollie [18], where the random effect associated with spatial autocorrelation, vi, is defined according to the conditional auto-regressive model (CAR) [14]. Note that the distribution of vi is conditional on geo- graphic location, whereby its expectation equals a local neighborhood average. The Bayesian model puts increas- ing emphasis on this term as the underlying population at location i decreases. The relative risk parameter θi is assigned a log-normal Although covariates can be incorporated into the log-lin- ear expression at the second level of the model, our inter- est is with estimating and mapping the relative risk, θi = exp(α + ui + vi). prior distribution, log(θi) ~ N(µi, ), where the expecta- tion and variance are defined by a linear function of a common value (intercept), α, and two independent ran- dom effects, a heterogeneous component, ui, that does not depend on geographic location (exchangeable) and an autocorrelated component, vi, that reflects local spatial structure by incorporating the influence of neighboring geographic units. Prior distributions are then assigned to these linear terms and consequent hyperprior distribu- σi 2 http://www.ij-healthgeographics.com/content/3/1/29 These results can help guide resource allocation for state cancer prevention and control efforts. While the smoothed results in Figure 2 present an advan- tage over mapping raw data, a limitation of smoothing is that the pattern we decipher is subject to confounding by spatially varying population sizes [30]. In other words, smoothed maps like in Figure 2 reveal high and low rela- tive risk in areas with larger populations, while areas with small populations tend to be smoothed towards the null value. While this means that areas with small populations that actually have abnormally high or low disease rates may be obscured, it is still well recognized that many While there are open areas for improvement in the meth- odology of hierarchical Bayes spatial modelling, it is a val- uable tool for geo-spatial assessment of disease patterns that can help identify differences among communities. This may in turn indicate patterns of health care access, Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 tions are assigned to the variance terms, thus creating a 4- level hierarchical model as follows. screening and diagnostic follow up and possibly indicate etiologic clues about causal relationships. Level 1, define the likelihood: Yi ~ Poisson(Eiθi) (1) Level 1, define the likelihood: Yi ~ Poisson(Eiθi) (1) Methods D Level 4, assign hyperprior distributions to precision terms: Level 4, assign hyperprior distributions to precision terms: τu = 1 / ~ Gamma(a,b) and τv = 1 / ~ Gamma(c,d) (5) σu 2 σv 2 (5) Page 9 of 12 (page number not for citation purposes) Choosing Gamma Hyperpriors While it is established that a vague prior is acceptable for the linear term α in Equation (2) (i.e. Ghosh et al [20]), the model should be evaluated for sensitivity to choice of the Gamma hyperprior distributions of the precision terms, as in Equation (5). Two very different hyperprior specifications that appear in the literature for this convo- Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 http://www.ij-healthgeographics.com/content/3/1/29 http://www.ij-healthgeographics.com/content/3/1/29 precision estimates, while also defining a vague hyperp- rior that allows more learning from the data. precision estimates, while also defining a vague hyperp- rior that allows more learning from the data. lution model were experimented with. Hyperparameters were specified for one model as Γ(1,1), which yields a probability of 99% that the precision lays between 0.01 and 4.6, and for the other model as Γ(0.5,0.0005), which yields a probability of 99% that the precision lies between 0.16 and 6635, with most of the probability concentrated towards 0. Note that these parameter choices also satisfy sufficient conditions for ensuring a proper joint posterior distribution of all the stochastic nodes [20]. For the statewide collection of New York ZIP code log(SIR)s to be smoothed in this paper, the sample precision equals 6.25 (variance = 0.16) and the precision of first order neighbor- hood means equals 20 (variance = 0.05). Therefore, it may be desired to retain the model with hyperprior specifica- tion of Γ(0.5,0.0005) to at least capture the sample-based Final smoothed results from each model are compared in Figure 6 where we see, in agreement with Bernardinelli et al [34], that it essentially makes no difference which hyperprior is used. Therefore, the Γ(0.5,0.0005) hyperp- rior specification was chosen. It is not the intention of this paper to perform a rigorous sensitivity analysis with respect to hyperprior specification; however, the two models assessed in Figure 6 represent very different distri- butions and therefore indicate that the fully Bayes hierar- chical model is quite robust with respect to hyperprior specification when smoothed relative risks are the objective. tion of Γ(0.5,0.0005) to at least capture the sample based objective. Choosing Gamma Hyperpriors Comparison of smoothed standardized incidence ratios for two different specifications of the hyperprior distributions Figure 6 Comparison of smoothed standardized incidence ratios for two different specifications of the hyperprior distri butions. Each point represents a ZIP code with two medians of the posterior distribution of standardized incidence ratios obtained from different models. Comparison of smoothed standardized incidence ratios for two different specifications of the hyperprior distributions Figure 6 Comparison of smoothed standardized incidence ratios for two different specifications of the hyperprior distri- butions. Each point represents a ZIP code with two medians of the posterior distribution of standardized incidence ratios obtained from different models. Comparison of smoothed standardized incidence ratios for two different specifications of the hyperprior distributions Figure 6 Comparison of smoothed standardized incidence ratios for two different specifications of the hyperprior distri- butions. Each point represents a ZIP code with two medians of the posterior distribution of standardized incidence ratios obtained from different models. http://www.ij-healthgeographics.com/content/3/1/29 International Journal of Health Geographics 2004, 3:29 International Journal of Health Geographics 2004, 3:29 Table 1: Deviance analysis. See text for explanation. Model Mean (-2 LL) Mean Deviance Saturated 8082.0 Convolution 8194.0 112.0 CAR only 8191.0 109.0 Exchangeable only 10800.0 2718.0 Table 1: Deviance analysis. See text for explanation. Running the Gibbs Sampler g p WINBUGS 1.4 [35] was used for running three independ- ent Markov Chains. Initial values of all stochastic nodes of the model were chosen to provide dispersed initial values without being excessively overdispersed. For the common intercept, α, and heterogeneous random effect, ui, zero (0) was used to initiate one chain, plus/minus four standard deviations of the statewide log(SIR) were used to initiate the other respective chains. Zero is the statewide average log(SIR) that provides a point estimate for α, plus it is the expected value of ui. For the random effect associated with local spatial clustering, vi, the initial values were based on the average plus/minus four standard deviations of the log of first order neighborhood average SIRs. For the precision terms τu and τv, the inverse of the sample variances of the log(SIR) and the log of the first order neighborhood aver- age SIRs were used respectively for one chain, plus lower and higher values were chosen for the other two chains in order to be well dispersed from the middle value, but not wildly so with respect to what is reasonably expected based on the observed spatial variability. information than the exchangeable random effect alone (Table 1), which assumes purely heterogeneous variation across the state. This agrees with findings by Spiegelhalter et al [38], who developed the Deviance Information Cri- terion as a penalized version of deviance and applied it to Scottish lip cancer data. The convolution model was therefore chosen, which incorporates both random effects. References 1. New York State Department of Health: Cancer surveillance improvement initiative, 1999–2001 [http:// www.health.state.ny.us/nysdoh/cancer/csii/nyscsii.htm] 2. Narain V, Cher ML, Wood DP Jr: Prostate cancer diagnosis, staging and survival. Cancer and Metastasis Reviews 2002, 21:17-27. 3. Stewart SL, King JB, Thompson TD, Friedman C, Wingo PA: Cancer mortality surveillance –– United States, 1990–2000. M M W R Surveill Summ 2004, 53:1-108. Acknowledgements The author sincerely appreciates the encouragement and dialogue with col- leagues throughout this project; namely, from the New York State Depart- ment of Health, Colleen McLaughlin and Maria Schymura of the New York State Cancer Registry, and Edward Fitzgerald, Syni-An Hwang, Thomas Tal- bot and Francis Boscoe of the Bureau of Environmental and Occupational Epidemiology, along with Thomas Richards of the US Centers for Disease Control. Convergence of relative risk for the three independent chains was confirmed by graphing their traces and observ- ing random mixing of all chains, which revealed white noise variation around a common value, with no trend. This was supported by observing Brooks-Gelman-Rubin diagnostics that clearly satisfied convergence criteria [36]. After a burn-in of 10,000 iterations, which was far more than actually necessary, the following 1000 iterations were sampled from each of the three chains by choosing every third iteration to help avoid possible autocorrela- tion within a chain. This large sample approximation of the stationary posterior distribution for each ZIP code rel- ative risk was then summarized in WINBUGS and brought into a Geographic Information System [37] for mapping. Contract/grant sponsor: This publication was made possible through a Cooperative Agreement between the Centers for Disease Control and Prevention (CDC) and the Association of Schools of Public Health (ASPH), award number S1355-20/22. Its contents are the responsibility of the author and do not necessarily reflect the official views of the CDC or ASPH. Model Selection y y y ] 2. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge p j J 33. McLaughlin CC: Confidentiality protection in publicly released central cancer registry data. J Registry Mngt 2003, 29:84-88. central cancer registry data. J Registry Mngt 2003, 29:84 88. 34. Bernardinelli L, Clayton D, Montomoli C: Bayesian estimates of disease maps: how important are priors? 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https://openalex.org/W4312885842	https://ejournal.sthd-jateng.ac.id/index.php/WidyaAksara/article/download/125/62	Indonesian	null	PERAN PASRAMAN ASTIKA SIDHI DALAM MENUMBUHKAN SRADHA DAN BHAKTI GENERASI MUDA HINDU DI KABUPATEN KLATEN JAWA TENGAH	Widya Aksara	2,020	cc-by-sa	6,485	Volume 25 Nomor 2 September 2020 PERAN PASRAMAN ASTIKA SIDHI DALAM MENUMBUHKAN SRADHA DAN BHAKTI GENERASI MUDA HINDU DI KABUPATEN KLATEN JAWA TENGAH THE ROLE OF PASRAMAN ASTIKA SIDHI IN GROWING SRADHA AND BHAKTI YOUNG HINDU GENERATIONS IN KLATEN DISTRICT, CENTRAL JAVA Oleh: Edi Sutrisno, Dewi Ayu Wisnu Wardani Sekolah Tinggi Hindu Dharma Klaten Jawa Tengah Dewidanendra3@gmail.com ABSTRAK Pendidikan memiliki peran yang sangat penting, bangsa besar pasti memiliki sistem pendidikan yang baik. Begitu pula dengan pendidikan agama, pemerintah sudah mengatur pendidikan agama dan pendidikan keagamaan dalam PP No 55 Tahun 2007 dan PMA No 56 Tahun 2014. Pasraman Astika Sidhi merupakan pasraman pemersatu dari pasraman-pasraman yang ada di daerah dan selalu diminati oleh generasi muda Hindu yang ada di Kabupaten Klaten. Pelaksanaan pasraman Astika Sidhi dilaksanakan di aula gedung pitamaha dan STHD Klaten Jawa Tengah untuk peserta kategori anak dan ditempat yang berbeda-beda setiap tahunnya untuk peserta kategori dewasa dan umum. Peran pasraman Astika Sidhi adalah turut serta mencerdaskan kehidupan bangsa dengan mendukung dan menerapkan PP No 55 tahun 2007 dan PMA No 56 tahun 2014, mencetak generasi muda Hindu yang memiliki jiwa kepemimpinan berkualitas, memberikan wadah untuk ajang berkumpul generasi muda Hindu di Klaten Jawa Tengah, dan sebagai ikon tahunan generasi muda Hindu di Klaten Jawa Tengah. Dampak positif dari kegiatan pasraman Astika Sidhi yaitu, membentuk kaderisasi pengurus organisasi PGHD, mewujudkan kerukunan generasi muda Hindu di Klaten, dan memotivasi generasi muda Hindu di Klaten. Harapan kedepan seluruh elemen agama Hindu di Indonesia pada umumnya dan elemen Hindu di Klaten Jawa Tengah pada khususnya untuk memaksimalkan pengelolaan pasraman mingguan di masing-masing daerah bahkan bisa menjadi pasraman formal, sehingga kita mampu bersaing dengan pendidikan keagamaan dari agama lain. Kata Kunci : Peran, Astika Sidhi, Generasi Muda Hindu. Kata Kunci : Peran, Astika Sidhi, Generasi Muda Hindu. ABSTRACT Education has a very important role.A big nation must have a good education system. Likewise with religious education, the government has regulated religion education and religious education in PP No. 55 of 2007 and PMA No. 56 of 2014. Pasraman Astika Sidhi is a unifying pasraman in the region and is always favored by the younger generation of Hindus. in Klaten Regency. Pasraman Astika Sidhi for child category participantsis held in the hall of the Pitamaha building and STHD Klaten, Central Javaand in different places each year for adult and general participants. Pasraman Astika Sidhi's role is to participate in educating the nation by supporting and implementing PP No. 55 of 2007 and PMA No. 56 of Jurnal Widya Aksara Vol. 25 No. 2 September 2020 208 2014, creating Hindu young generation who has a quality leadership spirit, providing a forum for gathering the Hindu young generation in Klaten, Central Java, and as an annual icon of the Hindu youth generation in Klaten, Central Java. The positive impact of Pasraman Astika Sidhi activities is forming cadresfor the boardof PGHD organization, creating harmony among Hindu young generation in Klaten, Central Java, as well as motivating them. Hopefully, in the future all elements of Hinduism in Indonesia in general and Hindu elements in Klaten, Central Java in particular, are able to maximize the management of the weekly pasraman in each region and even become formal pasraman, so that we can compete with religious education from other religions. Keywords: Role, Astika Sidhi, Hindu Young Generation. Keywords: Role, Astika Sidhi, Hindu Young Generation. Jurnal Widya Aksara Vol. 25 No. 2 September 2020 I. PENDAHULUAN 2 September 2020 209 agama Hindu masih ada yang mendapat alokasi waktu dan tempat yang tidak setara dengan siswa yang beragama lain, contohnya siswa yang beragama Hindu mendapatkan jam pelajaran agama Hindu secara serentak mulai dari kelas tujuh sampai kelas sembilan dan dalam ruang yang sama untuk kegiatan belajar mengajar, hal ini tentu menghambat kelangsungan pembelajaran materi agama Hindu. Untuk menanggulangi permasalahan tersebut siswa yang beragama Hindu dapat mengenyam dan mendalami materi agama Hindu melalui Pasraman. Pasraman merupakan sistem pendidikan non formal agama Hindu yang sangat bermanfaat dalam membentuk karakter yang bernafaskan Hindu, di dalamnya lebih menekankan penanaman sradha dan bhakti agar kelak menjadi generasi yang memiliki rasa bangga menjadi Hindu. Seperti yang telah dijelaskan dalam Peraturan Menteri Agama atau PMA No 56 Tahun 2014 tentang Pendidikan Keagamaan Hindu, mulai dari maksud dan tujuan, jenjang pendidikan, dibentuknya pasraman, pengelolaannya telah dijelaskan secara detail. Jadi sudah jelas, pasraman telah memiliki payung hukum yang kuat. Namun pada kenyataannya payung hukum tersebut belum dimanfaatkan secara maksimal, terbukti dengan belum meratanya keberadaan pasraman di Indonesia dan banyak yang belum maksimal dalam kegiatan belajar mengajarnya karena terbatasnya fasilitas pasraman seperti ruang kelas, tenaga pendidik, dan dukungan dari orang tua siswa pasraman. Beberapa pasraman yang sudah memiliki fasilitas memadahi seharusnya menjadi contoh dan motivasi bagi pasraman-pasraman yang lainnya, karena kegiatan belajar mengajarnya selain untuk Penanaman sradha dan bhakti, juga diajarkan materi umum, bahkan ada pasraman yang berkenan mengajari mengerjakan pekerjaan rumah dari sekolah umum. Kegiatan belajar mengajar hampir sama dengan kegiatan belajar mengajar yang diterapkan di sekolahan, yaitu ada guru yang mengajarkan materi dan siswa yang menyimaknya. Dalam segi fasilitas ruang kelas dan lainnya, tergantung pada kemampuan pangempon pura selaku pengelola Pasraman, jika pangempon pura mampu maka fasilitasnya juga mendukung, seperti beberapa pasraman di Kabupaten Klaten yang sudah memiliki ruang pasraman dan sarana prasarana yang baik. Guru atau tenaga pendidik biasanya guru agama Hindu yang mengajar di sekolah yang kebetulan menjadi pangempon pura di mana pasraman tersebut berada, namun ada juga pasraman karena keterbatasan tenaga pendidik, biasanya pemuda setempat yang dianggap memiliki kemampuan lebih lah yang bertugas sebagai tenaga pendidik Salah satu komunitas kepemudaan Hindu yang peduli dengan kemajuan Pendidikan Hindu khususnya di Kabupaten Klaten yaitu Persaudaraan Generasi Hindu Dharma (PGHD). Meskipun di masing- masing pura yang ada di Kabupaten Klaten hampir semua memiliki pasraman, namun PGHD Klaten memiliki inisiatif untuk melaksanakan sebuah pasraman kilat. I. PENDAHULUAN Pendidikan memiliki peran yang sangat vital dalam memajukan sebuah bangsa. Bangsa yang memiliki sistem pendidikan yang baik, peluang untuk menjadi bangsa yang besar. Termasuk negara tercinta kita, yaitu Indonesia terus menerus melakukan pembaharuan terhadap sistem pendidikan, agar mampu bersaing dengan negara-negara lainnya. Tidak berbeda dengan majunya sebuah agama, apapun agama tersebut jika mampu menerapkan sistem pendidikan yang baik, pasti akan memiliki generasi-generasi penerus yang mumpuni, solid, dapat diandalkan, dan mampu menjadi pemimpin, baik itu pemimpin kelompok agamanya maupun bangsanya. Mengenai pendidikan agama dan pendidikan keagamaan di Indonesia telah diatur secara lengkap dalam Peraturan Pemerintah Republik Indonesia Nomor 55 Tahun 2007 atau yang lebih dikenal dengan PP NO 55. Pada PP No 55 Tahun 2007 (BAB I Pasal 1) menjelaskan bahwa pendidikan agama adalah pendidikan yang memberikan pengetahuan dan membentuk sikap, kepribadian, dan keterampilan peserta didik dalam. Sedangkan pendidikan keagamaan adalah pendidikan yang mempersiapkan peserta didik untuk dapat menjalankan peranan yang menuntut penguasaan pengetahuan tentang ajaran agama dan/atau menjadi ahli ilmu agama dan mengamalkan ajaran agamanya. Pedidikan keagamaan agama Hindu diatur pada PP No 55 Tahun 2007 pasal 38/ 41, yang pada intinya Pendidikan keagamaan Hindu merupakan pendidikan berbasis masyarakat yang diselenggarakan dalam bentuk Pasraman, Pesantian, dan bentuk lain yang sejenis. Pendidikan Pasraman diselenggarakan pada jalur formal, dan nonformal. Pendidikan Pasraman diselenggarakan pada jalur formal setingkat TK disebut Pratama Widya Pasraman, yaitu tingkat Pratama Widya Pasraman A (TK A) dan tingkat Pratama Widya Pasraman B (TK B). Pendidikan pasraman pada jalur formal jenjang pendidikan dasar setingkat SD disebut Adi Widya Pasraman terdiri atas 6 (enam) tingkat. Pendidikan Pasraman pada jalur formal jenjang pendidikan dasar setingkat SMP disebut Madyama Widya Pasraman terdiri atas 3 (tiga) tingkat. Pendidikan Pasraman pada jalur formal jenjang pendidikan menengah setingkat SMA disebut Utama Widya Pasraman terdiri atas 3 (tiga) tingkat. Maha Widya Pasraman atau pendidikan keagamaan tinggi Hindu. Sedangkan Pendidikan keagamaan Hindu nonformal dilaksanakan dalam bentuk Pesantian, sad dharma yaitu dharmatulla, dharma sadhana, dharma wacana, dharma yatra, dharma gita, dharma santi atau dalam bentuk lain yang sejenis. Pendidikan keagamaan Hindu nonformal merupakan kegiatan pendidikan keagamaan Hindu secara berjenjang atau tidak berjenjang bertujuan untuk melengkapi pendidikan agama di sekolah formal dalam rangka meningkatkan sraddha dan bhakti peserta didik. Fakta yang ada di lapangan, meskipun di sekolah yang bersifat umum, mata pelajaran Jurnal Widya Aksara Vol. 25 No. Jurnal Widya Aksara Vol. 25 No. 2 September 2020 A. Sejarah Pasraman Astika Sidhi A. Sejarah Pasraman Astika Sidhi Menurut informasi dari tokoh pendiri pasraman Astika Sidhi, Pasraman Astika Sidhi merupakan suatu kegiatan yang dibentuk oleh organisasi kepemudaan Hindu yang bernama Persaudaraan Generasi Hindu Dharma (PGHD) pada tahun 2000. PGHD dibentuk pada tahun 1998 berkat inisiatif pemuda Hindu yang dipelopori oleh Basuki dan Dwi Hartanto, untuk menyatukan pemuda Hindu yang ada di Klaten dalam suatu wadah organisasi. Pada tahun 2000 pura yang ada di Kabupaten Klaten tidak semuanya mempunyai pasraman atau bentuk kegiatan kepemudaan yang lainnya, maka dari itu PGHD melalui devisi bidang Pemuda, Pendidikan, dan kaderisasi membuat gagasan untuk membuat sebuah wadah untuk menyatukan pemuda Hindu yang ada di Kabupaten Klaten dan terbentuklah Pasraman Astika Sidhi. Kegiatan ini mendapatkan respon yang baik dari pemuda Hindu yang ada di masing- masing pura di Klaten Jawa Tengah. Kegiatan Pasraman Astika Sidhi ini pertama kali dilaksanakan di Gedung Sekolah Tinggi Hindu Dharma (STHD) Klaten Jawa Tengah (yang sekarang menjadi gedung sekretariat Parisada Hindu Dharma Indonesia Kabupaten Klaten), dengan jumlah peserta 30 orang, karena belum sosialisasi secara all out, jadi dari panitia hanya mengajak pemuda dari asalnya masing-masing”. Jumlah ini belum memenuhi target awal dengan jumlah 50 orang. Selanjutnya kegiatan ini menjadi agenda tahunan oleh organisasi PGHD dan menetapkan pelaksanaan kegiatan ini pada saat saudara Muslim melaksanakan ibadah Puasa. Keputusan ini merupakan langkah berpolitik dari PGHD melihat pada saat siswa sekolah pada saat memasuki bulan Puasa, siswa yang beragama Muslim mengisi dengan kegiatan Pondok Romadon, sedangkan siswa yang lain tidak ada kegiatan atau diliburkan. Maka momen ini sangat tepat untuk melaksanakan Pasraman dengan tujuan menumbuhkan sradha dan bhakti generasi muda Hindu. Kegiatan apapun itu tidak lepas dari biaya, begitu pula dengan Pasraman Astika Sidhi. Pertama kali kegiatan ini dilaksanakan sepenuhnya ditanggung oleh panitia pelaksana dengan cara iuran sehingga belum bisa memfasilitasi peserta secara baik. Setelah tiga empat kali pelaksanaan kemudian mendapat „‟perhatian dari PHDI dan Depag (sekarang Pembimas Agama Hindu) dan terus berkembang hingga sekarang mendapat dukungan dari umat Hindu di Kabupaten Klaten bahkan sampai di luar Klaten. Dari hasil wawancara terdahulu, dapat disimpulkan bahwa terbentuknya pasraman Astika Sidhi karena melihat keadaan pada saat Pura yang ada di Kabupaten Klaten belum banyak terdapat kegiatan seperti pasraman, pasantian, dan kegiatan lainnya oleh pangempon Pura. Selanjutnya, antisipasi terhadap waktu luang karena adanya kegiatan dari saudara yang beagama Islam ketika memasuki bulan Ramadhan. I. PENDAHULUAN Di dalam pelaksanaan kegiatan pasraman kilat ini sedikit ada perbedaan dari pasraman-pasraman pada umumnya, dengan mengusung tema dan tempat yang berbeda setiap tahunnya sehingga membuat anak- anak tertarik dan dengan suka cita mengikuti kegiatan tersebut. Pasraman tersebut termasuk upaya mempersatukan generasi muda Hindu di Klaten dan regenerasi organisasi PGHD tidak terputus, dan terbukti hingga saat ini PGHD tetap eksis di Masyarakat Hindu khususnya di Kabupaten Klaten. Pasraman tersebut adalah pasraman Astika Sidhi, yang awal mulanya terbentuk dari obrolan ketika berdiskusi di alun-alun Klaten pada tahun 1999 oleh pengurus PGHD Klaten guna mengisi kegiatan pada saat umat muslim memasuki bulan Ramadan, dan terus mengalami perkembangan hingga sampai saat ini pada tahun 2018 ini sudah mencapai umur yang ke 19 tahun. Merupakan kegiatan rutin setiap tahun sekali yang diselenggarakan oleh Persaudaraan Generasi Hindu Dharma (PGHD) Klaten yang sudah tidak muda lagi dalam hal usia dan tentu sudah berpengalaman. Dengan terus diadakannya kegiatan tersebut setiap tahun, merupakan sebuah indikasi bahwa kegiatan tersebut mampu memberikan Jurnal Widya Aksara Vol. 25 No. 2 September 2020 210 dampak yang positif untuk pendidikan Hindu khususnya dalam hal menumbuhkan sradha dan bhakti. B. Peran Pasraman Astika Sidhi Dalam Menumbuhkan Sradha dan Bhakti Generasi Muda Hindu Di Kabupaten Klaten 1. Pelaksanaan Pasraman Astika Sidhi Menurut ketua Persaudaraan Generasi Muda Hindu Dharma (PGHD) Klaten periode 2017 / 2019 pasraman Astika Sidhi diselenggarakan setiap satu tahun sekali. Dalam pelaksanaannya para peserta dibagi menjadi dua kategori, yaitu peserta pasraman kategori anak dan peserta pasraman kategori dewasa dan umum. Untuk peserta kategori anak diikuti oleh anak PAUD, SD, dan SMP kelas VII dan VIII. Sedangkan untuk peserta kategori dewasa dan umum diikuti oleh siswa SMP kelas IX, SLTA, Mahasiswa, dan Generasi Hindu secara umum. Pengklasifikasian kategori umur ini dilakukan karena pola pikir dari peserta berbeda dan berpengaruh dalam metode pengajaran oleh pemateri. Lokasi yang digunakan untuk kategori anak setiap tahunnya sama yaitu di Aula Gedung Pitamaha dan Kampus Sekolah Tinggi Hindu Dharma (STHD) Klaten Jawa Tengah, sedangkan untuk kategori dewasa umum di laksanakan berbeda tempat setiap tahunnya, hal ini dilakukan agar peserta tidak bosan sehingga memiliki daya tarik setiap diselenggarakan kegiatan pasraman ini. Terselenggaranya kegiatan tahunan ini tidaklah mudah dan membutuhkan waktu yang cukup lama, mulai dari tahap pembentukan panitia, penggalangan dana, persiapan teknis kegiatan, pelaksanaan kegiatan, serta tahap pelaporan dan evaluasi. Seperti dalam penjelasan terdahulu, pasraman Astika Sidhi ada yang berbeda setiap pelaksanaannya mulai dari panitia pelaksana, tempat, dan tema yang diusung. G h i di l i d b k i i p p y p p , p , y g g Menurut Ketua PGHD tahap persiapan dimulai dengan rapat pembentukan panitia pasraman yang dilaksanakan oleh anggota PGHD dan beberapa Mahasiswa STHD Klaten Jawa Tengah, dari rapat tersebut menghasilkan susunan panitia pelaksana pasraman Astika Sidhi sebagai berikut, Setelah terbentuk panitia, kemudian menentukan tema yang akan diusung dan waktu pelaksanaan. Untuk menentukan waktu pelaksanaan dari pihak panitia bekerja sama dengan Kelompok Kerja Guru (KKG) Agama Hindu dan Musyawarah Guru Mata Pelajaran (MGMP) SLTP dan SLTA agama Hindu, karena yang paham mengenai kalender akademik adalah dari pihak guru. Pasraman Astika Sidhi XVII mengusung tema “Bersatu Menuju Kejayaan Hindu”. Diselenggarakan pada hari Sabtu s/d Minggu, 4 s/d 5 Juni 2016 di Aula Gedung PitaMaha dan STHD untuk kategori anak-anak. Sedangkan untuk kategori dewasa dan umum diselenggarakan pada Minggu s/d Selasa, 12 s/d 14 juni 2016 di Wisma Pesanggrahan PU, Jl. Astorenggo No 369 RT 08/ RW 19, Kaliurang Barat, Hargobinanun, Pakem, Sleman. Menurut ketua PGHD 2017/ 2019 Dwi Hantoro menentukan tema bersatu menuju kejayaan Hindu karena ingin merangkul generasi muda Hindu yang ada di Kabupaten Klaten untuk bersatu untuk meraih kejayaan seperti masa kejayaan pada masa Majapahit. B. Peran Pasraman Astika Sidhi Dalam Menumbuhkan Sradha dan Bhakti Generasi Muda Hindu Di Kabupaten Klaten 1. Pelaksanaan Pasraman Astika Sidhi 1. Pelaksanaan Pasraman Astika Sidhi Pasraman Astika Sidhi dilaksanakan setiap satu tahun sekali. Menurut ketua Persaudaraan Generasi Muda Hindu Dharma (PGHD) Klaten periode 2017 / 2019 pasraman Astika Sidhi diselenggarakan setiap satu tahun sekali. Dalam pelaksanaannya para peserta dibagi menjadi dua kategori, yaitu peserta pasraman kategori anak dan peserta pasraman kategori dewasa dan umum. Untuk peserta kategori anak diikuti oleh anak PAUD, SD, dan SMP kelas VII dan VIII. Sedangkan untuk peserta kategori dewasa dan umum diikuti oleh siswa SMP kelas IX, SLTA, Mahasiswa, dan Generasi Hindu secara umum. Pengklasifikasian kategori umur ini dilakukan karena pola pikir dari peserta berbeda dan berpengaruh dalam metode pengajaran oleh pemateri. Lokasi yang digunakan untuk kategori anak setiap tahunnya sama yaitu di Aula Gedung Pitamaha dan Kampus Sekolah Tinggi Hindu Dharma (STHD) Klaten Jawa Tengah, sedangkan untuk kategori dewasa umum di laksanakan berbeda tempat setiap tahunnya, hal ini dilakukan agar peserta tidak bosan sehingga memiliki daya tarik setiap diselenggarakan kegiatan pasraman ini. Terselenggaranya kegiatan tahunan ini tidaklah mudah dan membutuhkan waktu yang cukup lama, mulai dari tahap pembentukan panitia, penggalangan dana, persiapan teknis kegiatan, pelaksanaan kegiatan, serta tahap pelaporan dan evaluasi. Seperti dalam penjelasan terdahulu, pasraman Astika Sidhi ada yang berbeda setiap pelaksanaannya mulai dari panitia pelaksana, tempat, dan tema yang diusung. Menurut Ketua PGHD tahap persiapan dimulai dengan rapat pembentukan panitia pasraman yang dilaksanakan oleh anggota PGHD dan beberapa Mahasiswa STHD Klaten Jawa Tengah, dari rapat tersebut menghasilkan susunan panitia pelaksana pasraman Astika Sidhi sebagai berikut, Setelah terbentuk panitia, kemudian menentukan tema yang akan diusung dan waktu pelaksanaan. Untuk menentukan waktu pelaksanaan dari pihak panitia bekerja sama dengan Kelompok Kerja Guru (KKG) Agama Hindu dan Musyawarah Guru Mata Pelajaran (MGMP) SLTP dan SLTA agama Hindu, karena yang paham mengenai kalender akademik adalah dari pihak guru. Pasraman Astika Sidhi XVII mengusung tema “Bersatu Menuju Kejayaan Hindu”. Diselenggarakan pada hari Sabtu s/d Minggu, 4 s/d 5 Juni 2016 di Aula Gedung PitaMaha dan STHD untuk kategori anak-anak. Sedangkan untuk kategori dewasa dan umum diselenggarakan pada Minggu s/d Selasa, 12 s/d 14 juni 2016 di Wisma Pesanggrahan PU, Jl. Astorenggo No 369 RT 08/ RW 19, Kaliurang Barat, Hargobinanun, Pakem, Sleman. Menurut ketua PGHD 2017/ 2019 Dwi Hantoro menentukan tema bersatu menuju kejayaan Hindu karena ingin merangkul generasi muda Hindu yang ada di Kabupaten Klaten untuk bersatu untuk meraih kejayaan seperti masa kejayaan pada masa Majapahit. Pasraman Astika Sidhi dilaksanakan setiap satu tahun sekali. A. Sejarah Pasraman Astika Sidhi Setelah adanya kegiatan pasraman Astika Sidhi lambat laun mulai terbentuk kegiatan pasraman mingguan yang dilaksanakan di pura masing-masing daerah hingga sampai sekarang terus berkembang. Hal ini bisa menjadi bukti kemajuan dalam dunia pendidikan, khususnya pendidikan agama Hindu. 211 Jurnal Widya Aksara Vol. 25 No. 2 September 2020 B. Peran Pasraman Astika Sidhi Dalam Menumbuhkan Sradha dan Bhakti Generasi Muda Hindu Di Kabupaten Klaten 1. Pelaksanaan Pasraman Astika Sidhi Jurnal Widya Aksara Vol. 25 No. 2 September 2020 B. Peran Pasraman Astika Sidhi Dalam Menumbuhkan Sradha dan Bhakti Generasi Muda Hindu Di Kabupaten Klaten 1. Pelaksanaan Pasraman Astika Sidhi Pasraman Astika Sidhi XVIII pada tahun 2017 mengusung tema “Melalui Pasraman Kilat Kita Bekali Generasi Muda Hindu Dengan Sradha dan Bhakti Yang Berkearifan Lokal”. Terselenggara pada Senin sampai Selasa tanggal 12 s/d 13 Juni 2017 untuk kategori anak di aula gedung Pitamaha dan untuk kategori dewasa umum diselenggarakan pada Sabtu s/d Senin tanggal 17 s/d 19 Juni 2017 di Wisma Pasraman Pinandita Tawangmangu, Karanganyar, Jawa Tengah. Menurut ketua panitia Suwanto, pemilihan tema tersebut karena melihat generasi muda sedang mengalami penurunan sopan santun atau unggah-ungguh dalam bahasa jawa. Panitia juga mencoba mengkolaborasikan budaya jawa dan ajaran agama Hindu kepada generasi muda Hindu di Kabupaten Klaten. Pasraman Astika Sidhi XIX mengusung tema “Melalui Pasraman Astika Siddhi XIX Tingkatkan Soliditas Dalam Menyikapi Era Globalisasi”. Diselenggarakan pada Senin s/d Selasa, 4 s/d 5 Juni 2018 di Aula Gedung Pita Maha dan Sekolah Tinggi Hindu Dharma Klaten Jawa Tengah untuk kategori anak-anak. Untuk kategori dewasa dan umum Jurnal Widya Aksara Vol. 25 No. 2 September 2020 212 diselenggarakan pada Rabu s/d Jumat, 16 s/d 18 Mei 2018 di Wisma Pesanggrahan PU, Jl. Astorenggo No 369 RT 08/ RW 19, Kaliurang Barat, Hargobinanun, Pakem, Sleman. Menurut ketua panitia Suraji, penentuan tema tersebut atas dasar kemajuan teknologi yang semakin pesat, maka dari itu panitia mengajak peserta pasraman untuk juga mengikuti perkembangan dalam arti mengikuti perkembangan teknologi yang positif. Dari uraian tersebut diambil kesimpulan bahwa pembentukan panitia selalu berganti pada tiap tahunnya agar semua generasi Hindu yang ada di Klaten belajar untuk berorganisasi apapun posisi atau peran yang diterimanya. Pemilihan tempat yang berubah setiap tahunnya agar memiliki daya tarik bagi peserta. Selanjutnya adalah menyusun jadwal kegiatan, penentuan jadwal dilaksanakan dengan musyawarah panitia agar nanti dalam pelaksanaan bisa berjalan lancar dan tidak meleset dari tema yang ditetapkan. Panitia telah melaksanakan tugas dan kewajiban masing-masing tiba di hari pelaksanaan kegiatan pasraman. Pertama peserta melakukan pendaftaran terlebih dahulu. Menurut Ketua PGHD Pada tahun 2016 peserta pasraman untuk kategori anak-anak adalah 115 peserta terdiri dari SD dan SMP dan untuk kategori dewasa 65 peserta terdiri dari SLTA, Perguruan Tinggi, dan Umum. Menurut data yang didapatkan, jumlah peserta pasraman Astika Sidhi pada tahun 2017 untuk kategori anak-anak adalah 120 peserta terdiri dari SD dan SMP. Sedangkan kategori dewasa adalah 75 peserta terdiri dari SMP, SLTA, Perguruan Tinggi, dan Umum. Pada tahun 2018 jumlah peserta untuk kategori anak adalah 128 peserta terdiri dari beberapa pasraman yang ada di Klaten, SD, dan SMP. B. Peran Pasraman Astika Sidhi Dalam Menumbuhkan Sradha dan Bhakti Generasi Muda Hindu Di Kabupaten Klaten 1. Pelaksanaan Pasraman Astika Sidhi Sedangkan untuk kategori dewasa jumlah peserta adalah 41 peserta terdiri dari SLTA, Perguruan Tinggi, dan Umum. Khusus untuk tahun 2018 jumlah peserta mengalami penurunan, ada beberapa faktor yang mempengaruhinya, yaitu kegiatan pasraman berbarengan dengan pelaksanaan Ujian Semester Genap dan ada beberapa Sekolah yang masih melaksanakan PKL. Kegiatan yang dilakukan adalah mengisi materi yang telah disiapkan oleh panitia, yaitu belajar Dharma Gita dengan metode yang menyenangkan, upacara dan upakara persembahyangan, pendidikan karakter, kepemimpinan, dan outbond. Menurut salah satu pemateri Dharma Gita Jaka Purnama kegiatan pasraman sangat bagus, karena sebagian besar peserta pasraman Astika Sidhi belum mendapatkan materi semacam ini di pasraman mereka masing-masing. Ketika mengisi materi beliau menggunakan metode pembelajaran interaktif dan menyenangkan dengan bantuan media belajar audio visual, sehingga para peserta tidak mudah bosan, dan penyampaian materi benar-benar bisa dipahami. Namun akan lebih baik lagi panitia memberikan waktu yang cukup banyak untuk materi Dharma Gita, karena generasi muda kita masih sangat minim pemahamannya terhadap Kitab Suci Weda. Pemberian materi diklasifikasikan berdasarkan kategori jenjang pendidikan, adapun materi yang di berikan sebagai berikut : a. Kategori Anak-anak Materi yang diberikan meliputi penanaman sradha dan bhakti berupa pemberian materi tentang Panca Sradha atau lima keyakinan dalam ajaran agama Hindu. Pemateri adalah dari Kelompok Kerja Guru (KKG) agama Hindu Kabupaten Klaten, penyampaian materi harus menggunakan strategi yang berbeda dengan di sekolah, karena berbeda jumlah yang biasanya paling banyak di kelas tidak lebih dari sepuluh anak, di pasraman guru harus menghadapi jumlah peserta didik yang mencapai 100 lebih peserta didik. Selain menggunakan pengeras suara dan media belajar, pemateri juga harus bisa menarik perhatian peserta didik. Ketua PHDI Kabupaten Klaten menghimbau agar kegiatan pasraman ini wajib ada materi keagamaan, untuk yang lainnya terserah panitia mau dibuat sekreatif mungkin asal kegiatan a. Kategori Anak-anak a. Kategori Anak-anak Materi yang diberikan meliputi penanaman sradha dan bhakti berupa pemberian materi tentang Panca Sradha atau lima keyakinan dalam ajaran agama Hindu. Pemateri adalah dari Kelompok Kerja Guru (KKG) agama Hindu Kabupaten Klaten, penyampaian materi harus menggunakan strategi yang berbeda dengan di sekolah, karena berbeda jumlah yang biasanya paling banyak di kelas tidak lebih dari sepuluh anak, di pasraman guru harus menghadapi jumlah peserta didik yang mencapai 100 lebih peserta didik. Selain menggunakan pengeras suara dan media belajar, pemateri juga harus bisa menarik perhatian peserta didik. Jurnal Widya Aksara Vol. 25 No. 2 September 2020 B. Peran Pasraman Astika Sidhi Dalam Menumbuhkan Sradha dan Bhakti Generasi Muda Hindu Di Kabupaten Klaten 1. Pelaksanaan Pasraman Astika Sidhi Ketua PHDI Kabupaten Klaten menghimbau agar kegiatan pasraman ini wajib ada materi keagamaan, untuk yang lainnya terserah panitia mau dibuat sekreatif mungkin asal kegiatan Jurnal Widya Aksara Vol. 25 No. 2 September 2020 213 tersebut positif. Materi Tata Susila sangat penting untuk disampaikan pada usia dini dalam membentuk karakter anak. Tata Susila adalah salah satu bagian dari Tri Kerangka dalam agama Hindu yaitu, Tattwa, Susila, Upakara. Kegiatan ini mencangkup tiga aspek kerangka tersebut agar seimbang, karena untuk memahami agama Hindu tidak boleh salah satu atau salah dua saja dari ketiga kerangka tersebut. Di analogikan Tri Kerangka agama Hindu adalah sebutir telur yang mana Tattwa adalah kuning telur, Susila adalah putih telur, dan Upakara adalah cangkang telur. Telur yang baik dan akan menetas jika tiga bagian tersebut memiliki kualiatas yang baik. Jika telur memiliki cangkang dan putih telur baik, tetapi kuning telur jelek maka telur tersebut tidak akan bisa menetas, begitu pula dengan putih dan cangkang telur juga harus baik. Demikian juga dengan Tri Kerangka agama Hindu ketiganya harus dipelajari secara seimbang agar generasi muda Hindu khususnya yang ada di Klaten menjadi generasi yang berkualitas dan mampu bersaing di kehidupan bermasyarakat. Penyampaian materi tentang Upakara, selain peserta diajarkan bagaimana membuat banten yang sering digunakan setiap hari, peserta juga diberikan wawasan tentang banten yang baik, karena pemikiran tentang banten yang baik adalah banten yang mewah dan megah masih berkembang di kalangan umat Hindu. Melalui kegiatan ini generasi muda diberikan pengetahuan bahwa persembahan banten yang baik adalah persembahan yang didasari dengan hati yang tulus ikhlas, seperti yang dijelaskan dalam kitab Bhagawad Gita 9. 26 y g p y g j g Patram puspam phalam toyam Yo me bhaktya prayaccati Patram puspam phalam toyam Yo me bhaktya prayaccati Tad aham bhakti-upahrtam 2. Peran Pasraman Astika Sidhi 2. Peran Pasraman Astika Sidhi Dalam rangka menumbuhkan sradha dan bhakti generasi muda Hindu di Kabupaten Klaten pasraman Astika Sidhi tentu menekankan pada pendidikan agama Hindu baik secara teori dan penerapannya di kehidupan sehari-hari. Pasraman mingguan yang ada di Kabupaten Klaten masih banyak yang belum maksimal dalam memberikan pemahaman tentang Weda. Maka melalui pasraman Astika Sidhi generasi muda yang datang dari berbagai tempat pasraman mingguan akan secara merata di berikan pelajaran mengenai Weda yang menjadi wawasan baru bagi peserta yang belum diterima di pasraman mingguan. Pasraman Astika Sidhi turut mendukung Peraturan Pemerintah Nomor 55 Tahun 2007 atau PP NO 55 dan Peraturan Menteri Agama Nomor 56 Tahun 2014 atau PMA NO 56 tentang pendidikan agama dan pendidikan keagamaan. Hal ini menunjukkan bahwa pasraman Astika Sidhi turut serta mencerdaskan kehidupan bangsa. p g Peran pasraman Astika Sidhi untuk melahirkan generasi berkarakter kepemimpinan, melalui kegiatan outbond yang mana kegiatan tersebut belum banyak di pasraman mingguan yang menerapkannya. Selain itu peserta juga mendapatkan motivasi dari pembicara berkompeten yang sengaja didatangkan oleh panitia pasraman. Hal ini dilakukan untuk membentuk generasi muda Hindu di Kabupaten Klaten menjadi generasi yang solid kelak ketika menginjak dewasa dan memasuki jenjang grehasta memasuki jenjang rumah tangga. Pada jenjang ini sangat rentan dengan perpindahan keyakinan, karena itu penting dilakukan penguatan sradha dan bhakti. Menjadi ikon tahunan generasi muda Hindu di Kabupaten Klaten, kegiatan pasraman Astika Sidhi menjadi sesuatu yang ditunggu-tunggu oleh sebagian besar generasi muda Hindu di Klaten, bahkan ada pasraman mingguan yang berkenan menjalin MOU atau kerjasama untuk selalu berpartisipasi dalam mensukseskan kegiatan pasraman Astika Sidhi sehingga setiap tahunnya secara rutin mengirimkan peserta untuk mengikuti pasraman Astika Sidhi. Sesuai dengan pernyataan salah satu pengelola pasraman mingguan Brahma Govinda kami secara rutin mengirim peserta pasraman, karena banyak manfaat yang didapatkan setelah mengikuti kegiatan pasraman Astika Sidhi dan sisya pasraman kami menjadi lebih giat mengikuti pasraman mingguan. Tidak ketinggalan juga para senior pendiri pasraman yang tinggal di luar jawa pun menyempatkan diri untuk mengikuti kegiatan pasraman Astika Sidhi, seakan kegiatan tersbut memiliki magnet yang mampu menarik generasi muda Hindu dan tidak rela untuk ketinggalan mengikuti atau sekedar datang dan menyaksikan. Pasraman Astika Sidhi juga berperan sebagai wadah pemersatu generasi muda Hindu yang ada di Klaten. Dengan adanya kegiatan ini para peserta mendapat kenalan baru dan bisa bertukar pikiran mengenai keadaan di pura masing-masing dan berdiskusi bersama mengenai kegiatan kepemudaan. Asnami prayatatmanah “Mereka yang dengan penuh rasa bhakti mempersembahkan kepada-Ku, walaupun anya selembar daun, sekuntum bunga, satu buah, dan setetes air, jika semua itu dipersembahkan dengan penuh bhakti oleh mereka yang berhati suci murni, maka Aku akan menerimanya”. Dari pemaparan sloka tersebut dengan tegas menyebutkan bahwa persembahan banten yang megah tidak dapat digunakan sebagai jaminan, akan tetapi hati yang tulus ikhlas. Dari pemaparan sloka tersebut dengan tegas menyebutkan bahwa persembahan banten yang megah tidak dapat digunakan sebagai jaminan, akan tetapi hati yang tulus ikhlas. b K i D d U b. Kategori Dewasa dan Umum Seperti halnya pada kategori anak, materi keagamaan wajib disampaikan kepada peserta, akan tetapi dengan materi yang tingkatannya lebih tinggi. Setelah penyampaian materi, peserta akan dibentuk kelompok untuk melakukan diskusi Sebagai generasi muda kita harus peka dan kritis dengan lingkungan khususnya di lingkungan keumatan. Gambar di atas adalah kegiatan diskusi pemecahan berbagai masalah yang terjadi di pura atau wilayah masing-masing. Sebelum dibentuk kelompok, peserta wajib menuliskan masalah yang ada di wilayah masing-masing, setelah semua menuliskan masalah, dilakukan identifikasi terhadap masalah kemungkinan ada masalah yang sama bisa dijadikan satu. Setelah diidentifikasi masalah, kemudian dibentuk kelompok dan masing-masing kelompok berdiskusi menemukan solusi dari masalah tersebut. Materi selanjutnya yaitu belajar bekerja dalam kelompok dan kepemimpinan, materi ini akan memupuk jiwa sosial dan kepemimpinan pada peserta pasraman. Dengan kegiatan semacam ini secara tidak langsung peserta belaar bagaimana bekerja dalam sebuah kelompok, dan belajar membuang ego masing-masing untuk mencapai tujuan bersama, manfaat akan dirasakan setelah peserta berada di kehidupan bermasyarakat. Dari uraian data terdahulu penulis menarik kesimpulan bahwa kegiatan pasraman Astika Sidhi memiliki daya tarik tersendiri bagi generasi muda Hindu di Kabupaten Klaten. Setiap tahunnya peserta mengalami peningkatan jumlah kecuali ada kendala yang memang belum bisa dicarikan jalan keluar seperti jadwal pelaksanaannya berbenturan dengan kegiatan sekolah. Jurnal Widya Aksara Vol. 25 No. 2 September 2020 214 p p 1 Kaderisasi Pengurus Organisasi PGHD Sebuah organisasi pasti mengalami pergantian kepengurusan tergantung berapa lama masa jabatan yang ditentukan. Persaudaraan Generasi Hindu Dharma (PGHD) Klaten melakukan pergantian kepengurusan tiga tahun sekali. Melalui pasraman Astika Sidhi lahir calon-calon pemimpin penerus PGHD kedepannya. Sebagai contoh ketua PGHD Klaten periode 2017/ 2019 yaitu Dwi Hantoro, beliau mengikuti secara rutin pasraman Astika Sidhi setiap tahun mulai dari kelas 4 SD, selama 12 tahun mengikuti pasraman Astika Sidhi pada tahun 2017 beliau terpilih menjadi Ketua PGHD periode 2017/ 2019 rekam jejak selama kepemimpinan beliau pun dinilai baik berbagai kegiatan keumatan dan sosial berhasil diselenggarakan. Beberapa kegiatan besar yang telah dilaksanakan yaitu yoga masal pertama kali di Klaten, donor darah secara rutin tiga bulan sekali. Di dalam struktur organisasi PGHD semua adalah jebolan peserta pasraman yang rutin mengikuti sejak dini. Menurut ketua Parisada Hindu Dharma Indonesia Kabupaten Klaten Ir.Hendrata Wisnu, MMR selaku pembina PGHD Klaten menyatakan bahwa organisasi PGHD harus belajar hidup mandiri secara materi dalam menyelenggarakan kegiatan, PHDI bisa saja memberikan bantuan dalam bentuk materi untuk membuat kegiatan, namun dengan pendidikan yang diterima dalam pasraman Astika Sidhi Organisasi PGHD dapat berkembang sampai saat ini. Pada saat manusia masih muda, saat itulah masa yang tepat untuk mencari ilmu sebanyak mungkin, seperti yang telah dituliskan dalam kitab Sarasamuscaya sebagai berikut; Pūrvve vayasi yah çāntah sa çānta iti me matih, Dhātuşu kşiyamaņeşu çamah kasya na vidyate Sarasamuçcaya 28 j h Pūrvve vayasi yah çāntah sa çānta iti me matih, Dhātuşu kşiyamaņeşu çamah kasya na vidyate Sarasamuçcaya 28 T j h Terjemahan : Masa muda adalah waktu yang terbaik untuk mempelajari hakekat dari kebijakan/ kebenaran, usaha perolehan harta dan pengejaran ilmu pengetahuan. Berdasarkan kutipan sloka tersebut, sebagai generasi muda harus terus mengejar ilmu pengetahuan, salah satu contoh melalui pasraman Astika Sidhi. Bagi mereka yang dengan tekun mempelajari materi-materi yang diberikan di Pasraman maka akan menuai hasilnya. 2 Mewujudkan Kerukunan Generasi Muda Hindu Di Kabupaten Klaten Pasraman Astika Sidhi sebagai wadah atau tempat berkumpul generasi muda Hindu dari 47 pura yang ada di Klaten. Melalui pertemuan inilah terjadi komunikasi di antara generasi muda Hindu. Ketika ada salah satu pura di Kabupaten Klaten melaksanakan upacara persembahyangan piodalan, maka generasi muda yang ada di pura lain turut membantu membuat banten dan menata tempat untuk persembahyangan piodalan. Dari pertemuan di Pasraman Astika Sidhi tercetus kegiatan lain yaitu kunjungan bersama generasi muda Hindu ke pura yang ada di Klaten dan melakukan kerja bakti di pura yang dikunjungi. 2. Peran Pasraman Astika Sidhi Bagi peserta yang di daerahnya sangat sedikit umat Hindu khususnya generasi muda, melalui pasraman Astika Sidhi bisa merubah pola pikirnya bahwa mereka memiliki teman seumat, dari momentum ini maka terjadilah komunikasi yang berkesinambungan melalui sosial media. Melalui sosial media ini generasi muda Hindu saling berbagi kegiatan keumatan di daerah masing-masing sehingga terjadilah komunikasi yang harmonis dan solid. 3 Dampak Positif Pelaksanaan Pasraman Astika Sidhi Dalam Menumbuhkan Sradha dan Bhakti Generasi Muda Hindu Di Kabupaten Klaten Keberadaan pasraman Astika Sidhi memiliki dampak positif yang bisa dirasakan bagi generasi muda Hindu yang ada di Kabupaten Klaten. Upaya menumbuhkan sradha dan bhakti generasi muda Hindu melalui kegiatan yang telah dilaksanakan yaitu pemberian materi, motivasi, dan kepemimpinan menimbulkan dampak positif jangka panjang. Jurnal Widya Aksara Vol. 25 No. 2 September 2020 215 Dari uraian terdahulu tentang pelaksanaan dan peran pasraman Astika Sidhi penulis menarik beberapa poin dampak positif yang diterima bagi generasi muda Hindu di Kabupaten Klaten dari pelaksanaan pasraman Astika Sidhi. p p 1 Kaderisasi Pengurus Organisasi PGHD 3 Memotivasi Generasi Muda Hindu Di Klaten Setelah mendapatkan motivasi dari pembicara yang sudah berpengalaman tentang bagaimana menjadi generasi yang memiliki kualitas diri dari sudut pandang agama Hindu, para peserta menjadi lebih tekun dalam mempelajari ilmu agama dan ilmu pengetahuan lain yang positif. Hal ini terbukti para peserta lebih giat dan rajin mengikuti pasraman mingguan dan aktif mengikuti kegiatan keagamaan yang ada di Kabupaten Klaten. g g g y g p Kedudukan motivator dalam membakar semangat generasi muda Hindu sangat penting, karena dengan semangat dan kekompakan maka organisasi akan terus berkembang dan menuju kejayaan. Dengan tetap menjaga semangat kebersamaan PGHD tetap eksis di masyarakat, khususnya masyarakat Hindu yang ada di Kabupaten Klaten, bahkan keberadaan Peradah belum mampu menggeser eksistensi dari PGHD. Peserta pasraman Astika Sidhi yang ada di daerah yang termasuk kategori paling minor termotivasi menjadi lebih percaya diri karena mereka mengerti bahwa di luar daerahnya ternyata banyak generasi Hindu sehingga bisa mencegah terjadinya perpindahan agama karena pernikahan. Tidak dapat di pungkiri ketika anak muda sering bertemu lawan jenis maka akan timbul benih asmara, jika sering berkumpul dengan seumat peluang untuk mendapatkan pasangan hidup dengan umat semakin besar. Karena kasus perpindahan agama pada jenjang berumah tangga masih menjadi pekerjaan rumah petinggi agama Hindu di Indonesia. Apabila kegiatan seperti ini bisa dilakukan di daerah minor perpindahan agama melalui pernikahan akan sedikit berkurang dan harapannya kedepan tidak ada kasus perpindahan agama. p p 1 Kaderisasi Pengurus Organisasi PGHD Setelah melakukan kerja bakti generasi muda akan melakukan Dharma Tula dari sesepuh setempat dan ketua PGHD mengenai sejarah pura, pengetahuan agama Hindu, dan perkembangan generasi muda yang ada di Kabupaten Klaten. Kegiatan yang dihasilkan dari pertemuan di pasraman Astika Sidhi yaitu futsal putra dan putri umat Hindu yang ada di Klaten. Kegiatan ini adalah salah satu contoh telah terjalin kerukunan anatar generasi muda Hindu yang ada di Klaten. Jurnal Widya Aksara Vol. 25 No. 2 September 2020 216 III. KESIMPULAN Berdasarkan dari uraian tersebut dapat tarik kesimpulan sebagai berikut; Berdasarkan dari uraian tersebut dapat tarik kesimpulan sebagai berikut; 1.Pasraman Astika Sidhi menjadi kegiatan bersejarah di Kabupaten Klaten karena untuk pertama kali melaksanakan kegiatan pendidikan pasraman pada tahun 1999. 2.Persaudaraan Generasi Hindu Dharma (PGHD) Klaten adalah pencetus terbentuknya pasraman Astika Sidhi 1.Pasraman Astika Sidhi menjadi kegiatan bersejarah di Kabupaten Klaten karena untuk pertama kali melaksanakan kegiatan pendidikan pasraman pada tahun 1999. 2 Persaudaraan Generasi Hindu Dharma (PGHD) Klaten adalah pencetus terbentuknya 1.Pasraman Astika Sidhi menjadi kegiatan bersejarah di Kabupaten Klaten karen pertama kali melaksanakan kegiatan pendidikan pasraman pada tahun 1999. pertama kali melaksanakan kegiatan pendidikan pasraman pada tahun 1999. 2.Persaudaraan Generasi Hindu Dharma (PGHD) Klaten adalah pencetus terbentuknya pasraman Astika Sidhi. 3.Pasraman Astika Sidhi dibagi menjadi dua kategori yaitu kategori anak dan dewasa umum. Peran pelaksanaan pasraman Astika Sidhi dalam menumbuhkan sradha dan bhakti generasi muda Hindu di Kabupaten Klaten antara lain; 1.Turut serta mencerdaskan kehidupan bangsa dengan mendukung dan menjalankan Peraturan 1.Turut serta mencerdaskan kehidupan bangsa dengan mendukung dan menjalankan Peraturan Pemerintah Nomor 55 tahun2007 dan Peraturan Menteri Agama Nomor 56 Tahun 2014 2.Mempersiapkan calon pemimpin dari generasi muda Hindu yang memiliki kualitas bai 3.Menjadi wadah pemersatu generasi muda Hindu yang ada di daerah-daerah agar mereka merasa bahwa banyak teman dari pemuda Hindu. Menjadi ikon generasi muda Hindu di Kabupaten Klaten, yang selalu ditunggu pelaksanaannya. 3.Menjadi wadah pemersatu generasi muda Hindu yang ada di daerah-daerah agar mereka merasa bahwa banyak teman dari pemuda Hindu. Menjadi ikon generasi muda Hindu di Kabupaten Klaten, yang selalu ditunggu pelaksanaannya. Dari peran dan pelaksanaan pasaraman Astika Sidhi di Kabupaten Klaten menghasilkan beberapa dampak positif yang bisa dirasakan oleh generasi muda Hindu di Kabupaten Klaten, yaitu; 1.Kaderisasi pengurus organisasi Persaudaraan Generasi Hindu Dharma (PGHD) Klate p g g 2.Mewujudkan kerukunan generasi muda Hindu di Kabupaten Klaten. 3.Memotivasi generasi muda Hindu di Kabupaten Klaten untuk menjadi generasi yang memilik kualitas diri baik. 3.Memotivasi generasi muda Hindu di Kabupaten Klaten untuk menjadi generasi yang memilik kualitas diri baik. Jurnal Widya Aksara Vol. 25 No. 2 September 2020 217 DAFTAR PUSTAKA Agustiani, H. (2006). "Psikologi perkembangan pendekatan ekologi kaitannya dengan konsep diri dan penyesuaian diri pada remaja." Agustiani, H. (2006). "Psikologi perkembangan pendekatan ekologi kaitannya dengan konsep diri dan penyesuaian diri pada remaja." ani, L. (2014). "Peran Duta Wisata Dalam Mempromosikan Kebudayaan Dan ata Di Kalimantan Timur." 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W4234084555.txt	https://www.qeios.com/read/2COKBF/pdf	null		GNAI1 Gene	Definitions	2,020	cc-by	55	Qeios · Definition, February 7, 2020 Ope n Pe e r Re v ie w on Qe ios GNAI1 Gene National Cancer Institute Source National Cancer Institute. GNAI1 Gene. NCI T hesaurus. Code C38371. T his gene is plays a role in signal transduction and is involved in neurotransmission. Qeios ID: 2COKBF · https://doi.org/10.32388/2COKBF 1/1
https://openalex.org/W2010200938	https://europepmc.org/articles/pmc4245583?pdf=render	English	null	Increasing Iron and Zinc in Pre-Menopausal Women and Its Effects on Mood and Cognition: A Systematic Review	Nutrients	2,014	cc-by	11,504	nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients OPEN ACCESS nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients OPEN ACCESS Nutrients 2014, 6, 5117-5141; doi:10.3390/nu6115117 nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients Review Increasing Iron and Zinc in Pre-Menopausal Women and Its Effects on Mood and Cognition: A Systematic Review Karla A. Lomagno 1, Feifei Hu 2, Lynn J. Riddell 2, Alison O. Booth 2, Ewa A. Szymlek-Gay 2, Caryl A. Nowson 2 and Linda K. Byrne 1,* 1 School of Psychology, Deakin University, 221 Burwood Highway, Burwood, Melbourne, Victoria 3125, Australia; E-Mail: klomagno@deakin.edu.au 2 Centre for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, 221 Burwood Highway, Burwood, Melbourne, Victoria 3125, Australia; E-Mails: feifeih@deakin.edu.au (F.H.); lynn.riddell@deakin.edu.au (L.J.R.); alison.booth@deakin.edu.au (A.O.B.); ewa.szymlekgay@deakin.edu.au (E.A.S.-G.); caryl.nowson@deakin.edu.au (C.A.N.) * Author to whom correspondence should be addressed; E-Mail: linda.byrne@deakin.edu.au; Tel.: +61-3-9244-6424; Fax: +61-3-9244-6858. Received: 11 July 2014; in revised form: 15 October 2014 / Accepted: 16 October 2014 / Published: 14 November 2014 Abstract: Iron and zinc are essential minerals often present in similar food sources. In addition to the adverse effects of frank iron and zinc-deficient states, iron insufficiency has been associated with impairments in mood and cognition This paper reviews current OPEN ACCESS Nutrients 2014, 6, 5117-5141; doi:10.3390/nu6115117 1. Introduction Iron insufficiency, or mild iron deficiency (ID), in the absence of anaemia is defined as depleted iron stores. When the absence of iron stores affects erythropoiesis, this reduces haemoglobin (Hb) concentration and iron deficiency anaemia (IDA) occurs [1]. ID is the most prevalent single nutrient deficiency in the world, affecting not only those in economically developing countries, but also individuals in developed countries [2,3]. Pre-menopausal women are at greater risk of ID due to their regular menstrual blood loss and elevated iron requirements in pregnancy [4,5]. Iron and zinc are often present in similar dietary sources, such as whole-grain cereals, red meats, legumes, seeds and nuts, as well as seafood, and deficiencies in iron and zinc often co-occur [6–8]. However, data on the prevalence of zinc deficiency are lacking, due to the absence of a biochemical marker of total body zinc status. Plasma or serum zinc concentrations, the most widely used indicators of zinc status, are maintained within a narrow range (12–15 μmol/L) and require fasting blood samples for a meaningful test [7,9]. Hair zinc concentration is often used in combination with plasma zinc to provide a good indication of zinc status [10,11]. However, hair zinc alone is better for representing longer-term zinc status. Further, the use of hair zinc concentration as an index of zinc status suffers from limited reference data and variations due to age, sex, season, hair colour and hair growth rate [12]. Thus, alternative methods have been used to estimate zinc deficiency in populations based on reported dietary intake. For example, World Health Organization (WHO) [13] used a factorial approach to estimate the average basal and standard zinc requirements (i.e., minimum amount of zinc to cover losses in individuals adapted or not adapted to low usual dietary intakes of zinc) for population subgroups and then considered issues of zinc bioavailability from the typical diet in various regions of the world to derive estimates of the minimal dietary intake of zinc that would meet these requirements. Using this approach, WHO [14] estimates that zinc deficiency affects around one-third of the world’s population. Research suggests that ID is strongly linked to cognition, mood, zinc insufficiency and depression. However, the evidence of a causal link though intervention studies is still limited or the results have been mixed. Nutrients 2014, 6 Nutrients 2014, 6 indicates a positive effect of improving zinc status on enhanced cognitive and emotional functioning. However, further study involving well-designed randomized controlled trials is needed to identify the impact of improving iron and zinc status on mood and cognition. indicates a positive effect of improving zinc status on enhanced cognitive and emotional functioning. However, further study involving well-designed randomized controlled trials is needed to identify the impact of improving iron and zinc status on mood and cognition. Keywords: iron deficiency; zinc deficiency; pre-menopausal women; depression; cognition Keywords: iron deficiency; zinc deficiency; pre-menopausal women; depression; cognition Increasing Iron and Zinc in Pre-Menopausal Women and Its Effects on Mood and Cognition: A Systematic Review Karla A. Lomagno 1, Feifei Hu 2, Lynn J. Riddell 2, Alison O. Booth 2, Ewa A. Szymlek-Gay 2, Caryl A. Nowson 2 and Linda K. Byrne 1,* 1 School of Psychology, Deakin University, 221 Burwood Highway, Burwood, Melbourne, Victoria 3125, Australia; E-Mail: klomagno@deakin.edu.au 2 Centre for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, 221 Burwood Highway, Burwood, Melbourne, Victoria 3125, Australia; E-Mails: feifeih@deakin.edu.au (F.H.); lynn.riddell@deakin.edu.au (L.J.R.); alison.booth@deakin.edu.au (A.O.B.); ewa.szymlekgay@deakin.edu.au (E.A.S.-G.); caryl.nowson@deakin.edu.au (C.A.N.) * Author to whom correspondence should be addressed; E-Mail: linda.byrne@deakin.edu.au; Tel.: +61-3-9244-6424; Fax: +61-3-9244-6858. Received: 11 July 2014; in revised form: 15 October 2014 / Accepted: 16 October 2014 / Published: 14 November 2014 Abstract: Iron and zinc are essential minerals often present in similar food sources. In addition to the adverse effects of frank iron and zinc-deficient states, iron insufficiency has been associated with impairments in mood and cognition. This paper reviews current literature on iron or zinc supplementation and its impact on mood or cognition in pre-menopausal women. Searches included MEDLINE complete, Excerpta Medica Database (EMBASE), psychINFO, psychARTICLES, pubMED, ProQuest Health and Medical Complete Academic Search complete, Scopus and ScienceDirect. Ten randomized controlled trials and one non-randomized controlled trial were found to meet the inclusion criteria. Seven studies found improvements in aspects of mood and cognition after iron supplementation. Iron supplementation appeared to improve memory and intellectual ability in participants aged between 12 and 55 years in seven studies, regardless of whether the participant was initially iron insufficient or iron-deficient with anaemia. The review also found three controlled studies providing evidence to suggest a role for zinc supplementation as a treatment for depressive symptoms, as both an adjunct to traditional antidepressant therapy for individuals with a diagnosis of major depressive disorder and as a therapy in its own right in pre-menopausal women with zinc deficiency. Overall, the current literature 5118 2.1.2. Types of Studies Included studies were intervention, placebo-controlled trials with a control group assessing the effect of increasing iron or zinc on markers of mood or cognition. Studies were excluded if they investigated an unrelated disorder (e.g., iron/cognition/Alzheimer’s disease). The intervention period had to be at least 4 weeks in length, and the effects of the intervention on mood or cognition had to be assessed by a recognized assessment tool. 2. Method 2.1. Selection Criteria 2.1. Selection Criteria 2.1.1. Participants Participants were required to be adolescent girls and pre-menopausal women between the ages of 12 and 55 years. Participants were also required to exhibit evidence of either ID or IDA based on Hb concentration, Hb plus ferritin or other author-defined evidence of ID or IDA. No restrictions on participant levels of zinc deficiency were applied to studies evaluating increased zinc intake; however, participants were still required to be females aged between 12 and 55 years. Included studies were required to have a sample size larger than 10. Studies with a population younger than 12, or older than 55 years of age, or that included non-human subjects were also excluded. Studies that included some male participants were only included if the results for females were reported separately. 2.1.3. Outcome Measures Domains of cognitive functioning considered relevant for this review included attention, memory, learning, psychomotor skills and concentration. The mood domain of predominate focus in this review was depression, but also included anxiety, irritability and anger. 1. Introduction Furthermore, there is limited research assessing the effect of increasing the dietary intakes of both iron and zinc on markers of mood or cognition. This systematic literature review considers randomized controlled trials, investigating the effect of increasing either iron or zinc (either though supplementation or dietary methods) on markers of mood or cognition, primarily in pre-menopausal women. 5119 Nutrients 2014, 6 2.3. Screening Process After electronic searching was complete and duplicates removed, titles and keywords were scanned for relevance; articles were retrieved if these included a combination of iron and/or zinc and mood and/or cognition. Abstracts of the retrieved articles were further screened for relevance, removing papers that were reviews or commentaries. Full-text articles were read, and papers not meeting the inclusion criteria were removed. Nutrients 2014, 6 Nutrients 2014, 6 Nutrients 2014, 6 references of identified trials and relevant review articles were examined for eligible studies that may have been missed in the original search. references of identified trials and relevant review articles were examined for eligible studies that may have been missed in the original search. 2.4. Data Extraction The data extracted included the year of publication, randomization procedures, allocation concealment, blinding, setting, characteristics of participants (e.g., age, country and relevant demographics), characteristics of intervention, baseline iron/zinc status data, information regarding attrition/dropout rates and length of clinical trials. Furthermore, the characteristics of the main outcome measures, analytic methodology and relevant results were extracted for evaluation. Data were extracted by the review author and cross-referenced by another author (Linda K. Byrne). 3.1. Search Results A total of 2046 articles were retrieved from database searches, and a further nine were retrieved from additional sources (i.e., reference lists; academic colleagues). The titles and abstracts were screened for eligibility, and 30 full-text articles were assessed for inclusion. Eleven articles met the criteria for inclusion in this review. No articles in a language other than English were retrieved. In total, nine randomized controlled trials and two non-randomized controlled trials were included in the review (Figure 1). 3.2. Study Characteristics A detailed summary of the study characteristics is included in Table 1. Three studies were conducted in the United States [2,15,16]; two were in Poland [17,18], and one study was conducted each in Japan [19], India [20], South Africa [21], France [22], Switzerland [19] and New Zealand [23]. 2.2. Identification of Studies The original search was conducted in May, 2013 (Appendix 1). Originally, the focus of the review planned to include studies that used dietary methods to increase iron or zinc. However, due to a paucity of these types of studies, the search terms were modified to capture both dietary intake and oral iron supplementation of iron and zinc and to limit research to female participant groups (Appendix 2). Electronic searching was conducted using the following databases: ProQuest Health and Medical Complete, MEDLINE complete, Excerpta Medica Database (EMBASE), PubMed, psychINFO, psychARTICLES, Academic Search complete, Scopus and ScienceDirect between May 2013 and August 2013. Key terms (iron) AND (deficien* OR status OR intake OR “dietary intake” OR nutrition) and (zinc) AND (deficien OR status OR intake OR “dietary intake” OR nutrition) were searched in the title, abstract and keywords and key terms (mood OR affect OR depress) and (cognit), (wom?n OR “female”) in the subject. Iron and zinc were searched separately in relation to their effects on mood and cognition. The search was not limited by language, but included the use of a “peer review only” filter. Articles published between January 1995 and May 2013, were included in the search. The 5120 3.3. Participant Characteristics Eight studies included only pre-menopausal women [2,15,16,19,21,22,24]. Three included male participants [17,18,20]. Participants of one study included women who were six weeks postpartum [21], and two studies included participants that met the Diagnostic Statistical Manual of Mental Disorders— Fourth Edition Text Revision (DSM-IV-TR) criteria for major depressive disorder (MDD), differentiating these two populations from those of the rest of the included studies [17,18]. The sample sizes for studies were heterogeneous and ranged from between 20 and 219 participants. 5121 Nutrients 2014, 6 Figure 1. Flow chart of how the literature was selected for inclusion. 3.4. Design Of the 11 included studies, nine were randomized, placebo-controlled, double-bli procedures [2,15–17,19,21–24]. Of the remaining two studies, one was a non-randomize placebo-controlled trial and did not explain the blinding procedure [20], and the other did not explic the allocation process [18]. Three studies looked at the effects of oral iron supplementation cognition only [2,15,23], while three looked at the effects of oral iron supplementation on j mood [16,22,24]. A further two assessed both variables as outcomes [20,21]. No studies were found th examined the effect of increasing dietary intake of iron and any subsequent effect on mood or cognitio Records identified through database searching (n = 2046) Additional records identified through other sources (n = 9) Records after duplicates removed (n = 1619) Titles Screened (n = 1619) Records Excluded (n = 1416) Animals (n = 55) Age inappropriate (n=417) Confounding Disorder (n = 814) Abstracts Screened (n = 203) Articles excluded (n = 173) Reasons for Exclusion, non-research article, animal studies, participants male only, or out of age range Full-text articles assessed for eligibility (n = 30) Studies excluded (n = 19) Observational (n = 14) Published before 1995 (n = 2) Studies included in Systematic review (n = 11) Figure 1. Flow chart of how the literature was selected for inclusion. Figure 1. Flow chart of how the literature was selected for inclusion. 3.3. Participant Characteristics Additional records identified through other sources (n = 9) Records identified through database searching (n = 2046) Records after duplicates removed (n = 1619) Records Excluded (n = 1416) Animals (n = 55) Age inappropriate (n=417) Confounding Disorder (n = 814) Titles Screened (n = 1619) Abstracts Screened (n = 203) Studies excluded (n = 19) Observational (n = 14) Published before 1995 (n = 2) Full-text articles assessed for eligibility (n = 30) Full-text articles assessed for eligibility Studies included in Systematic review (n = 11) 3.4. Design 3.4. Design 3.6. Measures of Depression Outcome measures used to determine depression or depressive symptoms varied considerably between studies. A detailed summary of the outcome assessment tools used for each individual study is outlined in Table 2. Four studies measured depressive symptoms, pre-/post-intervention [17,18,22,24], while a further two assessed general mood state, using subscales of the measures to assess depressive symptoms [16,19,20]. One study used a measure that specifically assessed postpartum depression [21]. Nutrients 2014, 6 The iron status of participants varied between the studies, and Table 1 includes further information regarding the levels used to determine iron status in each individual study. Three studies included ID, IDA treatment and iron-sufficient control groups and examined differences in changes between all three groups [2,16,20]. One included participants with only IDA and excluded those with ID [21]. Four studies included only participants with ID, but failed to have iron-sufficient control groups [15,22–24]. Three intervention studies were found looking at the effects of zinc supplementation and depression. One of these looked specifically at the effect of zinc supplementation in depressive symptoms in pre-menopausal women [19]. The two papers remaining, focusing on zinc, looked at the effect of using zinc supplementation as an adjunct to antidepressant therapy. Additionally, both used participants that met the criteria for MDD [17,18]. Similar to the iron studies, no intervention studies were found that assessed the effect of dietary zinc on depressive symptoms. Furthermore, the search yielded no results for intervention trials that fit the inclusion criteria and that specifically examined the effects of zinc supplementation on cognitive functioning. 3.5. Measures of Cognition Outcome measures for cognitive function varied across studies, as did the domain of cognition assessed for each study. Most of the included studies measured multiple domains of cognitive functioning. These included attention, working memory and other aspects of memory (e.g., short-term memory (STM) and long-term memory (LTM)), learning and intellectual abilities. A detailed summary of cognitive outcome measures used in each study is described in Table 2. Three of the 11 included studies measured aspects of attention [2,15,23]. Five assessed aspects of memory [2,15,20,21,23]; three assessed intellectual ability [2,20,21], and two assessed learning [2,21]. 3.4. Design Of the 11 included studies, nine were randomized, placebo-controlled, double-blind procedures [2,15–17,19,21–24]. Of the remaining two studies, one was a non-randomized, placebo-controlled trial and did not explain the blinding procedure [20], and the other did not explicate the allocation process [18]. Three studies looked at the effects of oral iron supplementation on cognition only [2,15,23], while three looked at the effects of oral iron supplementation on just mood [16,22,24]. A further two assessed both variables as outcomes [20,21]. No studies were found that examined the effect of increasing dietary intake of iron and any subsequent effect on mood or cognition. 5122 3.7. Analytic Methodology Table 2 has a detailed summary of the statistical analyses applied in each study. Eight studies used analysis of variance (ANOVA) [2,15–18,20,21]; three used multiple linear regression [15,23,24]; one used Pearson’s correlation [16]; and one used a repeated measures analysis of covariance (ANCOVA), with intellectual ability (IQ) as a covariate [2]. One further study used Wilcoxon’s sign-ranked test [19] to assess differences. 5123 Nutrients 2014, 6 Nutrients 2014, 6 Table 1. Characteristic summary of the included studies (N = 11). ID, iron deficiency Table 1. Characteristic summary of the included studies (N = 11). ID, iron deficiency. Study n Population Iron or Zinc Intervention Baseline Iron or Zinc Status Study Duration and Dropouts Outcome Measures Bruner et al., 1996 [15] 81 American adolescent females, ID only, 13–18 years Daily dose of 1300 mg ferrous sulphate, equivalent to 260 mg elemental iron ID = serum ferritin <12 μg/L, with normal haemoglobin (Hb) 8 weeks 8 dropouts The Brief Test of Attention, Symbol Digits Modalities. 3.7. Analytic Methodology The Visual Search and Attention Test, The Hopkins Verbal Learning Test IDA ≤ Hb 115 g/L for African American girls or <Hb 120 g/L for caucasian girls Beard et al., 2005 [21] 95 South African mothers, 18–30 years Daily dose of 125 mg ferrous sulphate to anaemic mothers IDA = Hb 90–115 g/L + 2 of the following: mean corpuscular volume <80 femtoliters (fL), transferrin saturation <15%, serum ferritin <12 μg/L 9 months, postpartum 14 dropouts Edinburgh Post-Natal Depressive Scale, Ravens Coloured Matrices, Perceived Stress Scales Murray-Kolb and Beard, 2007 [2] 152 American women, 18–35 years 160 mg ferrous sulphate, containing 60 mg elemental iron ID = Hb ≥120 g/L and two or more other abnormal iron values 16 weeks 39 dropouts Cognitive Abilities Test IDA = Hb between 105 and 119 g/L and two or more other abnormal iron values McClung et al., [16] 219 Female American soldiers, 15–25 years 100 mg ferrous sulphate, 15.0 elemental iron ± 0.2 mg ID ≥ 2 of the following 3 indicators: serum ferritin <12 μg/L, transferrin saturation <16% or red blood cell distribution width >15% 8 weeks 48 dropouts Profile of Mood States IDA = ID + Hb of <120 g/L Verdon et al., 2003 [24] 144 French females, ID only, 18–55 years Ferrous sulphate, 80 mg elemental iron as pill ID = not transparent 4 weeks 12 dropouts Questionnaire bref d’auto-évaluation des dimensions dépressive, asthénique et anxieuse IDA = Hb <117 g/L 5124 Nutrients 2014, 6 Nutrients 2014, 6 Table 1. Cont. 3.9.1. Iron and Cognition Despite the heterogeneous nature of the included studies, all that looked at the effect of iron status on cognition found some form of improvement in cognitive functioning after iron supplementation. Of the five studies that assessed the effect of iron supplementation on measures of cognition, two examined the effect of iron status on attention at baseline. Murray-Kolb and Beard [2] reported that an increasing level of severity of ID was associated with decreased cognitive functioning. Specifically, they found that women with ID had a composite baseline score that was significantly worse on tests of attention, learning and memory when compared to iron-sufficient participants. Furthermore, the authors reported that women with IDA performed significantly worse on tasks of attention and memory in comparison to healthy controls. Similarly, Devaki et al. [20] reported that adolescents with ID and IDA had lower mean scores on tasks of memory and intellectual ability than healthy controls. Nutrients 2014, 6 5125 Nutrients 2014, 6 Nutrients 2014, 6 3.9. Results Table 3 includes a detailed summary of all of the relevant findings for the included studies. Six studies reported increased iron status, as measured by varied markers of iron status in participants treated with iron supplements [2,15,16,20–23]. It was unclear in one study whether iron status had increased in the iron-treated group [24]. Two zinc studies reported increased serum zinc concentrations, post-treatment with zinc supplementation [17,19]; however, this was unclear in the third zinc study [18]. 3.8. Evaluation of Validity Study validity is reported in Table 2. Randomization procedures were outlined in all but two studies [18,20]. The researcher was clearly blinded to intervention in nine studies; the outcome assessor was clearly blinded in four studies, and the participants were clearly blinded in nine studies. Masking was unclear in one study [21] and did not occur in one study [20]. There were dropouts in nine studies [2,15–18,21–24]. Reasons for the dropouts were reported for all but one study [19]. An intention-to-treat protocol was used in three studies [15,22,24]. It was unclear how all of the other studies dealt with missing data. Four important areas of bias, including selection, performance, detection and attrition biases, were informally assessed using a bias hierarchy checklist by Wright, Brand, Dunn and Spindler [25]. 3.7. Analytic Methodology Vaucher et al., 2012 [22] 198 Swiss females, ID only, 18–50 years 80 mg ferrous sulphate ID = Hb ≥120 g/L, and ferritin <50 µg/L 12 weeks 77 dropouts Current and Past Psychological Scale Lambert et al., 2002 [23] 121 Adolescent females from New Zealand, ID only, 12.5–17.9 years 105 mg elemental iron ID = Serum ferritin <12 μg/L, Hb >120 g/L 8 weeks 5 dropouts Hopkins Verbal Learning Test, Stroop Task Visual Search, Reading Span Task, Visual search task IDA = Serum ferritin <12 μg/L, Hb <120 g/L Devaki et al., 2009 [20] 120 Adolescent male and female students from India, 15–18 years Iron (III) hydroxide polymaltose complex, 100 mg elemental iron ID boys = Hb ≥115 g/L and transferrin saturation <16% 8 months dropouts not recorded Ravens Progressive Matrices, Weschler Adult Intelligence Scale, Emotional Quotient ID girls = Hb ≥105 g/L and transferrin saturation <16% IDA boys = Hb <115 g/L and transferrin saturation <16% IDA girls = Hb <105 g/L and transferrin saturation <16% Siwek et al., 2009 [17] 60 Polish males and females with major depressive disorder, 18–55 years 100–200 mg imipramine (antidepressant) + 25 mg zinc Serum zinc determined by flame atomic absorption spectrometry; baseline zinc deficiency was unclear 12 weeks 9 dropouts Hamilton Depression Rating Scale, Beck Depression Inventory, Clinical Global Impression Montgomery—Åsberg Depression Rating Scale Nowak et al., 2003 [18] 20 Polish males and females with major depressive disorder, 25–57 years clomipramine 125–150 mg, amitriptyline 125–150 mg, citalopram, 20 mg fluoxetine, 40–20 mg (all standard antidepressant treatment) + 25 mg zinc Unclear 12 weeks 6 dropouts Hamilton Depression Rating Scale, Beck Depression Inventory Sawada and Yokoi, 2010 [19] 60 Japanese females, non-anaemic, 18–21 years Multivitamins + 7 mg elemental zinc Serum zinc determined by inductively coupled argon plasma-mass spectrometer; no other details provided on this 10 weeks dropouts not recorded Profile of Mood States Table 1. Cont. Attention and Memory Of the five studies that assessed the effect of iron supplementation specifically on memory, only one found a significant effect. Murray-Kolb and Beard [2] reported that women who received iron supplementation and were considered serum ferritin responders, that is women who experienced a significant improvement in serum ferritin regardless of whether they initially had ID or IDA, also showed significant improvement on measures of attention. Furthermore, they reported that women who showed a significant improvement in Hb concentration completed tasks significantly faster than those who did not have a significant improvement in Hb. Neither of the studies by Bruner et al. [15] and Lambert et al. [23] detected significant improvement on measures of attention upon increasing iron status with iron supplementation. 5126 Nutrients 2014, 6 Nutrients 2014, 6 Table 2. Validity Characteristics of included studies. Table 2. Validity Characteristics of included studies. Selection Bias or Detection Bias Performance or Detection Bias Attrition Bias Controlled Variables Reviewer Notes/Possible Limitations al., 6] Randomization clear, double-blinded design Researchers and participants blinded to allocation Not all randomized included in outcomes; dropouts and reasons for dropouts reported Additional vitamins and minerals All participants had ID. There was no measurement of baseline differences between iron-sufficient and ID adolescents. Serum ferritin was a marker of iron status. Statistical analysis was by group assignment, rather than ferritin response. al., 1] Randomization clear, double-blinded design Participants unaware of group allocation Intention-to-treat protocol utilized to deal with missing data; dropouts reported; however, reasons for dropouts not reported Age, socioeconomic status, income, education, nutritional intakes Staff members administered supplementations at health clinics with instructions for the mothers on when to take and when to return, etc. Standardized outcome measures were used. Kolb rd, 2] Randomization clear, double-blind placebo-controlled design One researcher knew the group allocation; outcome assessors, as well as participants were blinded to group allocation Not all of those randomized included in outcomes; dropout rates reported; however, reasons for dropouts not reported Socioeconomic status, grade point average, level of physical activity and other demographics; other vitamins/minerals; inflammation, contraception and menstrual cycle; intellectual ability (IQ) was used as a covariate Standardized and validated measures of were cognition used. High reliability. Analysis was completed on women who were classified as ferritin responders or non-responders. Determination of the relationship between changes in iron status and changes in cognition. 16 weeks used to allow more time for brain iron concentrations to replenish. Attention and Memory Not all of those randomized included in outcomes; dropout rates reported; however, reasons for dropouts not reported 5127 Nutrients 2014, 6 Table 2. Cont. McClung et al., 2009 [16] Randomization reported, double-blind, placebo-controlled design Researchers, outcome assessors and participants all blinded to allocation Unclear if all of those randomized included in outcomes; dropout rates and reasons for dropouts reported Compliance monitored and assessed as a confounding variable. Research assistants administered capsules to participants each day. Treatment occurred on the background of battle combat training. Randomization procedures were not described. Verdon et al., 2003 [24] Randomization clear, double-blind design Researchers, outcome assessors and participants blind to allocation Intention-to-treat protocol completed to deal with missing data; dropout rates recorded and reasons for dropout recorded Other disorders that could explain fatigue Intervention period was relatively shorter than other included studies. A validated self-administered questionnaire was used, although fatigue was assessed on a visual analogue scale. Adherence to treatment was monitored by an electronic device that recorded the date and time that the pill container was opened Vaucher et al., 2012 [22] Multi-centre study, randomization clear, double-blind, placebo-controlled design Researchers, outcome assessors and participants blinded to allocation Intention-to-treat protocol completed to deal with missing data A validated self-administered questionnaire used. Intention-to-treat analysis was used, and attrition and compliance were less of an issue. Dropout rates were not reported Lambert et al., 2002 [23] Randomization occurred, but allocation procedures not described Researcher and participants blinded to allocation Unclear if randomized participants included in outcomes/dropout rates reported/reasons for dropouts reported Standardized and validated outcome measures were used. Unclear if all of those randomized included in outcomes; dropout rates and reasons for dropouts reported Researchers, outcome assessors and participants all blinded to allocation Researchers, outcome assessors and participants all blinded to allocation 5128 Nutrients 2014, 6 Table 2. Cont. Devaki et al., 2009 [20] No randomization described No blinding methods described Unclear if randomized participants included in outcomes; dropout rates and reasons for dropout reported Compliance was monitored as supplements were administered by a supervisor at breakfast and lunch. Potential for performance, selection and detection bias. Siwek et al., 2009 [17] Randomization clear Researchers and participants blinded to allocation Outpatient sample. Nutrients 2014, 6 Nutrients 2014, 6 Table 3. Summary of relevant results. HVLT, Hopkins Verbal Learning Test; MCV, mean corpuscular volume. 1: Iron Dependent Variable Domains Statistical Analysis Changes in Iron/Zinc Status Findings evant results. HVLT, Hopkins Verbal Learning Test; MCV, mean corpuscular volume. Table 3. Summary of relevant results. HVLT, Hopkins Verbal Learning Test; MCV Changes in Iron/Zinc Status Statistical Analysis Attention and Memory Nowak et al., 2003 [18] Randomization described Researchers and participants blinded to allocation Unclear if randomized participants included in outcome; dropout rates reported however reasons for dropout not reported Clinical sample, so it is difficult to generalize to wider population. Standardized and validated measures of depressive symptoms were used. Allocation procedures were not described. Sawada and Yokoi, 2010 [19] Randomization clear, A double-blind, double-blinded, placebo-controlled design Researchers and participants blinded to allocation Information around dropout rates and missing data was not reported Age, height, body mass, body weight Pilot study, first of its kind. There are missing data and information around how this was dealt with. Treatment was possibly confounded by the use of multivitamins, alongside zinc Table 2. Cont. 5129 Findings Significant increases occurred in ID iron treatment and IDA iron treatment groups for ferritin (p < 0.001 and p < 0.01, respectively) and body iron (p < 0.0001 and p < 0.01, respectively) The IDA iron-treated participants also had a significant increase in Hb (p < 0.0001), haematocrit (p < 0.001) and transferrin saturation (p = 0.018). Murray-Kolb and Beard, 2007 [2] Cognition IQ: Shipley Institute of Living Scale (20), Cognitive Abilities test (CAT) measuring 3 domains. Attention: Reaction Time Task. Memory: Reaction Time task (short-term memory (STM)), Probe Recall Task (recognition), Sternberg Memory Search Task (STM, retrieval and executive functioning). Learning: CAT Murray-Kolb and Beard, 2007 [2] Cognition IQ: Shipley Institute of Living Scale (20), Cognitive Abilities test (CAT) measuring 3 domains. Attention: Reaction Time Task. Memory: Reaction Time task (short-term memory (STM)), Probe Recall Task (recognition), Sternberg Memory Search Task (STM, retrieval and executive functioning). Learning: CAT ANCOVA, assessed differences between groups with IQ as a covariate; repeated-measures ANOVA, assessed change in cognitive variables over time Learning: Composite score on the learning domain showed that iron-sufficient group scored better than ID group, and there was a significant difference between iron-sufficient and IDA group (p = 0.013) and ID and Findings No significant effect on attention were found. Significant improvement on total recall scores of HVLT for girls who took iron treatment, compared to control group (p < 0.02). No significant differences were found between delayed recall and recognition. Learning: All participants recalled more words after each trial (F = 273.7), p < 0.001), although no significant differences were found between groups. Despite this, participants who took iron treatment performed better compared to the control group (F = 6.3, p < 0.02). Intention-to-treat analysis; multiple linear regression, assessment of post iron treatment cognitive scores; analysis of variance (ANOVA), assessed HVLT learning curve Iron girls: significantly higher serum ferritin concentration (18.2 (SD 12.6) vs. 3.5 (6.6) µg/L, p < 0.001) and higher mean Hb concentration (135 (8.0) vs. 127 (7.0) g/L, p < 0.001) Iron girls: significantly higher serum ferritin concentration (18.2 (SD 12.6) vs. 3.5 (6.6) µg/L, p < 0.001) and higher mean Hb concentration (135 (8.0) vs. 127 (7.0) g/L, p < 0.001) Iron treatment effected significant improvement on RPMs and Digit Symbol scores. Iron-treated mother’s scores were almost identical to non-anaemic mothers at 9 months. An association was found between —RPM scores and Hb, 10 weeks —Digit symbol test and MCV at 9 months Intellectual abilities: Raven’s Coloured Progressive Matrices (RPM). Memory: Digit Symbol Test Repeated measures ANOVA; Pearson’s correlation, strength of association between variables Results of EPDS were unclear. Pearson’s correlation found that scores on the EPDS Hb and MCV. Repeated Measures ANOVA; Pearson’s Correlation Nutrients 2014, 6 5130 Table 3. Cont. Cross-sectional baseline comparisons: Attention: Composite score significantly lower in IDA group, between iron sufficient and ID (p = 0.008; p = 0.003, respectively). Performance in attention significantly better than IDA group, in control group and iron-deficient groups (p = 0.047 and 0.008, respectively). Memory: Composite scores show that iron-sufficient and ID groups scored equally well and significantly better than IDA (p < 0.001). Performance in this domain showed the same pattern. Learning: Composite score on the learning domain showed that iron-sufficient group scored better than ID group, and there was a significant difference between iron-sufficient and IDA group (p = 0.013) and ID and Iron-treated participants were found to have significantly improved iron status. ANCOVA, assessed differences between groups with IQ as a covariate; repeated-measures ANOVA, assessed change in cognitive variables over time Ferritin level was increased for both placebo (t (58) = 3.73, p < 0.001) and iron group (t (56) = 9.16, p < 0.001). It was found that the extent of increase was larger in the iron group. A significant decrease in Hb concentration was found in the placebo group pre-/post-treatment (t (56) = 4.09, p < 0.001), and there was no change in Hb level for iron groups pre-/post-treatment (t (56) = 1.41, not significant). IDA group (p = 0.042). Longitudinal analysis: Significant improvement in serum ferritin (n = 66) was related to improvements in the attention and learning and memory domains, 5-/7-times, respectively, in women that were non-responders (n = 66) (p < 0.001). No significant correlation between the size of ferritin change and the cognitive improvement. Women who had significant change in Hb concentration (n = 33) completed the attention and memory tasks significantly faster than Hb non-responders (n = 80) (p < 0.001). 5131 Nutrients 2014, 6 Table 3. Cont. lung et al., 09 [16] Mood and physical performance General mood: Profile of Mood State (POMS) Two-way repeated measures ANOVA, treatment and time effects Participation in 8-wk battle combat training had effects on iron status, as shown by elevation (p < 0.05) of Hb, red blood cell distribution width and soluble transferrin receptor (sTfR) in both placebo and iron-treated groups. Serum ferritin was diminished (p < 0.05) in placebo, but not iron-treated groups. Group x Time interaction for vigour on the POMS: Iron supplementation had a significant effect on vigour, after Battle CombatTraining (BCT). After stratification by iron status at the beginning of BCT, the positive effects of time on mood stayed significant (p < 0.05) for all indicators, except anger, in the iron-deficient group don et al., 03 [24] Depression, anxiety and fatigue Depressive and anxiety symptoms: Questionnaire bref d’auto-évaluation des dimensions dépressive, asthénique et anxieuse Intention-to-treat protocol χ2 and linear regression Unclear The iron group showed the largest decrease in cumulative −4.6 (7.5) score for fatigue (−7.5 (8.0)), a difference of 3.0 points, 0.3 to 5.6, p = 0.03. Scores for depression were not significant between groups. Depression was not associated with any markers of iron status bert et al., 02 [23] Cognition Verbal Working Memory, free recall: Hopkins Verbal Learning Test. Attention: Stroop Task. Processing speed and attention: Visual Search. Working memory: Reading Span Task. Multiple regression analysis Ferritin level was increased for both placebo (t (58) = 3.73, p < 0.001) and iron group (t (56) = 9.16, p < 0.001). It was found that the extent of increase was larger in the iron group. A significant decrease in Hb concentration was found in the placebo group pre-/post-treatment (t (56) = 4.09, p < 0.001), and there was no change in Hb level for iron groups pre-/post-treatment (t (56) = 1.41, not significant). IDA group (p = 0.042). Hopkins Verbal Learning Test: For the iron group, significant improvement in recall of words from baseline to post-treatment (t (56) = 2.40, p = 0.01) in the second half list, but no significant relationship between iron treatment and the first half of the list. A significant relationship between Hb change and post-treatment performance on recall for the iron treatment group, second half of the list only (p < 0.004). Reading Span Test: Multiple regression found a significant relationship between change in serum ferritin and reading span (p < 0.01). Stroop Task: No relationship found between performance on this task with changed iron status or supplementation. Visual Search Task. No treatment x testing interaction found. Table 3. Cont. Table 3. Cont. Participation in 8-wk battle combat training had effects on iron status, as shown by elevation (p < 0.05) of Hb, red blood cell distribution width and soluble transferrin receptor (sTfR) in both placebo and iron-treated groups. Serum ferritin was diminished (p < 0.05) in placebo, b t t i t t d Group x Time interaction for vigour on the POMS: Iron supplementation had a significant effect on vigour, after Battle CombatTraining (BCT). After stratification by iron status at the beginning of BCT, the positive effects of time on mood stayed significant (p < 0.05) for all indicators, except anger, in the iron-deficient group Two-way repeated measures ANOVA, treatment and time effects Intention-to-treat protocol χ2 and linear regression Intention-to-treat protocol χ2 and linear regression Unclear Ferritin level was increased for both placebo (t (58) = 3.73, p < 0.001) and iron group (t (56) = 9.16, p < 0.001). It was found that the extent of increase was larger in the iron group. A significant decrease in Hb concentration was found in the placebo group pre-/post-treatment (t (56) = 4.09, p < 0.001), and there was no change in Hb level for iron groups pre-/post-treatment (t (56) = 1.41, not significant). Ferritin level was increased for both placebo (t (58) = 3.73, p < 0.001) and iron group (t (56) = 9.16, p < 0.001). It was found that the extent of increase was larger in the iron group. A significant decrease in Hb concentration was found in the placebo group pre-/post-treatment (t (56) = 4.09, p < 0.001), and there was no change in Hb level for iron groups pre-/post-treatment (t (56) = 1.41, not significant). g p treatment and the first half of the list. A significant relationship between Hb change and post-treatment performance on recall for the iron treatment group, second half of the list only (p < 0.004). Reading Span Test: Multiple regression found a significant relationship between change in serum ferritin and reading span (p < 0.01). Stroop Task: No relationship found between performance on this task with changed iron status or supplementation. Visual Search Task. No treatment x testing interaction found. 5132 Nutrients 2014, 6 Table 3. Cont. Table 3. Cont. Iron supplementation groups showed improvements in mean scores of STM, LTM WAIS and RPM, at four- and eight-month follow up, which also reflected changes in haematological parameters. Table 3. Cont. Treatment-resistant groups demonstrated lower levels of zinc than treatment- and non-treatment-resistant groups. A significant negative correlation between zinc levels and MADRS scores at Week 12 was found, when either all (p < 0.0001) or zinc supplemented patients were taken into the analysis Nowak et al., 2003 [18] Depression Depression Index: HDRS and BDI. Group differences were assessed using t-test and multiple ANOVA with two between-subjects factors (placebo vs. zinc treatment) Unclear Scores of the HDRS were significantly reduced over time: F (3, 28) = 5.091; p < 0.001, placebo, and F (3, 20) = 29.578 p < 0.001, zinc group. The group effect (F (1, 48) = 4.275, p = 0.049) and time effect (F (1, 48) = 21.683; p < 0.001) were statistically significant. There was no significant interaction effect. Zinc improved this reduction at Weeks 6 and 12, compared to treatment (ca. 55%). Zinc supplementation significantly improved the reduction in BDI scores at Week 12, compared to placebo (by 40%). Sawada and Yokoi, 2010 [19] Depression Somatic symptoms and mood feelings, like anxiety, sensitivity, anger and tension: Profile of Mood State (POMS) and Cornell Medical Index (CMI). Data were analysed with Wilcoxon’s signed-rank test Neither intervention showed a significant change in serum ferritin or Hb concentration. Multivitamin (MV) + zinc significantly increased serum zinc concentration, whereas MV alone did not. Women who took multivitamins with zinc showed a significant decrease in anger-hostility and depression-dejection scores on the POMS. Zinc-supplemented groups had a significant increase in serum zinc levels at Weeks 6 and 12. Non-zinc-supplemented groups saw an increase of zinc at Week 12. Treatment-resistant groups demonstrated lower levels of zinc than treatment- and non-treatment-resistant groups. Neither intervention showed a significant change in serum ferritin or Hb concentration. Multivitamin (MV) + zinc significantly increased serum zinc concentration, whereas MV alone did not. 2: Zinc Changes in Iron/Zinc Status Zinc-supplemented groups had a significant increase in serum zinc levels at Weeks 6 and 12. Non-zinc-supplemented groups saw an increase of zinc at Week 12. Treatment-resistant groups demonstrated lower levels of zinc than treatment- and non-treatment-resistant groups. A significant negative correlation between zinc levels and MADRS scores at Week 12 was found, when either all (p < 0.0001) or zinc supplemented patients were taken into the analysis A significant negative correlation between zinc levels and MADRS scores at Week 12 was found, when either all (p < 0.0001) or zinc supplemented patients were taken into the analysis Scores of the HDRS were significantly reduced over time: F (3, 28) = 5.091; p < 0.001, placebo, and F (3, 20) = 29.578 p < 0.001, zinc group. The group effect (F (1, 48) = 4.275, p = 0.049) and time effect (F (1, 48) = 21.683; p < 0.001) were statistically significant. There was no significant interaction effect. Zinc improved this reduction at Weeks 6 and 12, compared to treatment (ca. 55%). Zinc supplementation significantly improved the reduction in BDI scores at Week 12, compared to placebo (by 40%). Group differences were assessed using t-test and multiple ANOVA with two between-subjects factors (placebo vs. zinc treatment) Table 3. Cont. Changes in cognitive scores were much higher in the iron-deficient and iron-deficient anaemic group, compared to iron-sufficient, supplement group. Iron-sufficient, supplement group, ID and IDA groups showed significant increases in Hb, transferrin saturation (TS) and serum ferritin 8 months post-treatment (p < 0.01). Iron-sufficient, placebo group showed no increase in iron status. Devaki et al., 2009 [20] Cognition and Emotion (mood) Devaki et al., 2009 [20] Cognition and Emotion (mood) Cognition and Emotion (mood) 2 × 2 ANOVA with duration as one factor and study group as another 2 × 2 ANOVA with duration as one factor and study group as another 2 × 2 ANOVA with duration as one factor and study group as another No significant change in Emotion Quotient (EQ) score was seen for any group. After 6 weeks of iron treatment, significant effects on markers of iron status were found for iron-supplemented groups. Patients receiving iron supplement had a 3.5 point improvement (95% CI) in their fatigue score in current and past psychological scale compared to those in placebo group. Iron treatment had a significant effect on the global fatigue index from the Multidimensional Assessment of Fatigue Scale (p = 0.03) and its severity index (p = 0.03). Iron was not found to have a significant effect on anxiety or depression scores in this study. Hb: (3 g/L; p = 0.001). Ferritin: (6.8 μg/L; p = 0.01). MCV: (1.2 fL; p = 0.01). sTfR: (−0.4 mg/L; p < 0.001) transferrin saturation (6.6%; sTfR: (−0.4 mg/L; p < 0.001) transferrin saturation (6.6%; p < 0.001). Similar effects were seen after Week 12. 5133 Nutrients 2014, 6 Table 3. Cont. 2: Zinc Study Dependent Variable Domains Statistical Analysis Changes in Iron/Zinc Status Findings Siwek et al., 2009 [17] Depression Depressive Index scores on Clinical Global Impression (CGI), Montgomery-Åsberg Depression Rating Scale (MADRS), Hamilton Depression Rating Scale (HDRS), Beck Depression Inventory (BDI). Remission of depression was defined by a score of “very much” on CGI, plus scores of ≤7 on HDRS, ≤10 on MADRS or ≤9 on BDI. Linear model, mixed-design ANOVA, with repeated factor being test number, and between factors being treatment, antidepressant and treatment resistance. A non-treatment-resistant group and a treatment-resistant group formed. Zinc-supplemented groups had a significant increase in serum zinc levels at Weeks 6 and 12. Non-zinc-supplemented groups saw an increase of zinc at Week 12. Learning Two studies examined the effect of iron supplementation on learning. One study found an overall effect of learning on the Hopkins Verbal Learning Test (HVLT) scores between treatment and placebo group in groups of females with ID in the absence of anaemia [15]. Bruner et al. [15] noted that both iron and placebo groups recalled more words on the HVLT recall task after each successive trial; however, there was no significant difference between these groups. Although they found that both groups did better after intervention, it was noted that girls who received iron supplementation recalled more words on each trial than girls who received the placebo. Murray-Kolb and Beard [2] reported that serum ferritin responders showed significant improvement in their learning abilities. They reported that improvements in learning for ferritin responders were five- to seven-times greater than the improvement in women who did not have a significant increase in serum ferritin. Nutrients 2014, 6 All five studies assessing the effect of iron supplementation on memory found significant improvements on some aspects of memory regardless of whether the participants had been classified as having ID or IDA at the beginning of the clinical trial. Differences in dose ranged between 100 mg elemental iron once per day [14] to 130 mg elemental iron twice daily [7]. Further information on dosage can be found in Table 1. Murray-Kolb and Beard [2] administered a supplement including 160 mg ferrous sulphate with 60 mg of elemental iron once daily. Four studies found improvement on tasks of working memory after treatment with iron supplementation [2,15,21,23]. Additionally, Devaki et al. [20] reported a significant improvement on tasks of STM and LTM after iron intervention. Both Bruner et al. [15] and Lambert et al. [23] reported significant improvements on verbal working memory post-iron treatment, even in participants with ID. Murray-Kolb and Beard [2] found no significant differences between iron ID participants and iron-sufficient controls on measures of memory; however, participants who were serum ferritin responders did significantly improve on these tasks. Lambert et al. [23] also reported a relationship between changes in Hb concentration and improvement in recall of recently heard words, changes in serum ferritin and improvement in reading span task and post-treatment ferritin level and post-treatment reading-span performance. Similarly, Beard et al. [21] reported significant correlations between mean corpuscular volume (MCV) and performance on the digit symbols task at nine months postpartum. Unclear Neither intervention showed a significant change in serum ferritin or Hb concentration. Multivitamin (MV) + zinc significantly increased serum zinc concentration, whereas MV alone did not. Women who took multivitamins with zinc showed a significant decrease in anger-hostility and depression-dejection scores on the POMS. Data were analysed with Wilcoxon’s signed-rank test 5134 3.9.2. Iron and Mood Studies investigating whether increasing iron status effects changes on parameters of mood were mixed, and those presented within this literature review should be interpreted with caution. Two studies found no association between changes in iron status and depressive symptoms or other mood aspects, including anxiety [22,24]. McClung et al. [16] reported that battle combat training resulted in significant improvement in mood state over time on all subscales of the Profile of Mood State (POMS) and the total score for both groups. Despite this, the only subscale in which there was a significant group by time interaction was for vigour, indicating that this was the only subscale on which iron supplementation had any effect [16]. Alternatively, Beard et al. [21] reported finding a 25% improvement in previously iron-deficient mothers’ depressive scale following an iron supplement compared with a placebo- controlled group. Multivariate analysis suggested a strong link between the Edinburgh Post-Natal Depression Scale (EPNDS) and markers of iron status, specifically mean corpuscular volume and Hb concentration [21]. 3.9.3. Zinc and Depression Two studies assessed the efficacy of zinc supplementation as an adjunct to antidepressant treatment for depression. Nowak et al. [18] found statistically significant group effects (treatment vs. placebo) and time effects (pre-treatment vs. post treatment) for zinc supplementation as an adjunct to antidepressant therapy. It was observed that zinc supplementation significantly augmented the reduction in Hamilton Depression Rating Scale score at Week 12 of treatment [18]. Similarly, Siwek et al. [17] found the zinc supplementation as an adjunct to tricyclic antidepressant treatment significantly reduced depressive symptoms measured by the Montgomery–Åsberg depression rating scale (MDRAS). They reported this result in participants who were deemed treatment resistant. The same pattern was not observed in depressed individuals who were treatment responsive. Only one study was found that examined the effect of zinc supplementation on mood states. Sawada and Yokoi [19] discovered that individuals who were provided with multivitamin (A, D, B1, B2, B6, B12, niacin and folic acid) supplements containing 7 mg added of elemental zinc significantly decreased scores on anger-hostility and depression-dejection scales on the POMS (p < 0.05). Nutrients 2014, 6 5135 Nutrients 2014, 6 3.9.2. Iron and Mood Intellectual Ability Beard et al. [21] found significant improvements on Ravens Progressive Matrices (RPM) scores, for mothers receiving iron supplementation, and they noted that their scores were almost identical to those of control non-anaemic mothers at the end of the treatment period. They also report that anaemic mothers who did not receive iron had no change in cognitive performance over time. Furthermore, they noted a correlation between increased Hb concentration at 10 weeks and improved scores on the RPM. Similarly, Devaki et al. [20] observed that iron supplementation in adolescents with either ID or IDA was associated with significant improvement in intellectual ability in comparison to healthy controls receiving iron or healthy controls with placebo. Nutrients 2014, 6 lowered iron status. Memory, particularly working memory and intellectual ability, may be affected by states of iron insufficiency and are potentially improved as iron status is improved. Furthermore, conclusions about the influence of low iron on attention and learning in premenopausal women cannot be made based on this review alone, and further research is required to fully understand these results [2]. These findings are somewhat in contrast to those reported by a meta-analysis on a similar topic. Falkingham et al. [26] reported no evidence to suggest any overall effect on memory, psychomotor skills or scholastic achievement in either older children or pre-menopausal women, but did find effects of iron supplementation on aspects of attention and intelligence. In a recent review, Murray-Kolb [27] discussed a number of intervention studies on pre-menopausal women and reported on five that found improvements on differing measures of memory after iron supplementation. Furthermore, Murray-Kolb discussed a number of observational studies and reported a correlation between memory, attention, learning and spatial ability. There is still a relative paucity of research in this area highlighting the need for carefully-designed, randomized controlled trials. The effect of iron supplementation and its role in improving mood states is not clear, and the interpretation of studies to date is hindered by the range of instruments used to measure mood. Two of the four studies that looked at the effect of increasing iron on mood found no significant improvement of depressive symptoms after iron supplementation, despite a significant increase in iron status [22,24]. However, these studies were only exploring the effect of iron supplementation on depressive symptoms as a secondary outcome, and the inclusion of more comprehensive depressive scales may have yielded different results. On the other hand, a number of previous studies have observed a relationship between low iron status and mood, indicating a potential role for iron in the development of mild depressive symptoms [28,29]. The study by Beard et al. [21] reported a significant improvement in depressive symptoms after iron supplementation. The participants of this study did not have a diagnosis of MDD, and the researchers were looking specifically at the risks of ID in developing postpartum depression. Nonetheless, the results of this study appear to indicate that women with IDA may be susceptible to developing postpartum depression and that treatment with iron can reverse these symptoms. McClung et al. 4. Discussion The current systematic literature review found five studies that suggested reduced iron status, regardless of whether it is ID or IDA, has a detrimental impact on cognitive function in pre-menopausal women. All five studies found that increasing iron through supplementation had a positive influence on the performance on tasks measuring certain aspects of memory. Four of these studies found significant improvements on tasks of working memory [2,15,21,23], while one noted improvements on tasks of STM and LTM [20]. Only one of the five studies examining the effects of increasing iron status on cognitive functioning found a positive effect on attention [2]. Furthermore, some improvement was found in learning ability in women who were serum ferritin responders, but again, only in one study [2]. In addition, two studies found significant improvements in intellectual ability [20,21]. The results of the current literature review suggest that some, but not all, aspects of cognitive function are impacted by 5136 Nutrients 2014, 6 need to be interpreted with caution; however, they do suggest a role for zinc in the treatment of mild depressive symptoms in the absence of a confounding antidepressant treatment. That being said, both the treatment and placebo groups were provided with additional multivitamins, so intervention studies using only zinc as a treatment are required to clearly elucidate the role of zinc in the treatment of depression. While no studies were found looking at the association between low zinc status and cognitive function, there is evidence to suggest that there is a potential role for zinc in improving aspects of cognitive ability [31–33]. For example, Keller, Chu and Coffield [31] observed that pregnant women with zinc deficiency performed significantly worse on the Raven’s Progressive Matrices than those without zinc deficiency. Furthermore, Maylor et al. [34] in a randomized controlled trial on cognitive function in healthy middle-aged and older adults (>70) found a significant positive effect of zinc supplementation on spatial working memory, but a detrimental effect on one measure of attention. As with all of the variables included within this review, further well-designed randomized controlled trials in adults are warranted to better understand the causal relationship between zinc and cognition. Heterogeneity among the included studies with regard to population and outcome measures used make it difficult to compare the results completely. Only one study controlled for the potential effect of inflammation, as measured by C-reactive protein, on iron status in participants [2]. This is a useful variable to control for, as inflammation caused by chronic disease (e.g., tuberculosis or endocarditis), autoimmune disease (e.g., Crohn’s disease), obesity or exercise can negatively impact iron status and in itself can result in ID [35]. Furthermore, Murray-Kolb and Beard [2] were the only researchers to use IQ as a covariate when looking at learning ability and intellectual functioning. Despite this, most studies matched their participants for socioeconomic status, education, age and physical activity, and most of the studies had procedures to monitor compliance. Generally, all studies were at low risk of selection, performance, detection and attrition biases, as randomization or allocation procedures were made explicit; blinding after allocation was explicit and, for the most part, attrition rates were reported. However, it should be noted that the study by Devaki et al. Nutrients 2014, 6 [16] found a significant positive effect over time of iron supplementation on scores on the POMS for the subscale vigour, but failed to find any similar effect for the depression subscale. While results are mixed, previous research suggests that there is a relationship between low iron status and mood. For example, Vahdat Shariatpanaahi et al. [28] report finding that serum ferritin levels were significantly lower (11 μg/L) in individuals with depression in comparison to healthy controls. Furthermore, they reported that the incidence of ID was higher in depressed individuals by 15%. Similarly, a study on postpartum depression discovered a relationship between lowered Hb concentration and depressive symptoms in comparison to mothers with a normal Hb concentration postpartum [29]. This area of research warrants well-designed randomized controlled trials with a large population sample utilizing standardized and valid measures of depressive symptoms. Clinical intervention trials looking at the effect of zinc supplementation on aspects of mood are limited, and no intervention trials looking at the effect of zinc supplementation on improving cognition were found using the current search criteria. Results for zinc studies found in this review are similar to that of Lai et al. [30], as little research in this area has been conducted since then. Zinc has been found to be effective in the treatment of depressive symptoms, both as an adjunct to traditional antidepressant medication and as a stand-alone treatment. The findings by Sawada and Yokoi [19] are preliminary and 5137 5. Conclusions The current review has explored the effect of increasing elemental iron, or zinc, on aspects of mood and cognition. Some evidence has been found to suggest a positive effect of increasing iron status on cognitive functioning. It also seems that this positive effect occurs regardless of whether the participant was initially iron insufficient or iron deficient with anaemia. Results are mixed as to whether iron deficiency impacts on general mood; however, further research in this area is warranted. The current review also found some evidence to suggest that increasing zinc may have a positive impact on mood. Further study in the area, utilizing well-designed randomized controlled designs, is needed in order to strengthen the evidence suggesting a causal relationship. Given the risk posed for women of child bearing age for iron and zinc deficiencies, it would be useful to have further research specific to this population group. Furthermore, research exploring the effectiveness of using dietary approaches to increasing iron and zinc in pre-menopausal women to improve cognitive functioning and mood states is warranted. Acknowledgments The authors would like to acknowledge the contribution of the Meat and Livestock Association Ltd. (Sydney, Australia). Nutrients 2014, 6 [20] is at risk of selection, performance and detection biases, because randomization of participants did not occur, nor were the investigators or subjects blinded to the group in which they were. While all studies included control groups, Bruner et al. [15] split their ID participants into control or treatment groups and, thus, were unable to measure baseline differences between normal status and iron-deficient adolescents. Devaki et al. [20] had a supplemented control group; however, they neglected to have iron-deficient or anaemic participants take a placebo. Therefore, their study was not truly placebo controlled. The current search yielded no results looking at increasing zinc and its effects on cognitive functioning and limited results looking at zinc supplementation and its effects on depression. Secondly, no studies were found that used dietary methods to increase iron or zinc, and lastly, there was limited literature looking specifically at women of childbearing age. As a consequence, the search terms for this review were broadened to include oral iron supplementation, as well as male participants. This means that the results of the current review cannot be generalized specifically to pre-menopausal women, nor can it comment on the effectiveness of increasing dietary iron and zinc on improved mood or cognitive functioning, which was the original intended purpose of this review. Thus, studies investigating the relationship between dietary iron and zinc on markers of mood and cognition, and whole of diet interventions aimed at improving neuropsychological functioning are required. Nutrients 2014, 6 Nutrients 2014, 6 5138 Author Contributions All authors contributed to the conception and intellectual content of the paper. All authors contributed to the editing and final approval of the paper. Appendix 1. Initial Search Strategies From Proquest Health and Medical complete search. Iron/mood/cognition: (ab((iron) AND (deficien* OR status OR intake OR dietary OR nutrition)) AND peer(yes)) AND ((su((mood) OR (depress) OR (affect)) AND peer(yes)) OR (cognit AND peer(yes))) AND (su((wom?n) OR (“female”)) AND peer(yes)) Zinc/mood/cognition: ((all(“zinc deficien” OR “low zinc” OR “zinc store” OR “zinc status” OR “decree zinc” OR “deplete* zinc” OR “dietary intake of zinc”) AND peer(yes)) AND (ab(mood OR affect* OR depress) AND peer(yes))) OR ((all(“zinc deficien” OR “low zinc” OR “zinc store” OR “zinc status” OR “decree zinc” OR “deplete* zinc” OR “dietary intake of zinc”) AND peer(yes)) AND (ab(Cognit) AND peer(yes))) Appendix Appendix 1. Initial Search Strategies From Proquest Health and Medical complete search. Iron/mood/cognition: (ab((iron) AND (deficien OR status OR intake OR dietary OR nutrition)) AND peer(yes)) AND ((su((mood) OR (depress) OR (affect)) AND peer(yes)) OR (cognit AND peer(yes))) AND (su((wom?n) OR (“female”)) AND peer(yes)) Zinc/mood/cognition: ((all(“zinc deficien” OR “low zinc” OR “zinc store” OR “zinc status” OR “decree zinc” OR “deplete* zinc” OR “dietary intake of zinc”) AND peer(yes)) AND (ab(mood OR affect* OR depress) AND peer(yes))) OR ((all(“zinc deficien” OR “low zinc” OR “zinc store” OR “zinc status” OR “decree zinc” OR “deplete* zinc” OR “dietary intake of zinc”) AND peer(yes)) AND (ab(Cognit) AND peer(yes))) Appendix 2. Refined Search Strategies From Proquest Health and Medical Complete search. Iron/mood/cognition/women: (ab((iron) AND (deficien OR status OR intake OR dietary OR nutrition)) AND peer(yes)) AND ((su((mood) OR (depress) OR (affect)) AND peer(yes)) OR (cognit AND peer(yes))) AND (su((wom?n) OR (“female”)) AND peer(yes)) Appendix 1. Initial Search Strategies References 1. Food and Agriculture Organization of the United Nations/World Health Organization. Food-based approaches to meeting vitamin and mineral needs. In Human Vitamin and Mineral Requirements; Food and Agriculture Organization of the United Nations/World Health Organization: Rome, Italy, 2002; Chapter 2, pp. 7–25. 2. Murray-Kolb, L.E.; Beard, J.L. Iron treatment normalizes cognitive functioning in young women. Am. J. Clin. Nutr. 2007, 85, 778–787. 2. Murray-Kolb, L.E.; Beard, J.L. Iron treatment normalizes cognitive functioning in young women. Am. J. Clin. Nutr. 2007, 85, 778–787. 3. McLean, E.; Cogswell, M.; Egli, I.; Wojdyla, D.; de Benoist, B. Worldwide prevalence of anaemia, who vitamin and mineral nutrition information system, 1993–2005. Public Health Nutr. 2008, 12, 444–454. 4. McClung, J.P.; Murray-Kolb, L.E. Iron nutrition and premenopausal women: Effects of poor iron status on physical and neuropsychological performance. Annu. Rev. Nutr. 2013, 33, 271–288. 4. McClung, J.P.; Murray-Kolb, L.E. Iron nutrition and premenopausal women: Effects of poor iron status on physical and neuropsychological performance. Annu. Rev. Nutr. 2013, 33, 271–288. 5. Beutler, E. Disorders of iron metabolism. In Williams Hematology; Kaushansky, K., Lichtman, M.A., Beutler, E., Kipps, T.J., Seligsohn, U., Prchal, J.T., Eds.; McGraw-Hill: New York, NY, USA, 2010; pp. 565–606. 5. Beutler, E. Disorders of iron metabolism. In Williams Hematology; Kaushansky, K., Lichtman, M.A., Beutler, E., Kipps, T.J., Seligsohn, U., Prchal, J.T., Eds.; McGraw-Hill: New York, NY, USA, 2010; pp. 565–606. 6. Gibson, R.S.; Heath, A.L.; Ferguson, E.L. Risk of suboptimal iron and zinc nutriture among adolescent girls in Australia and New Zealand: Causes, consequences, and solutions. Asia Pac. J. Clin. Nutr. 2002, 11 (Suppl. 3), 543–552. 7. Lim, K.; Riddell, L.; Nowson, C.; Booth, A.; Szymlek-Gay, E. Iron and zinc nutrition in the economically-developed world: A review. Nutrients 2013, 5, 3184–3211. 8. Kordas, K.; Stoltzfus, R.J.; López, P.; Rico, J.A.; Rosado, J.L. Iron and zinc supplementation does not improve parent or teacher ratings of behavior in first grade Mexican children exposed to lead. J. Pediatr. 2005, 147, 632–639. 9. King, J.C. Assessment of zinc status. J. Nutr. 1990, 120 (Suppl. 11), 1474–1479. 10. Sanstead, H.H. Subclinical zinc deficiency impairs human brain function. J. Trace Elem. Med. Biol. 2012, 26, 70–73. 11. Sandstead, H.H.; Prasad, A.S.; Penland, J.G.; Beck, F.W.J.; Kaplan, J.; Egger, N.G.; Alcock, N.W.; Carroll, R.M.; Ramanujam, V.M.S.; Dayal, H.H.; et al. Zinc deficiency in Mexican American children: Influence of zinc and other micronutrients on T cells, cytokines, and antiinflammatory plasma proteins. Am. Nutrients 2014, 6 5139 Zinc/mood/cognition/women: ((zinc) AND (deficien OR status OR intake OR dietary)) AND (su((mood) OR (depress) OR (affect)) OR cognit) AND su((wom?n) OR (“female”)) Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. 12. Hotz, C.; Brown, K.H. Assessment of the Risk of Zinc Deficiency in Populations and Options for Its Control; International Nutrition Foundation: Tokyo, Japan, 2004. Appendix 2. Refined Search Strategies From Proquest Health and Medical Complete search. From Proquest Health and Medical Complete search. Iron/mood/cognition/women: (ab((iron) AND (deficien* OR status OR intake OR dietary OR nutrition)) AND peer(yes)) AND ((su((mood) OR (depress) OR (affect)) AND peer(yes)) OR (cognit AND peer(yes))) AND (su((wom?n) OR (“female*”)) AND peer(yes)) Nutrients 2014, 6 References J. Clin. Nutr. 2008, 88, 1067–1073. 12. Hotz, C.; Brown, K.H. 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Randomized, double-blind, placebo-controlled trial of iron supplementation in female soldiers during military training: Effects on iron status, physical performance, and mood. Am. J. Clin. Nutr. 2009, 90, 124–131. 17. Siwek, M.; Dudek, D.; Paul, I.A.; Sowa-Kućma, M.; Zięba, A.; Popik, P.; Pilc, A.; Nowak, G. Zinc supplementation augments efficacy of imipramine in treatment resistant patients: A double blind, placebo-controlled study. J. Affect. Disord. 2009, 118, 187–195. 18. Nowak, G.; Siwek, M.; Dudek, D.; Zieba, A.; Pilc, A. Effect of zinc supplementation on antidepressant therapy in unipolar depression: A preliminary placebo-controlled study. Pol. J. Pharmacol. 2003, 55, 1143–1147. 19. Sawada, T.; Yokoi, K. Effect of zinc supplementation on mood states in young women: A pilot study. Eur. J. Clin. Nutr. 2010, 64, 331–333. 20. Devaki, P.B.; Chandra, R.K.; Geisser, P. Effects of oral iron (III) hydroxide polymaltose complex supplementation on hemoglobin increase, cognitive function, affective behavior and scholastic performance of adolescents with varying iron status: A single centre prospective placebo controlled study. Arzneimittelforschung 2009, 59, 303–310. 21. Beard, J.L.; Hendricks, M.K.; Perez, E.M.; Murray-Kolb, L.E.; Berg, A.; Vernon-Feagans, L.; Irlam, J.; Isaacs, W.; Sive, A.; Tomlinson, M. Maternal iron deficiency anemia affects postpartum emotions and cognition. J. Nutr. 2005, 135, 267–272. 22. Vaucher, P.D.M.; Druais, P.-L.M.D.; Waldvogel, S.M.D.; Favrat, B.M.D. Effect of iron supplementation on fatigue in nonanemic menstruating women with low ferritin: A randomized controlled trial. Can. Med. Assoc. J. 2012, 184, 1247–1254. 23. Lambert, A.; Knaggs, K.; Scragg, R.; Metcalf, P.; Schaaf, D. 25. Wright, R.W.; Brand, R.A.; Dunn, W.; Spindler, K.P. How to write a systematic review. Clin. Orthop. Relat. Res. 2007, 455, 23–29. References Effects of iron treatment on cognitive performance and working memory in non-anaemic, iron-deficient girls. N. Z. J. Psychol. 2002, 31, 19. 24. Verdon, F.; Bonard, C.; Graft, M.; Michaud, A.; Bischoff, T.; de Vevey, M.; Studer, J.P.; Herzig, L.; Chapuis, C.; Tissot, J.; et al. Iron supplementation for unexplained fatigue in non-anaemic women: Double blind randomised placebo controlled trial. BMJ 2003, 326, doi:10.1136/bmj.326.7399.1124. 25. Wright, R.W.; Brand, R.A.; Dunn, W.; Spindler, K.P. How to write a systematic review. Clin. Orthop. Relat. Res. 2007, 455, 23–29. Nutrients 2014, 6 5141 Nutrients 2014, 6 26. Falkingham, M.; Abdelhamid, A.; Curtis, P.; Fairweather-Tait, S.; Dye, L.; Hooper, L. The effects of oral iron supplementation on cognition in older children and adults: A systematic review and meta-analysis. Nutr. J. 2010, 9, doi:10.1186/1475-2891-9-4. 26. Falkingham, M.; Abdelhamid, A.; Curtis, P.; Fairweather-Tait, S.; Dye, L.; Hooper, L. The effects of oral iron supplementation on cognition in older children and adults: A systematic review and meta-analysis. Nutr. J. 2010, 9, doi:10.1186/1475-2891-9-4. 27. Murray-Kolb, L.E. Iron status and neuropsychological consequences in women of reproductive age: What do we know and where are we headed? J. Nutr. 2011, 141, 747–755. 28. Shariatpanaahi, M.V.; Shariatpanaahi, Z.V.; Moshtaaghi, M.; Shahbaazi, S.H.; Abadi, A. The relationship between depression and serum ferritin level. Eur. J. Clin. Nutr. 2007, 61, 532–535. 29. Corwin, E.J.; Murray-Kolb, L.; Beard, J. Low hemoglobin level is a risk factor for post-partum depression. J. Nutr. 2003, 133, 4139–4142. 30. Lai, J.; Moxey, A.; Nowak, G.; Vashum, K.; Bailey, K.; McEvoy, M. The efficacy of zinc supplementation in depression: Systematic review of randomised controlled trials. J. Affect. Disord. 2012, 136, 31–39. 31. Keller, K.A.; Chu, Y.; Grider, A.; Coffield, J.A. Supplementation with l-histidine during dietary zinc repletion improves short-term memory in zinc-restricted young adult male rats. J. Nutr. 2000, 130, 1633–1640. 32. Massaro, T.F.; Mohs, M.; Fosmire, G. Effects of moderate zinc deficiency on cognitive performance in young adult rats. Physiol. Behav. 1982, 25, 117–121. 33. Stoecker, B.J.; Abebe, Y.; Hubbs-Tait, L.; Kennedy, T.S.; Gibson, R.S.; Arbide, I.; Teshome, A.; Westcott, J.; Krebs, N.F.; Hambidge, K.M. Zinc status and cognitive function of pregnant women in Southern Ethiopia. Eur. J. Clin. Nutr. 2009, 63, 916–918. 34. Maylor, E.A.; Simpson, E.E.A.; Secker, D.L.; Meunier, N.; Andriollo-Sanchez, M.; Polito, A.; Stewart-Knox, B.; McConville, C.; O’Connor, J.; Coudray, C. References Effects of zinc supplementation on cognitive function in healthy middle-aged and older adults: The zenith study. Br. J. Nutr. 2006, 96, 752–760. 35. Gaffney-Stomberg, E.; McClung, J.P. Inflamation and diminished iron status: Mechanisms and functional outcomes. Curr. Opin. Clin. Nutr. Metab. Care 2012, 15, 605–613. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2782159703	https://www.nature.com/articles/s41598-019-40335-w.pdf	English	null	Altering brain dynamics with transcranial random noise stimulation	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	7,581	Altering brain dynamics with transcranial random noise stimulation Received: 15 December 2017 Accepted: 14 February 2019 Published: xx xx xxxx Received: 15 December 2017 Accepted: 14 February 2019 Published: xx xx xxxx Onno van der Groen1,2, Jason B. Mattingley3,4,5 & Nicole Wenderoth1 Onno van der Groen1,2, Jason B. Mattingley3,4,5 & Nicole Wenderoth1 Random noise can enhance the detectability of weak signals in nonlinear systems, a phenomenon known as stochastic resonance (SR). This concept is not only applicable to single threshold systems but can also be applied to dynamical systems with multiple attractor states, such as observed during the phenomenon of binocular rivalry. Binocular rivalry can be characterized by marginally stable attractor states between which the brain switches in a spontaneous, stochastic manner. Here we used a computational model to predict the effect of noise on perceptual dominance durations. Subsequently we compared the model prediction to a series of experiments where we measured binocular rivalry dynamics when noise (zero-mean Gaussian random noise) was added either to the visual stimulus (Exp. 1) or directly to the visual cortex (Exp. 2) by applying transcranial Random Noise Stimulation (tRNS 1 mA, 100–640 Hz zero -mean Gaussian random noise). We found that adding noise significantly reduced the mixed percept duration (Exp. 1 and Exp. 2). Our results are the first to demonstrate that both central and peripheral noise can influence state-switching dynamics of binocular rivalry under specific conditions (e.g. low visual contrast stimuli), in line with a SR-mechanism. Noise is detrimental for the transfer of information in linear systems1. However, in nonlinear systems such as the brain, noise can enhance information transfer via a stochastic resonance (SR) mechanism1,2. SR can be experi- mentally observed when noise is added to a non-linear system which enhances (i) the output of the system e.g. by improving the signal-to-noise ratio (SNR)3,4, (ii) the signal amplitude5 or (iii) the degree of coherence within neu- ral networks6. In humans, the SR-effect has been observed in multiple sensory modalities when both signal and experimentally controlled noise are added to the peripheral nervous system7–10. Recently, we have demonstrated that central mechanisms of perception are also sensitive to an SR-effect3. We showed that transcranial random noise stimulation (tRNS), a type of non-invasive brain stimulation, applied over visual cortex can enhance the detection performance of weak subthreshold visual stimuli.h p Theoretical considerations predict that SR not only plays a role in signal enhancement but that it can also influence the dynamics of bistable-systems11. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 15 December 2017 Accepted: 14 February 2019 Published: xx xx xxxx Altering brain dynamics with transcranial random noise stimulation One paradigm that allows the observation and measurement of how the brain dynamically transitions between different perceptual states is binocular rivalry. Binocular rivalry is a perceptual phenomenon that occurs when different stimuli are simultaneously presented to each eye. During binocular rivalry visual awareness switches spontaneously between the two stimuli12 so that at any given time participants perceive either one of the two images (exclusive percept) or a combination of both (mixed percept).l Models of binocular rivalry propose that it reflects a competition between neural populations coding for each image13. The neural population coding for the dominant percept inhibits neurons that code for the suppressed image. However, over time the inhibition of the dominant population becomes weaker due to adaptation or fatigue, which allows the suppressed image to become dominant13,14. This results in a deterministic anti-phase oscillation of the firing rates of the two neuronal populations15. If adaptation was the only driving factor of bin- ocular rivalry, however, perception would change fairly regularly. In fact, the dynamics of binocular rivalry are highly nonlinear and stochastic, leading to the proposal that noise associated with the activity of the two neuronal populations causes a random distribution of dominance durations16. 1Neural Control of Movement Laboratory, Health Sciences and Technology, ETH Zurich, Zurich, 8057, Switzerland. 2School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, 6027, Australia. 3Queensland Brain Institute, The University of Queensland, St Lucia, Queensland, 4072, Australia. 4School of Psychology, The University of Queensland, St Lucia, Queensland, 4072, Australia. 5Canadian Institute for Advanced Research (CIFAR), Toronto, Canada. Correspondence and requests for materials should be addressed to O.v.d.G. (email: vandergroen@gmail.com) Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w 1 www.nature.com/scientificreports/ Noise is thought to play a crucial role in the occurrence of the perceptual switches, and it has been suggested to represent an essential driving force of rivalry dynamics17,18. Consequently, there are many successful models of binocular rivalry which contain a noise component to describe the rivalry dynamics15,19,20. While it has been demonstrated that rivalry dynamics can be influenced by adaptation21, the effect of adding noise directly to the brain during binocular rivalry has not been empirically tested. Rivalry dynamics can conceptually be represented by a double-well energy landscape, with each well representing one of the images. Altering brain dynamics with transcranial random noise stimulation Neuronal adaptation can be represented as changes in the energy-landscape: adaptation reduces the depth of the well so that the current state is less stable, thus making a switch to the competing percept more likely. Adding noise to such a system can change its dynamics in a specific way11,22. Here we investigated whether the attractor dynamics of binoc- ular rivalry can be modulated by adding noise to either the visual stimulus or directly to the visual cortex with tRNS23,24. Previously it has been demonstrated that 10 minutes of continuous tRNS increases cortical excitability for up to an hour after stimulation24. In the current study, however, we were interested in the direct online effects of noise on rivalry dynamics. Therefore, noise and no-noise conditions were randomly intermixed, and noise was applied non-continuously to reduce the likelihood of overall changes in neural excitability. Two experiments were performed: In Experiment 1, noise was added to low contrast or high contrast visual stimuli to test whether an SR-effect is induced when noise is added to the periphery. In Experiment 2, we added noise to the visual cortex with tRNS to test whether central mechanisms of perception are sensitive to an SR-effect. The results of these experiments suggest that rivalry dynamics can be influenced by noise when there are three stable states, namely, perception of the left-eye image, the right-eye image and a combination of both images. Since SR occurs prefer- entially for weak but not for strong stimuli3,7–10,25 we tested a low- and a high-contrast condition in both exper- iments. In order to make clear predictions as to which outcome parameters were most likely to be affected by adding noise to rivalry dynamics, we simulated different experimental conditions with a computational model19 prior to data collection. Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w Results C Computational modelling results. We used a conventional binocular rivalry model19 which relies on competition between neurons tuned to orthogonal orientations. Perceptual switches result from the interplay of mutual inhibition between these neurons and a noise component. We added a common noise term to the neu- ronal populations since tRNS is most likely to add correlated noise to many neuronal populations. When adding noise to the model, the duration of exclusive percepts decreased by 7% for the low contrast stimulus (low noise mean[s.d.]: 223.5[7.1] ms, high noise mean[s.d.]: 207.4[7.2]) ms) and increased by 3% for the high contrast stim- ulus (low noise mean[s.d.]: 246.3[15.5] ms, high noise mean[s.d.]: 253.3[14.4] ms, Fig. 1, left panel). The strongest effect of adding noise was observed as a substantial reduction of the mixed percept (Fig. 1, right panel) which was, however, different depending on contrast intensity: there was a stronger reduction of mixed percept durations for low contrast trials (−22% change, low noise mean[s.d.]: 1187.3[84] ms, high noise mean[s.d.]: 931[48.2]) ms) than for high contrast trials (1% change, low noise mean[s.d.]: 636.8[19.9] ms, high noise mean[s.d.]: 645.3[31.9]) ms). These modelling results are in line with previous research suggesting that mixed percept duration is affected by neuronal noise26–28. Based on these studies and our own modelling findings, the primary outcome measure for the subsequent binocular rivalry experiments was the mixed percept duration. In particular, we hypothe- sized that the mixed percept duration would be more strongly influenced by adding noise to low contrast stimuli than to high contrast stimuli. As a secondary outcome measure we further calculated the number of perceptual switches over the 12 minutes of modelled rivalry. The number of switches increased most with increased noise in the low-contrast condition (low noise mean[s.d.]: 310.3[11.41], high noise mean[s.d.]: 397.8[14.4]; 28% change), whereas there was no change in the number of switches in the high-contrast condition (low noise mean[s.d.]: 425.3[16.6], high noise mean[s.d.]: 423.8[12.6]; 0% change). Computational modelling results. Behavioural results. Experiment 1. Participants were seated in front of the monitor and viewed the images foveally through a mirror stereoscope (Fig. 2). The task for the participants was to continuously report on a key- board whether they perceived the left or right tilted grating, or a mixture of both. y pt g g g We investigated two separate cohorts (each n = 10) which performed the task either with low contrast or high contrast stimuli (see methods for further details). Adding noise to low contrast visual stimuli during binoc- ular rivalry significantly reduced the median dominance duration of the mixed percept (low-noise mean[s.d.]: 1.29[0.53]sec, high-noise mean[s.d.]: 1.08[0.45] sec; t(9) = 2.938, p = 0.017; Cohen’s d = 0.65; −16% change in dominance duration; Fig. 3). Adding noise to high contrast visual stimuli did not significantly affect the mixed dominance duration (low-noise mean[s.d.]: 1.07[0.38] sec, high-noise mean[s.d.]: 1.04[0.58] sec; t(9) = 0.356, p = 0.730; Cohen’s d = 0.08; −4% change in dominance duration). Note that we observed a medium effect size for the low contrast stimuli, whereas there was only a small effect for high contrast stimuli suggesting that the noise effect is larger for the former.h f g Additionally, we tested the number of perceptual switches as a secondary outcome parameter: The num- ber of perceptual switches was not significantly affected by peripheral noise for low contrast stimuli (low noise mean[s.d.]: 173.2[41.8], high noise mean[s.d.]: 181.7 [29.1]; t(9) = −1.284, p = 0.23; 5% change) or high contrast stimuli (low noise mean[s.d.]: 259.2[119.4], high noise mean[s.d.]: 258[113.9]; t(9) = 0.272, p = 0.79; 0% change). Experiment 2. In Experiment 2 we added noise directly to the visual cortex with tRNS. Consistent with previous investigations, no participant reported awareness of the tRNS stimulation during de-briefing29,30. Adding noise to visual cortex with tRNS yielded a similar pattern of results to Experiment 1. Specifically, the median mixed dom- inance duration of the low contrast visual stimulus was significantly reduced (low-noise mean[s.d.]: 2.05[1.53] sec, high-noise mean[s.d.]: 1.79[1.14] sec; t(14) = 2.263, p = 0.04; Cohen’s d = 0.41, −13% change in dominance Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w 2 www.nature.com/scientificreports/ Figure 1. Computational modelling results. We modelled the effect of adding noise to a rivalry model, and determined dominance durations for the exclusive and mixed percepts. Computational modelling results. The modelled results indicate that adding noise to the rivalry process reduces the duration of the mixed percept for low contrast visual stimuli by 22%. For the high contrast visual stimuli this reduction was 1%. For the exclusive percept, the dominance duration decreased with an increasing noise level for the low-contrast visual stimuli (7% reduction) and increased for the high-contrast visual stimuli (3% increase). Error bars represent standard deviations (s.d.). Figure 1. Computational modelling results. We modelled the effect of adding noise to a rivalry model, and determined dominance durations for the exclusive and mixed percepts. The modelled results indicate that adding noise to the rivalry process reduces the duration of the mixed percept for low contrast visual stimuli by 22%. For the high contrast visual stimuli this reduction was 1%. For the exclusive percept, the dominance duration decreased with an increasing noise level for the low-contrast visual stimuli (7% reduction) and increased for the high-contrast visual stimuli (3% increase). Error bars represent standard deviations (s.d.). duration). Adding noise to a high contrast visual stimulus did not significantly affect the mixed dominance dura- tion (low-noise mean[s.d.]: 1.51[1.03] sec, high-noise mean[s.d.]: 1.39[0.8] sec; t(14) = 0.930 p = 0.368, Cohen’s d = 0.16, −8% change in dominance duration). The number of perceptual switches was not significantly affected by peripheral noise for low contrast stimuli (low noise mean[s.d.]: 186.5[71.6], high noise mean[s.d.]: 194.2[61]; t(14) = −1.581, p = 0.136; 4% change) or high contrast stimuli (low noise mean[s.d.]: 184.7[49.9], high noise mean[s.d.]: 183.3[50.4]; t(14) = 0.428, p = 0.68; 0% change). Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w Discussion and Conclusioni The orange ball determines the current percept. Binocular rivalry is thought to be driven by adaptation and noise. Adaptation changes the landscape, meaning one of the wells becomes less shallow. Noise (arrow) causes the percept to change more quickly when the boundary between the two wells is low. (b) Experimental outline. In Experiment 1 noise (zero-mean Gaussian random noise) was applied to the visual stimulus for 5 seconds followed by 5.5–7 seconds of no stimulation. The same noise was applied to the left and right eye. The noise intensity was subthreshold for each individual participant. Experiment 2 followed the same protocol, except that the noise was applied to the visual cortex directly with tRNS (zero-mean Gaussian random noise, 100–640 Hz). Figure 2. (a) Representation of rivalry dynamics. Binocular rivalry can be represented by a double-well energy landscape. The orange ball determines the current percept. Binocular rivalry is thought to be driven by adaptation and noise. Adaptation changes the landscape, meaning one of the wells becomes less shallow. Noise (arrow) causes the percept to change more quickly when the boundary between the two wells is low. (b) Experimental outline. In Experiment 1 noise (zero-mean Gaussian random noise) was applied to the visual stimulus for 5 seconds followed by 5.5–7 seconds of no stimulation. The same noise was applied to the left and right eye. The noise intensity was subthreshold for each individual participant. Experiment 2 followed the same protocol, except that the noise was applied to the visual cortex directly with tRNS (zero-mean Gaussian random noise, 100–640 Hz). stimulation31. It is unclear how the intensity of “neural noise” induced by tRNS over visual cortex could be deter- mined at the level of the individual, or how it could be parametrized in the model. More sophisticated, biologi- cally plausible models might shed further light on these questions.h y p g g q The processes underlying the occurrences of mixed percepts during binocular rivalry are unclear, but one possible mechanism involves changes in the strength of mutual inhibition between the two neuronal pools, each coding for a different percept32,33. It is thought that stronger stimuli (stimuli with a higher signal-to-noise ratio) result in an increase in mutual inhibition, which leads to a reduction in the mixed percept duration33. Adding noise could have strengthened mutual inhibition due to an increase in the SNR of the stimulus representation in the brain. Discussion and Conclusioni Our results demonstrate for the first time that adding noise to a visual stimulus during binocular rivalry can significantly influence rivalry dynamics by reducing mixed percept durations. The same results were obtained when noise was added to the visual cortex directly with tRNS. This effect only occurred when the visual stimuli had a low contrast. These results are in line with the predictions made by the binocular rivalry model (see Fig. 1). Exclusive percept dominance durations in Experiments 1 and 2 were not affected by noise. f Adding noise to the visual stimuli (peripheral noise) resulted in the same behavioural results as adding noise to the brain directly with tRNS (central noise), namely, a significant reduction in mixed percept duration when the stimuli had a low contrast. No effect was observed when the stimuli were presented with a high contrast, suggesting that the stimulus contrast is a crucial parameter. Previously we have shown that noise added either peripherally or centrally can enhance performance on a contrast detection task3 for weak visual stimuli according to an SR-effect. An important difference between these two noise application methods is the location in the nerv- ous system at which they influence visual processing for the first time. The initial influence of peripheral noise is at the receptor level. tRNS, on the other hand, influences visual processing at the cortical level. By applying tRNS during binocular rivalry we found that cortical noise can causally influence mixed percept duration, as predicted by the model. d h b f l h h d l h h d l Our secondary outcome measurement, the number of perceptual switches, changed in line with the model prediction but the effect was small and did not reach significance in the behavioural experiment. Note that we adopted an existing model which was developed and validated in a different experimental context19. Since the model was not optimized for the current experiment, its predictions were qualitatively similar to our behav- ioural results but not quantitatively. For example, the absolute dominance durations predicted by the model were much shorter than those observed experimentally. This discrepancy might have arisen because we did not model inter-subject variability, which has been shown to be substantial in response to transcranial electrical Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w 3 www.nature.com/scientificreports/ Figure 2. (a) Representation of rivalry dynamics. Binocular rivalry can be represented by a double-well energy landscape. Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w Discussion and Conclusioni Related to this finding, a recent study demonstrated that alcohol intake enhances mixed percept duration34. It has been shown in cats that alcohol reduces the signal-to-noise ratio (i.e. increases noise) in primary visual cortex35. In our study, we likely increased the SNR in visual cortex, resulting in a reduction of mixed percept duration. Another possible mechanism that effects mixed percept duration is a change in the balance between excitatory and inhibitory neural activity36. An imbalance between cortical excitation and inhibition is an impor- tant factor in autism spectrum disorder (ASD) models28. However, it is still unknown whether lower biological noise levels are also involved in ASD37. Interestingly, our finding that only mixed percepts are sensitive to a noise effect is supported by a study of binocular rivalry in ASD28. Robertson and colleagues did not find any difference between healthy controls and people with ASD on exclusive percept duration, but did find an increase in mixed percept duration in people with ASD28.i p p p p Our rivalry stimuli were relatively large (4°), which is larger than the receptive fields of individual V1 cells. Therefore, participants could have experienced ‘piecemeal rivalry’, which occurs when patches of both images are perceived together38. We did not test how much piecemeal rivalry participants experienced, but previous research suggests that it would have been less prominent in the low contrast condition than in the high contrast condi- tion18. Previous research suggests that piecemeal rivalry is not affected by peripheral noise39.t gg yf y It is known that tRNS is able to enhance cortical excitability after 4 minutes of continuous stimulation40, which results in a change in the excitation-inhibition balance. Here we alternated tRNS and non-tRNS runs in random order across participants, making it unlikely that the observed effects were driven by an overall change of excit- ability. Additionally, tRNS runs in Experiment 2 applied the stimulation for relatively short periods of 5 seconds, and these were followed by 5.0 to 7.7 s with no stimulation to reduce effects on overall excitability. Besides this, adding noise to the visual stimulus resulted in similar effects, and as far as we know cortical excitability cannot Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w 4 www.nature.com/scientificreports/ Figure 3. Behavioural results from Experiments 1 (left) and 2 (right). Discussion and Conclusioni Adding noise to the visual stimulus significantly reduced the dominance duration of the mixed percept for low contrast visual stimuli by 16%, whereas the dominance duration for the high contrast visual stimuli was reduced by 4%. In Experiment 2 (right) the noise was added to the cortex with tRNS. This resulted in a reduction of 15% in median mixed percept duration. The dominance duration for the high contrast visual stimuli was reduced with 6% There was no significant effect of adding noise on the dominance duration of the exclusive percept. Error bars represent standard errors (s.e.m.). Figure 3. Behavioural results from Experiments 1 (left) and 2 (right). Adding noise to the visual stimulus significantly reduced the dominance duration of the mixed percept for low contrast visual stimuli by 16%, whereas the dominance duration for the high contrast visual stimuli was reduced by 4%. In Experiment 2 (right) the noise was added to the cortex with tRNS. This resulted in a reduction of 15% in median mixed percept duration. The dominance duration for the high contrast visual stimuli was reduced with 6% There was no significant effect of adding noise on the dominance duration of the exclusive percept. Error bars represent standard errors (s.e.m.). be modulated by peripheral noise. Therefore, our results are most likely explained by an SR-effect that modu- lates perceived stimulus contrast. It has been previously demonstrated that binocular rivalry is modulated by an SR-mechanism when evoked by peripheral noise41. In contrast to our study, Kim and colleagues did not add noise to the rivalry process, but changed the strength of the driving signal by changing the contrast of the two images in counter phase. The idea is that SR will occur when the driving signal matches biological noise levels. The reason we found no effect of noise on the dominance duration of exclusive percepts in our study could because the noise added might not have been optimal to introduce an SR-effect in a bistable system. The noise properties we applied were based on the findings of our previous study3 where we demonstrated that tRNS and peripheral noise are able to enhance performance on a contrast detection task. The fact that we might not have added an optimal noise level for everyone might explain why the effect sizes for the tRNS noise were relatively low. Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w Materials and Methods Th d d b h Participants were seated in front of the monitor and viewed the images through a mirror stereoscope from a distance of 45 cm while resting their head on a chin rest. The images were viewed foveally (through the stereoscope).h h Each participant performed 8 runs of binocular rivalry, each lasting 3 minutes. The task for the participants was to continuously report on a keyboard whether they perceived the left-eye or right-eye tilted grating or a mixture of both. In each experiment the noise was applied in 4 runs to either the screen (Exp. 1) or directly to the visual cortex with tRNS (Exp. 2). The order of the noise runs was randomized over participants. In these runs the noise was applied for 5 seconds followed by a 5.5–7 seconds’ interval of no stimulation, and in total 18 times per run. In each experiment participants received a total of 360 seconds’ visual noise (exp. 1) or tRNS (exp. 2). Participants performed one practice run without any noise before the start of the experiment. Experiment 1: Peri-noise condition. In this experiment, we tested whether adding noise (zero-mean Gaussian random noise) to the visual stimuli influences binocular rivalry dynamics. The same noise was applied to the left and right eye. Previous research demonstrated that a noise intensity corresponding to 60% of thresh- old effectively induces an SR-effect3. Therefore, we used a simple up-down method to estimate each individual’s 60% correct noise-threshold before the experiment started, where 75% correct reflects threshold. We tested two cohorts of participants, one with a low contrast visual stimulus (20% contrast, n = 10, mean age = 23 years) and the other with a high contrast visual stimulus (70% contrast, n = 10, mean age = 24 years). Experiment 2: tRNS-V1 condition. In this experiment, we tested the hypothesis that adding noise directly to the visual cortex with tRNS can influence rivalry dynamics. Noise was applied centrally with tRNS (100–640 Hz, zero-mean Gaussian random noise). Electrode placement was determined using the 10–20 system. The stimulation electrode was placed over the occipital region (Oz in the 10–20 EEG system) and the reference electrode over the vertex (Cz in the 10–20 EEG system). Electroconductive gel was applied to the contact side of the electrode (5 × 7 cm) to reduce skin impedance. Electrodes were held in place with a bandage. Materials and Methods Th d d b h The study was approved by the Kantonale Ethikkomission Zürich, Switzerland (KEK-ZH-Nr. 2014-0269) and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants before the start of the experiment. Computational model of rivalry dynamics. We applied a computational model to predict how noise influences rivalry dynamics19. We used a conventional binocular rivalry model which relies on competition between neurons tuned to orthogonal orientations. The model relies on mutual inhibition and it also includes a noise component. The model contains two different neuronal populations and calculates the difference in firing rate between these populations. Over time inhibition on the suppressed neuronal population weakens, which eventually results in the inhibited population becoming dominant and suppressing the other population. When one population is firing fully the other population will be inhibited, resulting in the percept related to the fully firing population being dominant. When the difference in firing rate between the two populations is small then there is no winning population, resulting in a mixed percept. We introduced a criterion for mixed percepts, which was a difference in firing rate between the two neuronal populations of smaller than 0.1. In calculating dominance durations, we only included dominance times longer than 150 ms. In the model, we changed the strength of the visual stimulus and the amount of noise according to our experimental parameters. All other model parameters were identical to the original model parameters19. The simulation was run 100 times in order to derive an estimate of the variability of the model. General procedures of Experiments 1 and 2. All experiments took place in a dark and quiet room. Visual stimuli (left and right tilted gratings) were generated using MATLAB version 2012b (MathWorks, Natick, USA) and the Psychophysics Toolbox53–55. Stimuli were controlled by a HP Elitedesk 800 G1 running Windows 7 (2009). Stimuli were presented on a Sony CPD-G420 colour monitor with a calibrated linearized output at a reso- lution of 1280 × 1024 pixels, with a refresh rate of 75 Hz. The two images were placed on the horizontal meridian to the left and right hand side of the screen on a uniform grey background (53 Cd/m2). The visual stimuli were orthogonal Gaussian gratings (orientation ± 45°, diameter 3 cm, contrast 20%, visual angle 4°, spatial-frequency 2.2 cycles per degree) surrounded by a white square (diameter 3.5 cm), to promote binocular fusion. Discussion and Conclusioni up new ways of manipulating noise levels within the brain, and provide a better understanding of the role noise plays in the brain when it is in a dynamical state. Discussion and Conclusioni y g p yf y In order to determine whether the tRNS-induced electrical noise reached the cortex, we have previously esti- mated that the applied field strength is too small to directly depolarize single neurons3. However, it has been pro- posed that the tRNS effect is amplified because thousands of neurons are modulated simultaneously42. Thereby even small depolarization may be enough to elicit action potentials (APs) in neurons that are close to their firing threshold, which results in a weak stimulus reaching the threshold for AP generation earlier. g g Although it is still debated where in the brain binocular rivalry is resolved, we targeted the visual cortex. In particular, we selected an electrode montage and chose an intensity which is suitable for stimulating visual cor- tex as indicated by previous modelling work3. The reason we targeted this region is because neuronal activity in primary visual cortex (V1) is linked to subjective percepts during binocular rivalry43–45. Changes in higher order brain areas, including frontoparietal cortex, are thought to reflect the consequences of perceptual alternations, rather than being the cause of them46. However, there is also evidence that frontoparietal activity might play a causal role47–49. Evidence that mixed percepts may be more influenced by low-level visual processes rather than higher order processes50,51 supports the interpretation that our effect is driven by stimulation of these low-level visual cortices. The results of our study show that the effect of noise on the rivalry dynamics occurs at a cortical level. It has been shown before that peripheral noise can enhance the perceived contrast of a visual stimulus at the receptor level10,52. Therefore, finding an effect of adding noise peripherally does not necessarily mean that rivalry dynamics are sensitive to an SR effect, because any such effect could be explained at the receptor level.i yf , yf p p In conclusion, our results are the first to demonstrate that mixed percept durations can be altered by applying noise to the visual cortex directly with tRNS, likely due to an enhancement of stimulus contrast. Our results open Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w 5 www.nature.com/scientificreports/ up new ways of manipulating noise levels within the brain, and provide a better understanding of the role noise plays in the brain when it is in a dynamical state. References Terney, D., Chaieb, L., Moliadze, V., Antal, A. & Paulus, W. Increasing human brain excitability by transcranial hi random noise stimulation J Neurosci 28 14147–14155 https://doi org/10 1523/JNEUROSCI 4248-08 2008 (2008) 24. Terney, D., Chaieb, L., Moliadze, V., Antal, A. & Paulus, W. Increasing human brain excitability by transcranial high-frequenc random noise stimulation. J Neurosci 28, 14147–14155, https://doi.org/10.1523/JNEUROSCI.4248-08.2008 (2008). y g y y g random noise stimulation. J Neurosci 28, 14147–14155, https://doi.org/10.1523/JNEUROSCI.4248-08.2008 (2008). p g aitoni, L., Hänggi, P., Jung, P. & Marchesoni, F. Stochastic resonance 25. Gammaitoni, L., Hänggi, P., Jung, P. & Marchesoni, F. Stochasti 26. Robertson, C. E., Ratai, E. M. & Kanwisher, N. Reduced GABAergic Action in the Autistic Brain. Curr Biol 26, 80–85 org/10.1016/j.cub.2015.11.019 (2016). g j 7. Freyberg, J., Robertson, C. E. & Baron-Cohen, S. Reduced perceptual exclusivity during object and grating rivalry in autism. J Vi 15, 11, https://doi.org/10.1167/15.13.11 (2015). p g 28. Robertson, C. E., Kravitz, D. J., Freyberg, J., Baron-Cohen, S. & Baker, C. I. Slower rate of binocular rivalry in autism. J Neurosci 33, 16983–16991, https://doi.org/10.1523/JNEUROSCI.0448-13.2013 (2013). p g 29. Ambrus, G. G., Paulus, W. & Antal, A. Cutaneous perception thresholds of electrical stimulation methods: Comparison of tDCS and tRNS. Clinical Neurophysiology 121, 1908–1914, https://doi.org/10.1016/J.Clinph.2010.04.020 (2010). p y gy p g J p ( ) 0. Fertonani, A., Pirulli, C. & Miniussi, C. Random noise stimulation improves neuroplasticity in perceptual learning. J Neurosci 31 15416 15423 htt //d i /10 1523/JNEUROSCI 2002 11 2011 (2011) p y gy p g p 0. Fertonani, A., Pirulli, C. & Miniussi, C. Random noise stimulation improves neuroplasticity in perceptual learning. J Neurosci 31 15416–15423, https://doi.org/10.1523/JNEUROSCI.2002-11.2011 (2011).f y gy g 30. Fertonani, A., Pirulli, C. & Miniussi, C. Random noise stimulation improves neuropl 15416–15423, https://doi.org/10.1523/JNEUROSCI.2002-11.2011 (2011).f 5416–15423, https://doi.org/10.1523/JNEUROSCI.2002-11.2011 ( g 31. Wiethoff, S., Hamada, M. & Rothwell, J. C. Variability in response to transcranial direct current stimulation of the motor cortex. Brain Stimul 7, 468–475, https://doi.org/10.1016/j.brs.2014.02.003 (2014). p g j 2. Klink, P. C., Brascamp, J. W., Blake, R. & van Wezel, R. J. Experience-driven plasticity in binocular vision. Curr Biol 20, 1464–1469 https://doi.org/10.1016/j.cub.2010.06.057 (2010).hf p g j 3. Hollins, M. The Effect of Contrast on the Completeness of Binocular-Rivalry Suppression. Perception & psychophysics 27, 550–556 https://doi.org/10.3758/Bf03198684 (1980). p g 4. Cao, D., Zhuang, X., Kang, P., Hong, S. W. & King, A. C. Acute Alcohol Drinking Promotes Piecemeal Percepts during Binocular Rivalry. Materials and Methods Th d d b h This setup has been demonstrated to be suitable for stimulation of the visual cortex3,56,57. More specifically, previous work has estimated the electrical field strength produced by this montage in visual cortex3. In brief, modelling the electrical field for specific frequencies between 100 and 500 Hz (50 Hz steps,) and the current flow across the brain revealed sufficiently focal stimulation of visual cortex. The induced electrical field was estimated to reach a maximum of 2 V/m with all frequencies being transmitted to the brain. Stimulation was delivered by a battery-driven electrical stimulator (DC-Stimulator Plus, neuroConn). An intensity of 1 mA was applied since it has been demonstrated that this intensity effectively induces an SR-effect in most subjects3. The maximum current density in this exper- iment was 28.57 μA/cm2, which is within current safety limits58. We again tested two cohorts of participants, one where the visual stimulus had a low contrast (20% contrast, n = 15, mean age = 23 years) and the other where the visual stimulus had a high contrast (70% contrast, n = 15, mean age = 24 years). We tested more participants in Experiment 2 because it was expected there would be less power to detect a reliable effect of tRNS relative to peripheral visual noise3. Data analysis and statistics. Statistical analyses were performed using SPSS (version 20.0, IBM). The same statistical procedures were applied to both experiments. For each participant, we calculated the median Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w 6 www.nature.com/scientificreports/ dominance duration for the exclusive percepts. We also calculated the mixed percept dominance durations. Times where no button was pressed, or when the dominance duration was shorter than 150 ms, were excluded from analysis. Dominance durations terminated by the end of a block were not included in the analyses. In each experiment, we also calculated the number of perceptual switches. To test for the effect of the added noise, dom- inance durations and number of perceptual switches were subjected to a two-sided within-subject t-test. The α-level was set to 0.05 for all tests. Data Availability Th d d y The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References 1. McDonnell, M. D. & Abbott, D. What is stochastic resonance? 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Acknowledgements g O.v.d.G was supported by the Swiss National Science Foundation (grant 165189 and 320030_149561), N.W. was supported by the Swiss National Science Foundation (grant 320030_149561), and J.B.M. was supported by the Australian Research Council (ARC) Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007) and by an ARC Australian Laureate Fellowship (FL110100103). g O.v.d.G was supported by the Swiss National Science Foundation (grant 165189 and 320030_149561), N.W. was supported by the Swiss National Science Foundation (grant 320030_149561), and J.B.M. was supported by the Australian Research Council (ARC) Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007) and by an ARC Australian Laureate Fellowship (FL110100103). www.nature.com/scientificreports/ 7. Davis, G. & Plaisted-Grant, K. Low endogenous neural noise in autism. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 8 Scientific Reports \| (2019) 9:4029 \| https://doi.org/10.1038/s41598-019-40335-w
https://openalex.org/W2817808795	https://europepmc.org/articles/pmc6051991?pdf=render	English	null	A dual-fMRI investigation of the iterated Ultimatum Game reveals that reciprocal behaviour is associated with neural alignment	Scientific reports	2,018	cc-by	12,160	A dual-fMRI investigation of the iterated Ultimatum Game reveals that reciprocal behaviour is associated with neural alignment Received: 29 December 2017 Accepted: 9 July 2018 Published: xx xx xxxx Dyadic interactions often involve a dynamic process of mutual reciprocity; to steer a series of exchanges towards a desired outcome, both interactants must adapt their own behaviour according to that of their interaction partner. Understanding the brain processes behind such bidirectional reciprocity is therefore central to social neuroscience, but this requires measurement of both individuals’ brains during real- world exchanges. We achieved this by performing functional magnetic resonance imaging (fMRI) on pairs of male individuals simultaneously while they interacted in a modified iterated Ultimatum Game (iUG). In this modification, both players could express their intent and maximise their own monetary gain by reciprocating their partner’s behaviour – they could promote generosity through cooperation and/or discourage unfair play with retaliation. By developing a novel model of reciprocity adapted from behavioural economics, we then show that each player’s choices can be predicted accurately by estimating expected utility (EU) not only in terms of immediate payoff, but also as a reaction to their opponent’s prior behaviour. Finally, for the first time we reveal that brain signals implicated in social decision making are modulated by these estimates of EU, and become correlated more strongly between interacting players who reciprocate one another. Many social interactions comprise a series of repeated exchanges between individuals. Within the constraints of social convention, such dynamic contexts demand a process of mutual reciprocity1; over the course of a repeated dyadic exchange, for example, both interactants modify their own behaviour in response to their partner’s in an attempt to steer the interaction towards a desired outcome. In this light, repeated exchanges unfold as a two-in-one process whereby each individual’s behaviour is simultaneously a consequence of and antecedent to that of their partner’s. Advancing our understanding of the brain processes underlying such reciprocity is there- fore central to social neuroscience research2, but this requires measurement of both interactants’ brains whilst they engage in naturalistic social exchanges.h y g g g The Ultimatum Game (UG3) presents a simple paradigm to investigate dyadic interaction. A Proposer is asked to choose from a range of options how they wish to divide a sum of money (the “pie”) between themselves and a Responder. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ One particular form of neural coupling is alignment – that is, correlated neural signals between brains21. This is analogous to a wireless commu- nication system, through which a sender and receiver become synchronised to a transmitted signal (e.g., light or sound22). The signals driving neural alignment vary in their level of abstraction, however; while sensory cortices will align to interactants’ physical movements, correlated signals in higher-order brain regions reflect a shared understanding of the intentions behind the actions18,21,23. g To investigate whether reciprocity during real-world, repeated dyadic exchanges elicits patterns of neural alignment, the present study performed functional magnetic resonance imaging on pairs of individuals simulta- neously (dual-fMRI) while they played a modified iUG designed to encourage reciprocity. Unlike other hyper- scanning methods (e.g., fNIRS), dual-fMRI affords direct localisation of inter-brain effects within cortical and subcortical regions. To capture behavioural reciprocity over multiple rounds of economic exchange, we developed a novel adaptation of a model from experimental economics24 that fits each player’s round-by-round behaviour (the proposed division or its acceptance/rejection) to an estimate of expected utility (EU) on a given exchange. Crucially, this estimate of EU considered not only the distribution of payoff between players, thereby incorpo- rating any social preferences (inequity aversion), but also the extent to which their choices reflect a reaction to their partner’s prior behaviour; if player A considers B’s past behaviour to have been fair then they will perceive greater utility in increasing B’s relative payoff, but if A believes B’s past behaviour to have been unfair they will take pleasure in decreasing B’s payoff in favour of their own (positive and negative reciprocity, respectively). By combining functional neuroimaging data with estimates of EU from our reciprocity model, we were then able to investigate whether brain responses map onto utility evaluations influenced by an opponent’s prior behaviour – that is, the neural coupling associated with reciprocal behaviour. We also assessed neural alignment specifically by measuring covariance in brain signals between interacting players21,25, and investigated whether this is related to their expression of reciprocity. We hypothesised that greater reciprocity would be associated with stronger co-activation between interacting players’ brains in regions implicated in fairness evaluations and strategic behav- iour on the UG – namely, AI and ACC/aMCC. www.nature.com/scientificreports/ cortices [aMCC; see7]; for meta-analytic reviews see8,9). In contrast, Proposers’ offers are believed to reflect stra- tegic behaviour; in an attempt to maximise their own payoff they avoid offers that are likely to be rejected, such as those with which they earn disproportionately more (advantageous inequity). Although fewer neuroimaging studies have investigated Proposers, the available evidence points to neural responses in frontal midline brain regions during offers that reflect such egoistic strategies (e.g.,10–12).ht g gfl g g ( g ) The UG is performed typically in a one-shot manner, however – a single round played, ending after the Responder accepts or rejects a proposed division. Whilst this simulates one-off social interactions, it fails to cap- ture the bidirectional and reciprocal property of repeated exchanges13; in such contexts, we do not simply react to another’s behaviour but we interact with them in an attempt to bring about a desirable outcome. Furthermore, previous studies have examined UG performance between individuals who are anonymous to one another, thereby removing the social context in which the majority of day-to-day interactions take place and limiting the applicability of resulting behaviours. Investigating the sequential and reciprocal nature of real-world dyadic inter- actions requires an iterated UG (iUG13–15) in which multiple exchanges occur between the same players. In this situation, both players can adapt to the behaviour of their opponent in order to maximise their own payoff over recursive rounds; Responders can encourage equitable offers by rejecting disadvantageous ones, and Proposers can increase acceptance by adapting to Responder behaviour16. Alternatively, either player can adopt an unwa- vering strategy; by offering or accepting only those proposals that benefit themselves maximally, players can force their partner into a compromise over fairness and ultimate payoff. In other words, both players can express varying degrees of reciprocity over multiple rounds with the same partner. y g g p y p p Reciprocity unfolds as an indirect chain of neural events; through neural coupling, one individual’s brain activity results in a behavioural output, which then elicits systematic neural responses in their interaction partner to initiate a behavioural reaction17. As such, only by measuring brain signals in both interactants simultaneously can we begin to elucidate the interpersonal neural processes underlying reciprocity18. By employing this “hyper- scanning” method, neuroscientific studies report spatially and temporally synchronised brain signals between interactants that vary with the nature of the social exchange (for reviews see19,20). A dual-fMRI investigation of the iterated Ultimatum Game reveals that reciprocal behaviour is associated with neural alignment The Responder then chooses whether to accept or reject the offer; if they accept it then the pie is divided accordingly, but if they reject it then neither player receives any payoff. Contrary to game theoretic pre- dictions of rational behaviour, modal offers are around 40% of the pie and Responders reject proposals of 20% approximately half the time4. Responders’ behaviour appears to reflect an aversion to inequity; they consider it unfair to be offered disproportionately less than their interaction partner (disadvantageous inequity), and reject such proposals as a challenge to subjugation5. Consistent with this notion, neuroimaging studies demonstrate that rejected offers elicit neural responses in brain systems implicated in subjective feeling states such as pain and disgust (anterior insula [AI]6) and social information processing (anterior [ACC] and anterior-mid cingulate 1Department of Psychology, Aston University, Birmingham, B4 7ET, United Kingdom. 2CEITEC – Central European Institute of Technology, Masaryk University, Brno, Czech Republic. 3Department of Economics, Faculty of Economics and Administration, Masaryk University, Brno, Czech Republic. 4Department of Public Economics, Faculty of Economics and Administration, Masaryk University, Brno, Czech Republic. 5Institute of Psychology, Academy of Sciences of the Czech Republic, Brno, Czech Republic. Correspondence and requests for materials should be addressed to D.J.S. (email: d.j.shaw@aston.ac.uk) Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 1 www.nature.com/scientificreports/ Results Results We report the results of non-parametric statistical analyses if Kolmogorov–Smirnov tests revealed that normality was violated by at least one of the assessed variables. Values present means (±SD). We report the results of non-parametric statistical analyses if Kolmogorov–Smirnov tests revealed that normality was violated by at least one of the assessed variables. Values present means (±SD). Behaviour. All players made their choices (the selection of a division to offer, and the decision to accept or reject the proposed division) within the 4 second limit on all exchanges. Interestingly, however, response times (RTs) differed between players and conditions; a mixed-plot ANOVA, with the within-subject factor Condition (PR vs. PP) and between-subject factor Player (Proposer vs. Responder), revealed that RTs were greater for Proposers compared with Responders (2007.28 [±439.89] vs. 1106.99 [±429.72] ms; F[1,36] = 46.73, p < 0.001, ηp2 = 0.57) and on PR relative to PP exchanges (1641.71 [±637.81] vs. 1472.56 [±608.78] ms; F[1,36] = 13.43, p = 0.001, ηp2 = 0.27). There was no Player-by-Condition interaction (F[1,36] = 1.07, p = 0.308, ηp2 = 0.029). This revealed that (a) both players took longer to make decisions when faced with a choice between advantageous and disadvantageous inequity, but (b) Responders had already begun to evaluate the choice set before an offer was made. Next we assessed the pattern of proposals and decisions across choice sets, both within and between the PR and PP conditions. We focused on two aspects of player behaviour; specifically, the proportion of offers that benefited the Proposer maximally (i.e. those with maximal advantageous inequity; MAXOffer), and the propor- tion of these offers that were accepted by the Responder (MAXAccept). Figure 1A presents the distribution of each behavioural measure. This indicates higher proportions of both MAXOffer and MAXAccept for choice sets com- prising the PR relative to the PP condition, which was confirmed with non-parametric comparisons (respec- tively, Z[19] = 3.82, p < 0.001; and Z[19] = 2.11, p = 0.033). The number of MAXOffer and MAXAccept also appeared to decrease with higher payoff for the Proposer or, in turn, lower payoff for the Responder. To quantify this, for each choice set we computed the payoff for each player presented by the division with maximal advantageous ineq- uity. Results Spearman correlations confirmed that with increasing payoff for the Proposer, both MAXOffer and MAXAccept decreased (respectively, ρ[18] = −0.53, p = 0.016; and ρ[18] = −0.64, p = 0.003). Since Proposers selected an offer from two alternatives, the degree of payoff in the division with maximal advantageous inequity could also be expressed relative to the other option. To investigate whether MAXOffer and MAXAccept differed according to this relative measure, we compared the payoff to each player between the two divisions of each choice set; higher val- ues represented a relative increase in the Proposer’s payoff or, in turn, a decrease in the Responder’s payoff for the division with maximal advantageous inequity (see Table S1). No significant relationships were observed between this relative measure of payoff and MAXOffer or MAXAccept (respectively, ρ[18] = 0.39, p = 0.089; and ρ[18] = 0.29, p = 0.217). Together, these results imply that neither player’s choices were driven solely by absolute or relative measures of payoff, which was confirmed in subsequent modelling procedures (see below).l p yfi q g p We then applied our adapted reciprocity model to estimate the degree to which each player’s choices reflected reciprocal reactions to their partner’s prior behaviour, and from this we modelled each player’s round-by-round EU. For Proposers, greater values of EU represent higher utility for the division with least advantageous inequity (the generous offer); for Responders, it represented greater utility in accepting a proposed division. As shown in Fig. 1B, the probability of MAXOffers and MAXAccepts varied according to EU; indeed, estimates of EU correctly predicted these behaviours on 73.46 (±7.58 [AIC = 2345.5; BIC = 2454.4; Log-likelihood = −1153.7]) and 85.57 percent of rounds (±10.14 [AIC = 1272.2; BIC = 1381.1; Log-likelihood = −617.1]), respectively. Reciprocity parameters, α, were lower for Proposers (0.06 [±0.03]) than Responders (0.41 [±0.37]; Z[18] = 5.04, P < 0.001), suggesting that the latter players’ decisions reflected stronger reactions to their partner’s offers. Estimated values of α for Proposers were correlated negatively with MAXOffers across the PR (ρ[17] = −0.78, p < 0.001) and PP condi- tion (ρ[17] = −0.88, p < 0.001), however; the more reciprocity they showed, the less likely there were to offer divi- sions that benefited themselves maximally (see Fig. 1C). Results No such relationship was observed between Responders’ α and MAXAccepts for either the PR (ρ[17] = −0.15, p = 0.551) or PP condition (ρ[17] = −0.07, p = 0.786). Finally, Proposer α estimates correlated positively with the amount of time they took to decide the division they wished to offer on PR (ρ[17] = 0.58, p = 0.009) but not PP rounds (ρ[17] = −0.22, p = 0.377). Across all Proposers the optimal Memory parameter was 73, identifying the range of preceding rounds that maximized the accuracy of Proposers’ predictions of their opponents’ decisions. There was only a slight benefit beyond a range of 20, however; for round-by-round estimates of EU, estimates of each player’s reciprocity parameter, and the accuracy in predicting Proposers’ choices, correlations were highly similar for models with a Memory parameter of 20 and upwards (see Table S2). To evaluate our adapted reciprocity model we compared it against a variety of alternatives (see Supplementary Materials for full model specifications). First we tested a nested model by fixing the reciprocity parameter to α = 0 for both players. This self-regarding model evaluated the assumption that both players care only about their own onetary payoff. The likelihood ratio (L) demonstrated that our reciprocity model outperformed this mself-regarding model when applied to both Proposers (L[19] = −1037.2 [AIC = 2074.5]; p < 0.001) and Responders (L[19] = −1553.0 [AIC = 3106.0]; p < 0.001). Second, given the relatively low estimates of Proposer’s reciprocity, we tested whether the reciprocity utility function should be applied only to Responders. We achieved this by evaluating the change in model fit by fixing only the Proposer’s reciprocity parameters to α = 0; the same reciprocity model was applied to Responders. Again, our model fitted Proposers’ choices more accurately than this nested self-regarding model (L[19] = −1609.3 [AIC = 3219.5]; p < 0.001). Finally, we assessed whether choices reflect learning processes over multiple rounds rather than reciprocal reactions. To do so, we modelled each player’s behavioural data with a three-parameter extension of the reinforcement learning model30. This model contains a forgetting parameter ϕ, an experimentation parameter ε, and a strength parameter s (the simple one-parameter reinforcement learning model is a special case of this three-parameter extension, with ϕ = 1 and ε = 0). www.nature.com/scientificreports/ y Interestingly, when selecting a division from two alternatives (the choice set), Proposers are more likely to offer the fairer of the two when the other option becomes more selfish26. Furthermore, even if the fairer of the two options rewards the Responder with disproportionately less than their opponent, they are more likely to accept it because they consider it justified27,28. This contextual effect appears to reflect each player’s consideration of their opponent’s motivation: As a means of risk aversion, Proposers avoid the most selfish option because it is more likely to be rejected, decreasing its utility; and Responders accept offers that reward them disproportionately less if the alternative division incurs a greater cost to the Proposer. Since these decisions will be influenced by an eval- uation of the other player’s prior behaviour, thereby eliciting more reciprocity, our modification of the iUG per- mitted comparisons between two different contexts: On Proposer-Responder (PR) rounds, the choice set required Proposers to choose between a division that presented themselves with either advantageous or disadvantageous inequity (e.g., 60:40 vs. 40:60). Since the cost to the Proposer was far greater for the latter (very generous) divi- sion, both players were more likely to regard the former (selfish) option as justified28,29. Moreover, very generous offers on PR exchanges indicate that the Proposer perceived greater utility in increasing the Responder’s relative payoff at a cost to themselves, signalling a high degree of co-operative intent. In contrast, on Proposer-Proposer (PP) exchanges the Proposer had to choose between two divisions that differed only in magnitude of advanta- geous inequity (e.g., 60:40 vs. 70:30). Since the relative increase in cost to the Proposer by offering the least selfish division was reduced, a very selfish offer on PP exchanges indicated low co-operative intent. Thus, choices on PR and PP rounds were intended to elicit strong expressions of positive and negative reciprocity by both players. We predicted greater neural alignment during the former, in which more shared (co-operative) intentionality would be elicited. Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 2 www.nature.com/scientificreports/ Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 Results Each parameter was fitted to maximize the log-likelihood function, separately for Proposers (ϕ = 0.93, ε = 0.31, s = 3.2) and Responders (ϕ = 0.27, ε = 0.15, s = 3.2). The fit of this reinforcement learning model against Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 3 www.nature.com/scientificreports/ Figure 1. Patterns of player behaviour. (A) Mean (±SD) proportions of offers of ma inequity (MAXOffers; blue) and their acceptance (MAXAccepts; red) across all dyads for This indicates more MAXOffers and MAXAccepts across PR relative to PP rounds, which comparisons (see text). Error bars present standard deviations. (B) Probabilities of M plotted as a logistic function of expected utility (EU), as estimated with the reciproc proportion of MAXOffers in each condition plotted as a function of Proposers’ recipro reciprocity model. This reveals that with increasing reciprocity, Proposers were less that benefited themselves maximally. (D) Cumulative frequencies of MAXOffers and M rounds, for three example dyads. For the leftmost dyad, estimates of reciprocity wer (αP) and Responder (αR). This is reflected in high number of MAXOffers and MAXAcce to offer divisions that benefited themselves maximally because the Responder did no with rejections (negative reciprocity). In the middle dyad, the Responder did challen Figure 1. Patterns of player behaviour. (A) Mean (±SD) proportions of offers of maximal advantageous inequity (MAXOffers; blue) and their acceptance (MAXAccepts; red) across all dyads for each of the 10 choice sets. This indicates more MAXOffers and MAXAccepts across PR relative to PP rounds, which was confirmed with direct comparisons (see text). Error bars present standard deviations. (B) Probabilities of MAXOffers and MAXAccepts plotted as a logistic function of expected utility (EU), as estimated with the reciprocity model. (C) The proportion of MAXOffers in each condition plotted as a function of Proposers’ reciprocity (α), as estimated by the reciprocity model. This reveals that with increasing reciprocity, Proposers were less likely to offer the division that benefited themselves maximally. (D) Cumulative frequencies of MAXOffers and MAXAccepts over all 120 UG rounds, for three example dyads. For the leftmost dyad, estimates of reciprocity were low for both the Proposer (αP) and Responder (αR). This is reflected in high number of MAXOffers and MAXAccepts; the Proposer was free to offer divisions that benefited themselves maximally because the Responder did not challenge such proposals with rejections (negative reciprocity). In the middle dyad, the Responder did challenge MAXOffers and this is Figure 1. Results This confirmed that each player’s choices were driven largely by evaluations of utility that incorporated a reaction to their opponent’s prior behaviour. As an exploratory analysis, we assessed whether performance of either player on the iUG was related to per- sonality variables measured with the Action Control Scale (ACS-90)31; and Interpersonal Reactivity Index (IRI)32. Given the exploratory, post-hoc nature of these analyses, however, we do not present the results in the main body of text; instead the reader can consult them in Table S3 of the Supplementary Material. In brief, neither MAXOffers nor MAXAccepts were associated with scores on either personality instrument. Neuroimaging. Despite the behavioural differences, the PRMOD > PPMOD and PPMOD > PRMOD contrasts revealed no significant differences in EU-modulated brain responses between the PR and PP conditions for either player. When collapsing across the two conditions, both players exhibited two similar patterns of BOLD signal expressing the UGMOD > CTRL contrast: In the first, brain responses were modulated positively by EU – that is, they were greater when Proposers saw more utility in offering the least advantageously inequitable (more generous) division, and Responders saw greater utility in accepting the proposed division. This pattern encom- passed primary striate and ventro-medial prefrontal cortex (vmPFC) in both players, and the superior temporal sulcus (STS) in Proposers (Fig. 2A). The second pattern represents BOLD signals modulated negatively by EU – these brain responses became stronger in Proposers with lower EU for the more generous division, and when Responders saw less utility in accepting the offered division. This second pattern encompassed primary and sec- ondary striate cortices, aMCC and supplementary motor cortex (SMA), lateral prefrontal cortices and the insulae in both players; and, in Proposers, the thalamus (Fig. 2B). Clusters of brain regions exhibiting these two opposing patterns are listed in Table S4. Despite the apparent difference between players in EU modulation within the STS and thalamus, direct comparisons between player roles revealed stronger positive modulation for Proposers only in the left primary motor cortex. This was true even after more lenient thresholding (pFWE < 0.01). Results Patterns of player behaviour. (A) Mean (±SD) proportions of offers of maximal advantageous inequity (MAXOffers; blue) and their acceptance (MAXAccepts; red) across all dyads for each of the 10 choice sets. This indicates more MAXOffers and MAXAccepts across PR relative to PP rounds, which was confirmed with direct comparisons (see text). Error bars present standard deviations. (B) Probabilities of MAXOffers and MAXAccepts plotted as a logistic function of expected utility (EU), as estimated with the reciprocity model. (C) The proportion of MAXOffers in each condition plotted as a function of Proposers’ reciprocity (α), as estimated by the reciprocity model. This reveals that with increasing reciprocity, Proposers were less likely to offer the division that benefited themselves maximally. (D) Cumulative frequencies of MAXOffers and MAXAccepts over all 120 UG rounds, for three example dyads. For the leftmost dyad, estimates of reciprocity were low for both the Proposer (αP) and Responder (αR). This is reflected in high number of MAXOffers and MAXAccepts; the Proposer was free to offer divisions that benefited themselves maximally because the Responder did not challenge such proposals with rejections (negative reciprocity). In the middle dyad, the Responder did challenge MAXOffers and this is Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 4 www.nature.com/scientificreports/ reflected in a higher reciprocity estimate. These rejections did not sway the Proposer, however; they continued to propose such offers, reflected in a low reciprocity estimate. In the rightmost dyad, estimates of reciprocity were high for the Proposer and Responder. This is reflected in a low number of MAXOffers and MAXAccepts; the Responder was unwilling to accept such offers, and the Proposer reacted to this with less advantageously inequitable proposals. reflected in a higher reciprocity estimate. These rejections did not sway the Proposer, however; they continued to propose such offers, reflected in a low reciprocity estimate. In the rightmost dyad, estimates of reciprocity were high for the Proposer and Responder. This is reflected in a low number of MAXOffers and MAXAccepts; the Responder was unwilling to accept such offers, and the Proposer reacted to this with less advantageously inequitable proposals. both Proposer and Responder behaviour was substantially poorer than that of our reciprocity model, according to both AIC and BIC criteria (Proposers = 2866.2 and 2883.4; and Responders = 1922.74 and 1939.9; p < 0.001). Results In other words, these seemingly unique EU-modulated brain responses appear to be present in both players but to subtly (non-significantly) different degrees.i gi yf g As specified in Table 1, intra-dyad correlations (IDC) in brain responses measured across all rounds of each experimental condition revealed greater inter-brain alignment between interacting players on both PR and PP relative to the CTRL condition. For the PPIDC > CTRLIDC contrast, greater IDC was observed in bilateral occipital and extra-striate cortices, and right inferior parietal cortex. In addition to these posterior sites, the PRIDC > CTRLIDC contrast revealed increased IDC in lateral prefrontal cortices, aMCC, posterior cingulate cortex, and bilateral AI. The PRIDC > PPIDC contrast revealed the extent of this differential IDC between experimental conditions, with stronger alignment over PR compared with PP rounds in right aMCC, AI, and lateral temporal cortex, and bilateral inferior occipital cortices. To investigate whether the strength of IDC was related to the degree of reciprocity, we performed an ROI analysis at the location in which IDC expressed the PRIDC > PPIDC contrast maximally (aMCC; x = 4, y = 34, z = 28). This revealed that greater IDC in the PR condition within right aMCC was correlated positively with estimates of reciprocity in Proposers (ρ[17] = 0.65, p = 0.003) but not in Responders (ρ[17] = 0.40, p = 0.089). Interestingly, no relationships were observed between IDC expressing this contrast in the right AI (x = 34, y = 26, x = −4) and reciprocity estimates of either Proposers (ρ[17] = 0.44, p = 0.062) or Responders (ρ[17] = 0.26, p = 0.291). Results from the PRIDC > PPIDC contrast and the relationship with Proposers’ α estimates are illustrated in Fig. 3. Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 Discussion Statistically equivalent patterns of EU- modulated brain responses were observed in Proposers (left) and Responders (right). are influenced by their emotional reaction to the prior behaviour of their interaction partner: If the Proposer predicts that their opponent is likely to reject the more selfish division, and they consider the Responder to have reacted reasonably to past offers, they see more utility in increasing their payoff at a cost to their own. Conversely, if the Proposer believes that the Responder has behaved uncooperatively in the past, they will be unwilling to change their egoistic motives despite their predictions (positive and negative reciprocity, respectively). Likewise, the Responder is more likely to accept a division that disadvantages themselves disproportionately more than their opponent if they consider the Proposer to have behaved fairly (generously, or with justified selfishness) in the past; but they see greater utility in rejecting such offers as a means of retaliating against prior unacceptable offers. Our adapted reciprocity model outperformed a self-regarding model without any estimate of reciprocity (a model that considered only inequity aversion for Responders and adaptive risk aversion for Proposers) and a reinforcement-learning model30.il g We interpret the superior fit of our model to reflect the novel aspects of our experimental design, which allowed a more accurate simulation of real-world social decision making. In our two-choice iUG (a) repeated exchanges were made between the same two individuals, allowing both players to adapt and express their reac- tions to the opponent’s behaviour; (b) Responders saw the choices from which Proposers selected their offer, and Proposers saw the decisions of the Responder; and (c) two conditions were implemented to encourage stronger reciprocity between players. This differs from other paradigms in which the social context is largely removed; many studies employ a one-shot version of the UG whereby the offers on each round are made by different anon- ymous Proposers28,33,35, players’ intentionality is masked28, or feedback about their choices is concealed from their opponent26. Fairness evaluations have been shown to adapt over multiple rounds, however; Responders compare offers against normative reference points that change over successive rounds in response to Proposers’ behaviour, and both subjective feelings and affective neural responses are sensitive to violations of these adaptive norms33,36. Proposers also adapt to their opponent, but on a strategic level; a lower frequency of generous offers on the Dictator Game is taken as evidence that such proposals reflect strategic self interest37. Discussion By scanning the brains of two individuals simultaneously while they are engaged in recursive economic exchanges with one another, we have explored brain processes associated with the bidirectional reciprocity characterising real-world, repeated dyadic interactions. This revealed three important findings: First, by modelling EU in a way that incorporates the degree of reciprocity displayed by each interactant, we show that both players’ choices on the iUG were influenced not only by their own payoff or social preferences, but also by their reactions to their oppo- nent’s prior behaviour. Second, both players exhibited opposing patterns of neural response modulated positively or negatively by these estimates of EU. Such modulation reveals neural coupling, whereby the brain of one inter- actant responds to the behaviour of their interaction partner. Third, neural signals within right AI and aMCC are correlated between interacting players, particularly during exchanges that require choices between advantageous and disadvantageous inequity – those in which decisions are more likely to be driven by reciprocal tendencies. Interestingly, this pattern of inter-brain alignment was stronger with more reciprocating Proposers. l’ 24 d l b d f d d l fi Cox et al.’s24 reciprocity model estimates EU by considering a range of individual-specific parameters. By weighing each player’s payoff against that of their opponent, it incorporates any risk aversion shown by Proposers26 or norm-seeking behaviour33 and avoidance of subjugation by Responders5,34. In our adaptation, however, we incorporated the additional probability that the Responder would accept an offer given their previous decisions. As such, risk aversion shown by the Proposer is modelled as a flexible adaptation to the Responder’s behaviour updated on a round-by-round basis. The reciprocity model also considers the extent to which a player’s choices Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Brain responses modulated parametrically by estimates of EU. Results of the UGMOD > CTRL contrast (pFWE < 0.001) revealed brain regions in which BOLD response across UG rounds was modulated positively (A) or negatively (B) by EU, as estimated with our reciprocity model. Statistically equivalent patterns of EU- modulated brain responses were observed in Proposers (left) and Responders (right). Figure 2. Brain responses modulated parametrically by estimates of EU. Results of the UGMOD > CTRL contrast (pFWE < 0.001) revealed brain regions in which BOLD response across UG rounds was modulated positively (A) or negatively (B) by EU, as estimated with our reciprocity model. Discussion The table lists peak voxels within sign clusters expressing condition-specific intra-dyad correlations, as revealed by random-effects paired-sam t-tests for each contrast. Coordinates are specified in MNI space (2 mm3 resolution). P-values are given family-wise error (FWE)-correction. Coordinates of the voxel around which a sphere was formed for analysis (see text). Table 1. Brain regions expressing intra-dyad correlations (IDC). The table lists peak voxels within significant clusters expressing condition-specific intra-dyad correlations, as revealed by random-effects paired-samples t-tests for each contrast. Coordinates are specified in MNI space (2 mm3 resolution). P-values are given after family-wise error (FWE)-correction. Coordinates of the voxel around which a sphere was formed for ROI analysis (see text). unfair in response to, respectively, unforgiving or tolerant Responders. Likewise, Billeke et al.11 report that while some Proposers adapt their offers to Responder’s behaviour, others adopt more unwavering strategies. We extend these findings by accurately modelling choices over multiple exchanges with the same known partner, permitting expressions of reciprocity during reputation building; players with low reciprocity appeared to try and maximise their advantage by establishing a “tough” reputation with unwavering unfair offers, and/or occasionally rejecting lower fair offers39.i f Our modification of the iUG might also explain the lack of association between self-reported trait empa- thy and proposals or acceptances/rejections observed in our exploratory analyses. Barraza and Zak40, for example, report more generous proposals after empathy induction in participants high on trait empathy. This relationship between Proposer behaviour and empathy was observed in a one-shot version of the UG between anonymous players, however, a context in which reciprocal tendencies cannot influence adaptive behaviour over repeated exchanges. Similarly, Shamay-Tsoory et al.41 observed that patients with lesions to the vmPFC who reported less empathy (perspective taking) were more likely to reject offers, but these Responders played against a pre-programmed computer. Finally, Lockwood et al.42, report that reward-based learning of self- and other-reward was related to individual’s empathy, but this learning was observed in response to non-social sym- bolic stimuli. Over the course of our modified iUG, players had the opportunity to maximise their payoff by learning how best to adapt to the behaviour of their opponent. The fact that expressions of reciprocity were unrelated to trait empathy suggests that such adaptation reflects self-oriented strategies or affectivity, rather than other-oriented considerations (e.g., guilt). Discussion Further, Winter and Zamir38 demonstrate that Proposers’ strategies emerge during the course of the game, becoming more fair or Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 6 www.nature.com/scientificreports/ Label PFWE Voxels T x y z PPIDC > CTRLIDC R Inferior parietal lobule <0.001 58 5.47 32 −66 48 L Fusiform gyrus <0.001 1042 7.07 −34 −80 −18 R Lingual gyrus <0.001 891 7.34 20 −86 −6 PRIDC > CTRLIDC L Frontal pole 0.002 61 7.22 −38 56 0 R aMCC 0.001 64 5.36 8 36 32 R Middle frontal gyrus <0.001 159 7.51 42 32 30 L AI 0.001 73 5.80 −32 20 −2 R AI <0.001 220 6.06 34 24 −10 R PCC <0.001 518 5.66 32 −74 56 R Inferior parietal lobule 0.003 60 5.07 28 −64 46 L Middle occipital gyrus <0.001 3044 8.27 −28 −86 2 R Inferior occipital gyrus <0.001 2552 9.94 36 −86 −4 PRIDC > PPIDC R aMCC 0.022 42 5.74 4 34 28 * R AI <0.001 75 5.57 34 26 −4 R Medial temporal gyrus 0.028 40 6.33 60 −28 −12 L Inferior occipital gyrus <0.001 100 5.79 −42 −80 −8 R Inferior occipital gyrus <0.001 405 6.76 42 −80 −8 L Cerebellum 0.016 52 6.07 −26 −72 −24 Label PFWE Voxels T x y z PPIDC > CTRLIDC R Inferior parietal lobule <0.001 58 5.47 32 −66 48 L Fusiform gyrus <0.001 1042 7.07 −34 −80 −18 R Lingual gyrus <0.001 891 7.34 20 −86 −6 PRIDC > CTRLIDC L Frontal pole 0.002 61 7.22 −38 56 0 R aMCC 0.001 64 5.36 8 36 32 R Middle frontal gyrus <0.001 159 7.51 42 32 30 L AI 0.001 73 5.80 −32 20 −2 R AI <0.001 220 6.06 34 24 −10 R PCC <0.001 518 5.66 32 −74 56 R Inferior parietal lobule 0.003 60 5.07 28 −64 46 L Middle occipital gyrus <0.001 3044 8.27 −28 −86 2 R Inferior occipital gyrus <0.001 2552 9.94 36 −86 −4 PRIDC > PPIDC R aMCC 0.022 42 5.74 4 34 28 * R AI <0.001 75 5.57 34 26 −4 R Medial temporal gyrus 0.028 40 6.33 60 −28 −12 L Inferior occipital gyrus <0.001 100 5.79 −42 −80 −8 R Inferior occipital gyrus <0.001 405 6.76 42 −80 −8 L Cerebellum 0.016 52 6.07 −26 −72 −24 Table 1. Brain regions expressing intra-dyad correlations (IDC). Discussion The scatter plot shows that greater IDC in the PR condition was correlated positively with Proposer reciprocity estimates (αProp.). There is accumulating evidence that the gyral aspect of the ACC processes the rewards for others, whereas the sulcus seems more sensitive to first-person reward7,44. Together with our findings, this indicates that the aMCC might compute the difference between self- and other-reward, whereas AI performs an emotional evaluation of any reward discrepancy that drives an affective (reciprocal) response to unfair reward discrepancy. This functional dissociation between aMCC and AI might explain the specificity of relationship between Proposer reciprocity estimates and inter-brain coupling in the aMCC during rounds that require decisions between advantageous and disadvantageous inequity; in our procedure, both players saw the choice set from which Proposers had to make decisions, and would have processed the difference in inequity presented by the two constituent divisions. In contrast, any affective reaction to the offered division would have been greater in Responders than Proposers, resulting in less covariance between players. Future studies should attempt to delineate the functions of these two brain regions during economic exchanges by modelling responses of the aMCC and/or AI as mediators of reciprocal choices – specifically, offers of unfair divisions and their rejections. Alternatively, our findings could be extended by investigating whether the parameters used in our model to estimate reciprocity – specifically, param- eters representing players’ emotional state (θ) and choice stochasticity (ϵ) – serve to modulate intra-subject brain responses differentially. This might dissociate between neural processes associated with emotional reactivity and error processing during economic choices. p g g Brain regions encompassed by the two opposing patterns of EU-modulated responses have been implicated in different forms of learning during social decision making. The reinforcement learning framework suggests that decision making is driven by differences between predicted and actual reward outcomes (prediction errors). The ACC (particularly the sulcal aspect) is involved in reward prediction errors45, especially those concerning the rewards that others will receive46. In contrast, the lateral temporal cortex is engaged during social prediction errors45 and its response profile differentiates between individuals according to their social learning strategy16 – it is engaged more in individuals who predict rewards on the basis of their interaction partners’ behaviour, relative to those who act according to more simple learned associations between their own actions and rewards. Discussion Importantly, Proposers’ offers on each round were restricted to those presented by specific choice sets, which were known to the Responder. As such, on some rounds they were free to propose selfish offers without feeling remorse. p pif g Turning now to our neuroimaging results, both players expressed two opposing patterns of brain response modulated by EU estimates: Firstly, in the ventro-medial prefrontal cortex (vmPFC) of both players, stronger neural responses were elicited for divisions with higher EU – that is, more generous divisions for the Proposer and their acceptance by Responders. This converges with and extends existing findings; fair offers have been shown to engage the vmPFC more than unfair offers8, and patients with vmPFC lesions are more inclined to accept unfair offers36. Hutcherson et al.43 propose that vmPFC combines various information to weigh value for one’s self against that for another (player), and uses the information to generate adaptive responses. Our findings demonstrate that this brain region is implicated specifically in positive valuations. In contrast, throughout the aMCC and AI the responses of both players’ brains were stronger for divisions with lower EU – in other words, selfish divisions and offers that were rejected. The pattern of negatively modulated brain responses also advances previous findings; meta-analyses8,9 show consistent engagement of ACC/aMCC and AI of Responder’s brains in response to unfair offers, and neural responses in dorsal ACC and bilateral AI of Responders’ brains were found to be modulated by the degree of payoff inequity in unfair offers35. Given the accuracy of our reciprocity model in predicting players’ choices, these two patterns of EU-modulated brain responses appear to reflect opposing eval- uations of utility driven by reactions to the behaviour of our interaction partner(s), which then drive opposing behavioural adaptations – specifically, positive or negative reciprocal responses. Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 7 www.nature.com/scientificreports/ Figure 3. Intra-dyad correlations (IDC). Brain images illustrate greater IDC in the PR compared with the PP condition (PRIDC > PPIDC; pFWE < 0.05) in the right anterior insula (AI; left) and anterior midcingulate cortex (aMCC; right). The scatter plot shows that greater IDC in the PR condition was correlated positively with Proposer reciprocity estimates (αProp.). Figure 3. Intra-dyad correlations (IDC). Brain images illustrate greater IDC in the PR compared with the PP condition (PRIDC > PPIDC; pFWE < 0.05) in the right anterior insula (AI; left) and anterior midcingulate cortex (aMCC; right). Discussion The differential sensitivity of these two brain systems that we have observed points to a dissociable contribution of reward-based and social learning processes in utility evaluations of generous (more costly) and selfish offers during complex, repeated interactions.h g p , p The equivalence of EU-modulated brain responses observed between the PR and PP conditions suggests that players engaged in similar evaluations of utility in both types of exchange. Wang et al.12 also report similar brain responses between players in dorsal ACC. This might also explain why both players expressed highly similar patterns of EU modulation; Responders took less time to accept/reject an offer than Proposers took to make the proposal on both conditions, suggesting that Responders had already begun a similar evaluation of the choice sets before an offer was made. Moreover, both players were slower to make their choices on rounds in which choice sets involved decisions between advantageous and disadvantageous inequity, suggesting that such decisions were more cognitively demanding. Since the proportions of selfish offers and their acceptance were greater on these PR rounds, these decisions appear to involve a mutual appreciation of the intention behind selfishness. Interestingly, estimates of Proposers’ reciprocity were correlated positively with their response times and negatively with the number of selfish offers made on PR exchanges – the more adaptive they were, the less selfish their offers. Finally, greater inter-brain alignment in aMCC on PR compared with PP rounds was associated strongly with the degree of Proposers’ reciprocity. Taken together, we propose that inter-brain alignment reflects a mutual effort of players to adapt to their interaction partner by inferring the intentions behind their actions – a process that involves an evaluation of their prior behaviour. Consistent with this interpretation, the response of the aMCC has been asso- ciated with task complexity, uncertainty, predicted value, and social decision making7,44,47. Furthermore, hyper- scanning research consistently demonstrates neural coupling within the ACC48–50, which is suggested to reflect accuracy in individuals’ representations of their interaction partners’ intentions48 and estimates of their behav- iour19,49. Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 Methods Participants. C h R bli Participants. The initial sample comprised 40 males recruited from various faculties of Masaryk University, Czech Republic, who participated for monetary compensation. These individuals were paired to form 20 age- matched dyads (mean age difference = 1.2 years), the members of which had never met prior to the experiment. Male-male dyads were measured exclusively to avoid potentially confounding factors of mixed-sex interactions. Neuroimaging data from both participants comprising one dyad were omitted due to excessive head motion (see below). The 38 males comprising the remaining 19 dyads were all right-handed, had a mean age of 24.6 years (standard deviation [SD] = 3.7; range = 19.8–38.0), reported normal or corrected-to-normal vision and no history of neurological diseases or psychiatric diagnosis. All participants provided informed consent prior to the experimental procedure, which was approved by the Research Ethics Committee of Masaryk University. All methods were carried out in accordance with the declaration of Helsinki. Procedure. Participants were introduced to one another for the first time on the day of scanning, during which they exchanged names and shook hands before being sent to one of two scanners located in adjacent rooms (see Imaging Protocol). Player roles were assigned randomly at the start but remained fixed throughout the procedure – one participant played the role of Proposer and the other Responder on all rounds. Fixing roles in this way allowed players the opportunity to learn about and adapt to their partner’s behaviour over a relatively short period. Players were told explicitly that throughout the experiment they would play with the same indi- vidual to whom they had just been introduced, and confirmed that they believed this to be true throughout the experiment. Each dyad underwent two functional runs performed successively in a single scanning session. In an event-related fashion, the two runs together comprised 120 rounds (events) of the iUG divided equally among two types of exchange (see Stimuli) and 60 rounds of a control condition (CTRL). Each UG round started with the Proposer being given four seconds to choose one of two divisions of the pie (the choice set; see Stimuli) between themselves and the Responder (Choice period). After this fixed period, the Proposer’s offer was highlighted for four secs (Offer period), during which the Responder could either accept or reject the proposal. After this four-sec period the Responder’s decision was then presented for a final four secs (Decision period). Methods Participants. C h R bli The exact same proce- dure was followed on CTRL rounds, but the choice set comprised two alternative divisions of colour between the players; rather than dividing a pie, Proposers were required to choose the colour they preferred for themselves and the colour that should go to the Responder, and the Responder then accepted or rejected that offer. Both play- ers were instructed that CTRL rounds had no monetary consequence. Each round ended with a jittered inter-trial interval, with a fixation cross presented pseudo-randomly for 2–4 (mean = 3) secs. An example UG and CTRL round is illustrated in Fig. 4. The same fixed sequence of choice sets (one for each run) was used for all pairs, which was defined by a genetic algorithm for design optimisation55 set to maximise contrast detection between conditions (see below). All stimuli were presented to both players simultaneously – Responders saw the initial choice set from which Proposers selected their offer, and Proposers saw the Responder’s accept/reject decision. Players were instructed at the start that they would receive the outcome of six rounds selected at random, and the mean payout was 270 CZK (approx. €10). At no point was any information given to participants on the number of rounds remaining in the task. g As pilot data for a future study, this sample also completed two personality instruments: the ACS-90 and the IRI. Since no a priori hypotheses were formulated concerning these data, they are not presented in this paper. Instead, the reader is referred to the Supplementary Material for further information. UG Stimuli. On each round of the iUG, players were presented with a choice of two possible divisions of 100 CZK (approx. €4) that differed in the degree of inequity (the “choice set”), and Proposers were required to select one division to offer the Responder. An example choice set is illustrated in Fig. 4, and Supplementary Table S1 lists all the choice sets used in the experiment. To encourage positive and negative reciprocity, we selected 10 choice sets for which repeated proposals and acceptances of minimally inequitable divisions were lowest in behav- ioural piloting. The choice sets took two forms: On Proposer-Responder (PR) rounds, one division presented the Proposer with advantageous inequity while the second presented them with disadvantageous inequity – in other words, greater relative payoff was achieved by the Proposer for one division but the Responder in the other (e.g., 70:30\|30:70). Discussion Such an interpretation fits our pattern of results nicely: On PR rounds, which require decisions between advantageous and disadvantageous inequity, we observed greater inter-brain alignment within aMCC among dyads comprising more reciprocating Proposers – the player who initiates each exchange.ff y p g p g p p y g Our adapted iUG paradigm affords multiple applications: For example, inter-brain effects could represent ffective neuromarkers for the quality of social communication1, providing assessment of social dimensions 8 www.nature.com/scientificreports/ along which certain psychiatric illnesses might be described51. It is important to acknowledge the limitations of our study that can be addressed in future research, however: Firstly, we have not considered some important factors that might influence the degree of reciprocity shown by either player. Behaviour during the UG appears to be influenced partly by variability in strategic reasoning52 and prosocial predispositions53. We observed large variability in the estimates of reciprocity among our sample of Proposers that appeared to be unrelated to trait empathy, and future studies should examine if and how individual differences in other personality variables influ- ence reciprocal behaviour during iUG. Furthermore, by examining only all-male dyads we have investigated a very specific type of dyadic interaction, and we have not explored potential sex differences in inter-brain effects54. Secondly, our design did not include choice sets that present a fair allocation of payoff, so it was not possible to contrast this behaviour with that following unfair options. Future studies should incorporate this condition to study all possible outcomes of repeated interaction. Finally, while our measure of brain-to-brain alignment across entire rounds permitted us to examine the degree of neural coupling through a bidirectional exchange, this crude measurement offers no insights into directionality. Neural coupling during sequential exchanges will necessarily be circular – just as the Responder’s brain synchronises to the offer of the Proposer, their decision to accept or reject will lead to systematic neural responses in the Proposer that influence future offers. To investigate such cir- cular brain-to-brain coupling, methods for assessing directed between-brain dependencies should be developed for hyperscanning research (e.g., inter-brain psychophysiological interactions). Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 Methods Participants. C h R bli Conversely, on Proposer-Proposer (PP) rounds both divisions presented a greater relative payoff Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 9 www.nature.com/scientificreports/ Figure 4. Event sequence. Timing of stimulus presentation for an example UG (left) and CTRL round (right), comprising 4-second Choice, Offer and Decision periods. In these examples, the offer made by the Proposer on a PR round is rejected by the Responder, while the offer made on the CTRL round is accepted. Figure 4. Event sequence. Timing of stimulus presentation for an example UG (left) and CTRL round (right), comprising 4-second Choice, Offer and Decision periods. In these examples, the offer made by the Proposer on a PR round is rejected by the Responder, while the offer made on the CTRL round is accepted. for the Proposer, differing only in magnitude (e.g., 70:30\|60:40). Presenting a choice between advantageous and disadvantageous inequity on PR rounds was intended to encourage greater expressions of positive or negative reciprocity from both players. Reciprocity Model. Unlike other distributional preference models that take into account only the final rel- ative payoff between players34,56, Cox et al.’s24 reciprocity model attempts to fit the behavioural observation that choices depend not only on the final monetary distribution but also on any available alternatives. Furthermore, this model also considers that player choices are influenced largely by their emotional reactions to their partner’s prior behaviour – specifically, whether their proposal or decision to accept or reject reflects positive or negative reciprocity. Finally, unlike higher beliefs equilibrium models29,57 the reciprocity model is tractable and enables the estimation of behavioural parameters. In our adaptation, for each player the EU of each division of the pie was specified as: (1) U x x x x ( , 100 ) ( )(100 ) (1) θ − = + + −  here, x is the player’s portion of the division, θ is a scalar representing their emotional state, and ϵ represents random shock with standard logistic distribution. Random shock represents an unobserved component of the utility function – a random variable that adds stochasticity to each player’s choice behaviour (e.g., unintended responses). The emotional state was formulated as: here, x is the player’s portion of the division, θ is a scalar representing their emotional state, and ϵ represents random shock with standard logistic distribution. Methods Participants. C h R bli Functional imaging was performed in two runs, both comprising 690 volumes (23 mins). Four dummy volumes were acquired at the beginning of each run to allow the gradients to reach steady state. For localisation and co-registration, a high-resolution T1-weighted struc- tural MR image was acquired prior to the functional runs (MPRAGE, TR/TE = 2300/2.34 msec; flip angle = 8°; matrix = 240 × 224 × 224, 1 mm3 voxels). For a given dyad, volume acquisition was synchronised between scan- ners (mean asynchrony = 1.13 [SD = 3.83] msec) with use of a programmable signal generator (Siglent SDG1025, www.siglent.com; mean acquisition delay = 10 [SD = 3.49] msec). Pre-processing. For every subject, each of the two time-series were pre-processed separately using a variety of tools packaged within FMRIB’s software library (FSL58), full details of which are provided in the Supplementary Materials. Importantly, both players from one pair exceed our exclusion criterion of 1 mm of movement in any direction for either run, and were omitted from all subsequent analyses. General linear modelling. All fMRI data modeling was performed in the same platform – SPM12 (http:// www.fil.ion.ucl.ac.uk). General linear modelling was performed on the pre-processed time-series in a two-step process: At the individual level, within-subject fixed-effects analyses were used for parameter estimation across both runs. Event-related responses were modelled with durations determined by the participants’ response time in each period of interest (see below), convolved with the canonical hemodynamic response function provided by SPM12: to capture brain responses that reflect reciprocal reactions in each player to their partner’s prior behaviour, for Proposers we modelled the Choice periods of each round until an offer was selected, while for Responders it covered the Offer period until a decision had been made to accept or reject the proposed division. This resulted in three task regressors for each participant, corresponding to the mean effect of the respective period in PR, PP or CTRL rounds. The remaining part(s) of the rounds were modelled as regressors of no interest. For the PR and PP task regressors we added parametric modulators that expressed the round-by-round EU esti- mated with the reciprocity model (PRMOD and PPMOD); and by collapsing across the PR and PP conditions we also examined the modulatory effect throughout all UG rounds (UGMOD). Methods Participants. C h R bli To examine brain responses in Proposers and Responders separately, statistical evaluation of parameter estimates from these first-level analyses were per- formed in the following group-level whole-brain random-effects contrasts using one-sample t-tests: PRMOD vs. PPMOD, UGMOD > CTRL. Comparisons between players were then performed with independent-sample t-tests of the same contrasts. Cluster-wise thresholding was applied at p < 0.001, with family-wise error (FWE) correction for multiple comparisons. Intra-dyad Correlations. Brain-to-brain alignment on the iUG was measured with condition-specific intra-dyad correlations. To achieve reliable correlation coefficients, we combined the three phases of each round to maximise the number of volumes over which BOLD signals were correlated (6 volumes [12-sec rounds] × 60 rounds [2 runs × 30 rounds per condition]). Although this prevented us from examining alignment in each phase separately, it allowed us to capture the overall degree of alignment that occurred throughout the course of each bilateral exchange; highly correlated brain responses should be observed throughout all phases only when players share an understanding of the intentions behind each other’s actions. Brain signals should be aligned during the Choice phase when both players have similar evaluations of the options comprising a choice set, and should remain synchronised during the Offer and Decision phases when the intention behind a proposal and its acceptance/rejection is appreciated by both opponents. For each Proposer-Responder dyad, using the grey-matter mask provided in SPM12 (ICBM-151) we cal- culated the condition-specific correlation in BOLD signal between spatially corresponding voxels of each player’s concatenated time-series, before converting correlation coefficients to z-scores using the Fischer trans- formation. This resulted in 19 whole-brain z-maps for each condition, with each voxel expressing the degree of condition-specific intra-dyad correlation (IDC) between a given Proposer and Responder at that spatial location (see Supplementary Fig. S1). We then compared the degree of IDC between conditions by performing the follow- ing group-level, whole-brain contrasts with random-effect paired-samples t-tests (cluster-level correction with FEW; pFWE < 0.05): PRIDC > CTRLIDC, PPIDC > CTRLIDC, PRIDC > PPIDC, and PPIDC > PRIDC. Methods Participants. C h R bli Random shock represents an unobserved component of the utility function – a random variable that adds stochasticity to each player’s choice behaviour (e.g., unintended responses). The emotional state was formulated as: x x ( ) (2) i 0 θ α = − (2) Equation (2) incorporates a player-specific reciprocity parameter, α, which serves to weight a comparison of the player’s share, x, against a fairness reference point, x0, by the extent to which a player’s choices are influenced by their partner’s prior behaviour. The reference point, x , 0 is a parameter estimated with α – it is different for each choice set. Using this utility function, we modelled round-by-round EU for both players. The Responder accepts a proposal if: x x ( )(100 ) 0 (3) θ + + − >  (3) The Proposer offers the least advantageously inequitable (more generous) division if: The Proposer offers the least advantageously inequitable (more generous) division if:   θ θ + + − > + + − P x x P x x ( ( )(100 )) ( ( )(100 )) (4) 1 1 1 2 2 2 (4) Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 10 www.nature.com/scientificreports/ In equation (4), x1 and x2 represent the division with minimal (or disadvantageous) and maximal advantageous inequity, respectively, and Pi represents the probability that the Responder will accept a division given their prior behaviour. In other words, the Proposer makes an offer that benefits themselves maximally only if they believe the offer is likely to be accepted. The Supplementary Material gives a full description of the procedures with which the various parameters were estimated. Imaging Protocol. For each individual, functional and structural MR data were acquired with one of two identical 3T Siemens Prisma scanners and 64-channel bird-cage head coil. Players were allocated to one of the two scanners in a counterbalanced fashion, ensuring an even number of Proposers and Responders were scanned in each. Blood-oxygen-level dependent (BOLD) images were acquired with a T2*-weighted echo-planar imaging (EPI) sequence with parallel acquisition (i-PAT; GRAPPA acceleration factor = 2; 34 axial slices; TR/ TE = 2000/35 msec; flip angle = 60°; matrix = 68 × 68 × 34, 3 × 3 × 4 mm voxels). Axial slices were acquired in interleaved order, each slice oriented parallel to a line connecting the base of the cerebellum to the base of orbitofrontal cortex permitting whole-brain coverage. Data Availability. The datasets generated and analysed during the current study are available from the cor- responding author on reasonable request. Data Availability. The datasets generated and analysed during the current study are available from the cor- responding author on reasonable request. Data Availability. The datasets generated and analysed during the current study are available from the cor- responding author on reasonable request. References 1 Koike T Tanabe H C & Sadato N Hyperscanning neuroimaging technique to reveal the ‘two in one’ system in social interactions References Falk, A., Fehr, E. & Fischbacher, U. On the Nature of Fair Behaviour. Econ. Inq. 41, 20–26 (2003). 28. Inaba, M., Inoue, Y., Akutsu, S., Takahashi, N. & Yamagishi, T. Preference and strategy in proposer’s prosocial giving in the ultimatum game. PLoS One 13, e0193877 (2018).h 29. Falk, A. & Fischbacher, U. A Theory of Reciprocity. Games Econ. Behav. 54, 293–315 (2006). h 30. Erev, B. I. & Roth, A. E. Predicting How People Play Games: Reinforcement Learning in Experimental Games with Unique, Mixed Strategy Equilibria. Am. Econ. Rev. 88, 848–881 (1998). 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Sex differences in brain activation to emotional stimuli: A meta-analysis of neuroimaging studies. Neuropsychologia 50, 1578–1593 (2012). p y g ( ) 5. Wager, T. D. & Nichols, T. E. References g p p p y 42. Lockwood, P. L., Apps, M. A. J., Valton, V., Viding, E. & Roiser, J. P. Neurocomputational mechanisms of prosocial learning and links to empathy. Proc. Natl. Acad. Sci. 113, 9763–9768 (2016). Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 12 www.nature.com/scientificreports/ Acknowledgements g We acknowledge the core facility MAFIL of CEITEC, supported by the Czech-BioImaging large RI project (LM2015062 funded by MEYS CR), for their support with obtaining scientific data presented in this paper. This work was supported financially by project GA16-18261S. g We acknowledge the core facility MAFIL of CEITEC, supported by the Czech-BioImaging large RI project (LM2015062 funded by MEYS CR), for their support with obtaining scientific data presented in this paper. This work was supported financially by project GA16-18261S. g We acknowledge the core facility MAFIL of CEITEC, supported by the Czech-BioImaging large RI project (LM2015062 funded by MEYS CR), for their support with obtaining scientific data presented in this paper. This work was supported financially by project GA16-18261S. Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-29233-9. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific REPOrtS \| (2018) 8:10896 \| DOI:10.1038/s41598-018-29233-9 13
https://openalex.org/W2168229704	https://storage.googleapis.com/jnl-lasa-j-erlacs-files/journals/1/articles/9701/submission/proof/9701-1-19685-1-10-20140814.pdf	es		Crisis de identidad y nuevos roles institucionales de los partidos	Revista europea de estudios latinoamericanos y del caribe/European review of Latin American and Caribbean studies	2,003	cc-by	11,058	Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 3 Crisis de identidad y nuevos roles institucionales de los partidos políticos en México Margarita Jiménez Badillo ‘Toda identidad política se constituye en referencia a un sistema temporal en el que la interpretación del pasado y la construcción del futuro deseado se conjugan para dotar de sentido a la acción presente’. (G. Aboy Carlés) El triunfo de Vicente Fox como candidato del Partido Acción Nacional (PAN) en las elecciones presidenciales del 2 de julio de 2000, fue un hito histórico para México porque por primera vez se produjo la alternancia en el poder después de que gobernara el Partido Revolucionario Institucional (PRI) durante setenta y un años consecutivos. Es un horizonte que abrió expectativas para consolidar el tan esperado orden democrático que no acaba de institucionalizarse toda vez que los partidos políticos enfrentan una crisis de su propia identidad como actores que tienen para sí el rol de consolidar la democracia en el país. El PRI dejó de ser la instancia principal de negociación y representación de los intereses políticos en la medida que fue perdiendo terreno ante la inclusión de fuerzas opositoras en el sistema político.1 Se trata de un signo de diversidad y modernización en el que los intereses corporativos que ciñeron la vida política del país, han dejado de ser la pauta para disipar el conflicto. Hoy, el sólo hecho de contar con un gobierno de oposición, es motivo suficiente para detenerse en los primeros cambios del funcionamiento institucional y advertir los nuevos rumbos que se imprimen en la arena política. Las reflexiones que aquí se presentan que van de diciembre 2000 a marzo 2002, tienen como finalidad analizar las tensiones existentes entre los partidos políticos, institución presidencial y Congreso Federal en este contexto de pluralismo en el que el PAN asume un rol de partido gobernante. Se muestra que lejos de la firmeza con que cada fuerza política ostentó identidades políticas definidas, en este momento histórico, sus roles institucionales exhiben divisiones internas y una crisis de identidad hacia los liderazgos cupulares, manifestada en actitudes y prácticas políticas de resistencia para desahogar la agenda pública. Por ello, oportuno es reflexionar si el gobierno de Fox asume un rol protagónico para consolidar la democracia y si los actores en juego están dispuestos a asumir este reto o por el contrario, la crisis de identidad empieza a ser una carga para encumbrar la idea de que el nuevo régimen conduce al país hacia su consolidación democrática, dejando traslucir la configuración de un nuevo régimen con nuevas personas en el poder, pero con viejos vicios y personalismos que actúan en nombre de la democracia. 4 \| European Review of Latin American and Caribbean Studies 74, April 2003 Geografía político-electoral México ha avanzado en la construcción democrática a partir de hacerse efectivos algunos de los atributos básicos que consideró Dahl (1989, 15) para caracterizar la poliarquía: elecciones libres y limpias, sufragio universal y derecho a competir por los cargos públicos. Hasta hace pocos años, O’ Donnell (1996, 7) había agrupado a México conjuntamente con la República Dominicana y Haití como países no poliárquicos por reflejar irregularidades en los procesos electorales, sin embargo, aún careciendo de otros requisitos, hoy prevalece un pluralismo político y multipartidista, lo que ha favorecido la alternancia en el poder local, y recientemente federal, que permiten sostener una evolución positiva en ese sentido. Efectivamente, los cambios profundos se advierten en el tránsito de un sistema de partido casi único a uno más plural en el que concurren tres partidos fundamentales2 y otros de ascendencia minoritaria o de reciente creación que favorecen la competencia electoral. En este nuevo contexto también se observan nuevos roles de la institución presidencial, considerada como otro de los pilares que constituyen el sistema político mexicano. Partido y presidencia no cuentan ya con la misma laxitud con que operaron por décadas; ha pasado a la historia la concentración del poder de que gozaba el Presidente, así como el gran capital electoral que tenía asegurado el partido en el gobierno, cuando éste mantenía el control del Parlamento con una mayoría absoluta. Las últimas dos legislaturas en la Cámara de Diputados dan cuenta de esos cambios en los que se advierte una ascendente posición del PAN y PRD con relación al otrora partido hegemónico3 (Tabla 1). Al momento de las elecciones, el PRI obtuvo 211 escaños y el PRD 51. La composición que se muestra en la tabla es la vigente a marzo 2002. Tabla 1: Resultados electorales para Diputados Federales 1988-2000 (en número total de escaños y porcentajes) Partidos 1988 1991 1994 1997 2000 207,0 41,4 Diferencia 1988-2000 + 106,0 + 21,2 Diferencia 1997-2000 + 86,0 + 17,2 PAN 101,0 20,2 90,0 18,0 119,0 23,8 121 24,2 PRI 260,0 52,0 321,0 64,2 300,0 60,0 239 47,8 210,0 42,0 - 50,0 - 10,0 - 29,0 - 5,8 PRD 139,0 27,8 40,0 8,0 71,0 14,2 125 25 52,0 10,4 - 87,0 - 17,4 - 73,0 - 14,6 Fuente: Para 1988 a 1994, datos tomados de Lujambio (1996); para 2000, http://www.cddhcu.gob.mx/servicios/bancoleg/datosyci.pdf La amplia presencia opositora de 1988, marca el agotamiento de la hegemonía priísta, prolongado declive que continúa al perder la mayoría absoluta en 1997. El PAN, de tercera fuerza electoral en ese año, pasa a ser en 2000 el partido en el gobierno sin mayoría parlamentaria y con gran proximidad en escaños al PRI opositor. El PRD después de su punto de inflexión más bajo en 1991, se recupera controlando apenas un 10,4 por ciento de escaños, aunque su eficiencia electoral se reconoce al obtener por segunda vez el gobierno del Distrito Federal. Este escenario más plural y equilibrado, es reafirmación de un sistema tripartita Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 5 que fue avanzando durante los años noventa, y da lugar a un Congreso más autónomo al no estar ya supeditado a las decisiones del Presidente en turno. A su vez, las elecciones de 2000 no dieron el triunfo a ninguna fuerza política para obtener la mayoría absoluta, como tampoco lo tuvo la anterior Legislatura (1997-2000). En ambas, el jefe del Ejecutivo se ve obligado a enfrentar intensas negociaciones con el Congreso para la aprobación de sus iniciativas, aunque la gran diferencia está en que con el último Presidente proveniente del PRI, Ernesto Zedillo (1994-2000), sí tenía mayoría en el Senado, mientras que el panista Vicente Fox (2000-2006), no la tiene en ninguna de las dos Cámaras, razón por la que las relaciones de conflicto entre Ejecutivo y Legislativo tensan más los procedimientos para mantener la gobernabilidad democrática. Una visión de la distribución territorial del voto refleja que el PRI sigue siendo una fuerza decisiva no sólo por su posición favorable en el Congreso Federal sino porque gobierna 19 de 31 entidades, por lo que es hoy el principal partido opositor. Según estudios del Banco Nacional de México (2001, 538), de las seis elecciones para gobernador efectuadas después del 2 de julio de 2000, el PRI controla un 40 por ciento de la votación en esas entidades; el PAN 61 por ciento y el PRD 18 por ciento, resultados que, no obstante provenir de alianzas entre partidos, confirman que las tres fuerzas políticas juegan un papel decisivo en el escenario político actual. Ahora bien, los comicios de julio 2000 fueron inéditos también no sólo por el triunfo de la oposición, sino porque ésta constituyó coaliciones partidistas para fortalecer su presencia política entre el electorado, un fenómeno que se venía practicando a nivel local pero no en elecciones nacionales. Tabla 2: Resultados de las Elecciones Federales de julio/2000 (votos efectivos en porcentajes) Partidos o coaliciones Alianza por el Cambio* Partido Revolucionario Institucional Alianza por México** Partido Convergencia Democrática Partido Auténtico de la Revolución Mexicana Democracia Social Candidatos no registrados Votos nulos Total Presidente 42,52 36,11 16,64 0,55 Diputados mayoría relativa 38,24 36,92 18.68 1,15 Senadores mayoría relativa 38,11 36,75 18,85 1,39 0,42 1,58 0,08 2,10 100,00 0,73 1,88 0,07 2,32 100,00 0.74 1,80 0,08 2,28 100,00 Fuente: Elaboración propia con datos de la Dirección Ejecutiva de Organización Electoral, Instituto Federal Electoral, 2000. * Partido Acción Nacional y Partido Verde Ecologista de México. Partido de la Revolución Democrática, Partido de los Trabajadores, Convergencia Democrática, Partido de la Sociedad Nacionalista y Partido Alianza Social. La Tabla 2 muestra que el triunfo de Vicente Fox como Presidente de la República, fue con el 42,5 por ciento de la votación total, lo que significa que es el primer Presidente que llega al poder con menos de la mayoría (50 por ciento) de los electores (Aguayo 2000, 251). Se muestra también la debacle hegemónica del PRI4 ante la coalición de la Alianza por el Cambio, que llegó al poder con 42,5 por ciento, aunque dicha coalición PAN-PVEM se disolvió porque la legislación electoral 6 \| European Review of Latin American and Caribbean Studies 74, April 2003 federal, impide que se mantengan alianzas permanentes en el Congreso, lo cual obliga al PAN establecer acuerdos y negociaciones con los grupos parlamentarios opositores para el desahogo de la agenda legislativa. En el siguiente epígrafe, se recupera la noción de identidad política en la óptica de dar un nuevo sentido al comportamiento de los actores políticos de referencia, un devenir que tiene que ver con el orden democrático. Identidad política y nuevas pautas de comportamiento La incorporación de actores excluidos – la oposición – al proceso democrático se puede ver como un requisito más para un sistema poliárquico, el cual, en México se ha venido dando gradualmente, por extensión o profundización democrática, en términos del modelo de Garretón (1997, 20-21), en donde no hay un momento formal de cambio de régimen o de inauguración democrática sino que se da como un proceso de extensión o profundización en esta dimensión. Es decir, México transita de prácticas discrecionales que las elites en el poder reproducían en el régimen autoritario, a arreglos institucionales que tienen como base la negociación entre los intereses existentes, favoreciendo con ello, mayores garantías para la reproducción de las estructuras democráticas. Es en esos términos que México se encuentra en un proceso en el que no acaba de morir el viejo régimen pero tampoco está bien definido el nuevo. Es decir, la concentración del poder en la figura presidencial y un PRI hegemónico como ejes que dieron forma a un presidencialismo fuerte, han visto disminuidas sustancialmente, sus estructuras de poder en los últimos años. Las reformas electorales que introducen normativas más equilibradas para la competencia política, y que llevan a los triunfos electorales de la oposición, son apenas algunos elementos que contribuyeron al desgaste del presidencialismo fuerte y que dan pauta a cambios sustanciales. El PRI agotó su capacidad para reproducirse en el poder cuando su fractura interna llevó a miembros prominentes a salir de sus filas en 1987, para crear una fuerza política de izquierda, – primero como un Frente democrático con varios partidos, y más tarde como PRD – llegando a ser primera fuerza de oposición en la Cámara de Diputados por lograr 139 de 500 escaños en 1988. Este hecho, además de la inclusión de los partidos de oposición al escenario político – léase participación electoral y parlamentaria – son el pivote que apuntó al desarrollo de las fuerzas opositoras, y el punto de inflexión para el PRI, quien paulatinamente comenzó a relajar la disciplina que le caracterizaba para mantenerse como un partido cohesionado, pues sus militantes habían dejado de tener garantías de que, guardando lealtad a la figura presidencial, serían recompensados para continuar su carrera política. Estos cambios se suceden no sólo en el sistema de partidos sino incluso en el tipo de presidencialismo llevado al exceso en el pasado, y que cobra un perfil más atenuado con el gobierno de Ernesto Zedillo (1994-2000),5 de quien su arribo al poder tuvo como fondo, un escenario de incertidumbre por los acontecimientos de 1994.6 De ahí se explica que cada actor político asuma un nuevo rol que tiene como fondo, los arreglos institucionales mediante el consenso democrático: partidos, institución presidencial y Congreso Federal están llamados a asumir un rol más Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 7 inclusivo con relación al ‘otro’ en el régimen que se aparta de las prácticas autoritarias, adaptando sus fines a los nuevos tiempos, modificando sus estrategias programáticas y de negociación, rediseñando sus vínculos, lo que en otras palabras, le significa redefinir sus identidades en la construcción del nuevo orden político. Hablar de identidad, es evitar la pretericidad, es decir, la pura reproducción de instituciones (Aboy 2001, 64-74), enfocándole mejor en términos de un devenir, que haga visible la transformación de éstas. El mismo autor recupera las nociones de alteridad y diferencia, expresando que no hay identidad si no hay límites que la definan; esto es, no hay identidad fuera de un sistema de diferencias, límites que se configuran como operación hegemónica, por ello la identidad se constituye a través del antagonismo, es decir, se trata de una alteridad en tanto hay límites de una identidad política, del mismo modo que no hay política sin representación como es la constitución de los liderazgos o la conformación de una ideología política o bien la relación con ciertos símbolos como elementos cohesivos de una identidad (2001). El mismo autor, así como Palermo y Novaro (1996, 393-394), distinguen entre identidades por alteridad e identidades por escenificación sustentadas en la imagen del líder. Alteridad y escenificación son dos dimensiones constitutivas de toda identidad política: la alteridad corresponde a un liderazgo que opera con un alter intersubjetivo que distingue entre los ‘amigos’ y los ‘enemigos’, es decir, una lógica de antagonismo; la escenificación en cambio, es la dimensión representativa inherente a la constitución de toda unidad gregaria de acción; unifica lo heterogéneo, es punto de unificación de una sociedad polimorfa. En otras palabras, existe identidad cuando existe la alteridad, esto es, la exclusión. La identidad por alteridad provoca un conflicto más excluyente; mientras que la identidad por escenificación, conlleva a que las líneas de conflicto se diluyan, esto es, las oposiciones son más moderadas. Si bien, ‘toda sociedad está integrada por una diversidad de grupos que se forman en torno a identidades que pueden ser complementarias o antagónicas, permanentes o variables (Loaeza 1996, 33), los partidos políticos también se definen por una identidad que les ha otorgado un reconocimiento de ser agrupamientos necesarios para articular el conflicto en tanto actores relevantes para la consolidación democrática. En este sentido, se plantea aquí la necesidad de su desprendimiento de la lógica clientelar y autoritaria como visión del pasado, derivada del arraigo a la lealtad como única vía para mantenerse en la élite del poder; el comprometerse con sus principios ideológicos más que con los intereses cupulares, pues sin una sólida plataforma programática parecen desgastarse entre sí mismos al fragmentarse en distintas corrientes confrontadas entre sí. Su identidad se deriva también de la visión del futuro, en la búsqueda de una renovación como cuerpos políticos que encaran nuevos retos que tienen que ver directamente con la democratización del régimen político. Ahora bien, para explicar como contribuyen partidos, Presidencia y Parlamento, a la consolidación democrática, pertinente es el esquema teórico de Russo (1995, 116) cuando identifica dos vías para ese fin: a) consolidación democrática conflictual, en donde predomina el desacuerdo y conflicto, consolidándose el régimen a través del conflicto; y b) consolidación democrática consensual, que se da mediante el acuerdo y la cooperación, creando mecanismos de negociación entre 8 \| European Review of Latin American and Caribbean Studies 74, April 2003 las elites para dirimir los conflictos y poder establecer vínculos de cooperación. La primera, se caracteriza por un predominio de confrontación entre oposición y gobierno y por la postergación de decisiones sobre problemas relevantes. La segunda, se caracteriza porque el consenso es la alternativa más económica para los actores relevantes; y se adoptan las decisiones de mayor alcance para el régimen político, con la participación del gobierno y de la oposición. Estos dos extremos son en realidad, espacios de competición política en tanto los actores respetan las reglas institucionales, y optan por concurrir abiertamente a luchar por el poder político, luego entonces, la competencia es también parte de su propia identidad como miembros de una agrupación política en competencia. El polo por el que están transcurriendo los acontecimientos en México decididamente es el consensual porque existen incentivos para la negociación entre partidos y gobierno y porque ninguna oposición es excluyente, en tanto los tres partidos han sido y son gobierno a nivel municipal y estatal y su propia identidad y acción política les ha otorgado tal status. El criterio para diferenciar ambos procesos, es la relación existente entre oposición y gobierno porque si ésta se transforma en gobierno se convierte en un agente clave de la consolidación democrática (Russo 1995, 116-119). Es en este sentido que el rol de la oposición ocupa un papel preponderante, pues depende de ésta, la vía que siga la consolidación democrática. Para determinar la relación existente entre oposición y gobierno, se tiene en cuenta la fortaleza o debilidad del Parlamento frente al Ejecutivo, es decir, si el jefe del Ejecutivo tiene una gran concentración de poder y el Parlamento muestra su debilidad en la arena de negociación política, entonces para fines operativos se le denomina aquí como oposición reactiva. Si por el contrario, la oposición en el Parlamento da muestras de alterar su inercia y asume un rol más protagónico, se le puede identificar como activa.7 Definir este comportamiento político, lleva a considerar los recursos institucionales con los que cuenta, es decir, su posición en número de escaños en el Congreso Federal. El PRI por ejemplo, pese a su derrota como partido en el poder, y no obstante su fractura interna por los distintos brotes de desplazamiento a otro partido, transfugismo o divisionismo entre tradicionalistas y tecnócratas, o la creación de una nueva fuerza política, sigue siendo un partido relevante, y una fuerza parlamentaria activa que relativamente, mantiene aún su disciplina de votación. Diferente al PRD, que ve disminuida su posición en escaños, restándole fuerza para lograr éxito en su agenda legislativa, por lo que su rol debe ser ahora más activo que en las Legislaturas pasadas, y a pesar de sus fracturas internas, tiene el potencial de constituir alianzas de votación con otros partidos representados en el Congreso. El incentivo con que cuentan los partidos en su quehacer político parlamentario, para permanecer como actores protagónicos en el sistema político ha sido su identidad, en cuanto que ésta hace posible la distinción entre cada partido, quienes luchan por la defensa de su ideología, por reproducir sus estrategias que les han funcionado para ampliar su representatividad social entre el electorado. Esto tiene que ver con el cuerpo de valores y concepciones que sustenta cada uno para plantear soluciones a las problemáticas en el país, es decir, la identidad les otorga un capital político, para diferenciarse entre sí, lo que en última instancia les otorga capacidad para establecer negociaciones y compromisos políticos. Estos elementos Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 9 decididamente son influyentes en la configuración del proceso de construcción democrática. Cuando esta identidad no es firme y sus miembros dejan de apoyarla, entonces la situación crítica en los partidos afecta su orientación, lo que les lleva a modificar su comportamiento estratégico hacia nuevos ángulos, que en el caso de México, aparecen con resistencias para superar los vicios del régimen autoritario. Oportuno es ahora, ocuparse del nuevo rol político de los actores en juego, en donde se advierte una crisis de identidad en los tres partidos políticos: el PRI deja de ser gobierno y desprendido de la tutela presidencial, se convierte en oposición; el PAN, que de oposición se transforma en gobierno, pervive bajo una doble crisis de identidad: 1) falta de coordinación con el Presidente, quien proviene del PAN; y 2) necesidad de reafirmarse como partido en el gobierno. En el caso del PRD que gobierna a más de 20 millones de habitantes en el Distrito Federal, también pasa por una crisis de identidad en cuando carece de unidad para definir su línea ideológico-política, y al igual que el PRI y el PAN, el divisionismo interno está minando su capacidad de influencia política. El PRI aprendiendo a ser oposición Sin el Presidente de la República como jefe máximo del partido y como su eje articulador, el PRI navega en el ámbito político con una ‘nueva identidad’ que se resiste a aceptar: la de ser oposición. Le ha implicado un replanteamiento de todas sus estructuras institucionales, ideológicas y de acción política. Enfrenta el reto de aprender a ser oposición en un régimen que agota los rasgos autoritarios en nombre de la institucionalización democrática. Su comportamiento político en estos marcos, ha derivado en el retorno a sus principios nacional-revolucionarios y a prácticas populistas para reencontrar el apoyo de la ciudadanía y recuperar el poder. El rechazo al neoliberalismo que tanto defendió, es ahora señalado como la razón de su propio declive (Crespo 2000, 54). Sin embargo, frente a la pervivencia de conflictos y disputas internas, carece hoy de una línea homogénea de partido que pone en cuestionamiento su propio funcionamiento institucional. El PRI está dividido. Por un lado, prevalecen intereses dinosáuricos8 reacios a la modernización del partido; por otro lado, hay grupos más críticos con una propuesta de renovación interna,9 más su dilema actual es que tiene que aprender que ya no es gobierno, y concurrir en la competencia electoral sin los recursos (de gobierno, humanos, financieros) que le proporcionaban un amplio margen de ganancia; hoy prevalece su desconcierto por carecer de incentivos al no contar con la figura a la que se guardaba lealtad para ser recompensados; ya no le resulta redituable mantener su tradicional unidad y disciplina, la crudeza del despojo se asimila lentamente, aunque aún no se de su propio reencuentro. La reconquista del poder es el aliciente, pero para lograrlo requiere reconstruir sus filas, renovar sus métodos de cooptación de voto, modificar su plataforma programática. Después del resultado electoral de julio 2000, sus esquemas organizativos han experimentado indefinición política, empezando por la falta de unidad para determinar sus propios liderazgos. Los priístas, no mantienen una alianza formal con sus líderes cupulares. Este signo de fractura identitaria, impide que existan acuerdos para su renovación interna, dificultando la reforma de sus estatutos y la definición 10 \| European Review of Latin American and Caribbean Studies 74, April 2003 de los nuevos métodos de selección del Comité Ejecutivo Nacional. Tal fue el caso en la Asamblea Nacional que celebró en noviembre de 2001, donde se acordó aplazar la elección de su dirigencia nacional a febrero 2002.10 La elección interna se llevó a cabo en esa fecha, pero con abiertas prácticas de manipulación del voto. El líder del PRI nombrado en febrero de 2002, tiene un amplio poder porque no sólo preside el Comité Ejecutivo Nacional sino también el Consejo Político Nacional, es decir, las principales estructuras de decisión. Con este nombramiento quedan aplazadas las expectativas de transformación o refundación en el PRI; y en los cambios que se han producido, no es prioritaria la vida democrática del país, máxime porque se trata de un liderazgo regional con un controvertido desempeño por el uso indiscriminado de recursos públicos y prácticas de impunidad en el quehacer político. Tampoco es signo para alentar una identidad por escenificación que de pauta a la disminución de la heterogeneidad del partido. La tendencia apunta a la reproducción de los mecanismos tradicionales corporativos que prevalecieron en el PRI hegemónico, y su rendimiento de cuentas a la ciudadanía estará en las elecciones para renovar el Congreso Federal en el año 2003. El PAN aprendiendo a ser gobierno El PAN, como agrupación de centro-derecha y de gran arraigo entre los grupos conservadores del país, llegó al poder en coalición con el Partido Verde Ecologista de México (PVEM), postulando para Presidente de la República, a Vicente Fox, un empresario, incorporado a Acción Nacional hace poco más de una década, aunque su militancia fue más de carácter autónomo que de miembro activo en el partido. De ahí su escaso arraigo como miembro panista. El partido fue modificando su corpus doctrinario; de origen se pronunció por el rechazo a las acciones estatistas de los gobiernos revolucionarios, por sus intereses ‘socializantes’, y fue evolucionando hasta concebir las ‘formas individualizadas del liberalismo político’ (Loaeza 1997). Se mantienen intactos los principios doctrinarios básicos del partido, tales como concebir que el fundamento de la sociedad es el individuo y el bien común es la causa que debe defender el Estado. Un humanismo político abierto a todo aquel que coincida con la naturaleza real de la nación y con la dignidad de la persona humana. En cuanto a la democracia, el PAN considera que su impulso debe provenir de la vía electoral y mediante la educación cívica de los individuos (Jiménez 2001, 422). Tal es el perfil político del gobierno de Vicente Fox que sustituye al PRI. Fox representa, por su orientación y alianzas, el proyecto social de la derecha histórica de nuestro país. Desde el inicio de su gobierno, han sido reiterativas las normas morales y expresiones de carácter religioso en los ámbitos del quehacer público, no obstante estar impedidas en el funcionamiento institucional del gobierno. Este es el ámbito en el que el PAN se ‘estrena’ como partido en el gobierno y su reto es aprender a ser gobierno en un régimen que está agotando los rasgos autoritarios que lo sustentaron para dar paso a una institucionalización democrática. El voto ciudadano otorgado a este partido fue porque se apostó por éste para preservar la democracia política, sin embargo, el principal factor que se lo impide es además de su proclividad derechista, su crisis de identidad manifestada en las tensiones entre partido y Presidente, quien, desde su campaña electoral, mantuvo – Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 11 y ahora todavía – un comportamiento distante respecto al PAN. Si bien, a diferencia del PRI o el PRD, el PAN guarda una mayor unidad – resultado de su victoria – pero aún no encuentra la fórmula para coordinarse con el Presidente y ser eficaz.11 Esta crisis de identidad que le impide una coordinación más estrecha con el Ejecutivo es una dimensión que dificulta la coordinación entre poderes y por tanto, su protagonismo como actores del cambio. Un rasgo que evidencia la crisis de identidad panista es el no sentirse como partido en el gobierno,12 pues en el gabinete de Fox están ausentes los panistas en los cargos de primer nivel; está constituido fundamentalmente por hombres de empresa, sin trayectoria panista y con una filosofía democristiana. Esta nueva élite política por su tendencia de involucrar religión y valores morales con sus funciones propias de la administración pública, ha dado pauta para advertir la incompetencia y debilidad del equipo. En este sentido, el PAN, tiene su primer resentimiento respecto al Presidente por ese desdén de no ser incorporado al gobierno. El agravio proviene porque además de ser un mínimo los funcionarios de extracción panista que forman parte del gabinete,13 éstos tienen cargos intermedios, y peor aún, que en la estructura burocrática hay priístas que hoy constituyen parte del aparato gubernamental, cuando es precisamente al PRI al que han derrotado en las urnas. Las prácticas tradicionales para conformar las elites de gobierno van modificándose. En este gobierno se ha seguido el criterio personalista, elegir a individuos con capacidades empresariales, a diferencia de los gabinetes priístas, que estaban constituidos por representantes de ‘grupos políticos’ que iban haciendo carrera en la administración pública, siempre bajo el amparo presidencial. El liderazgo actual del partido, ratificado en la elección interna de marzo 2002, tiene como punto central en su agenda, crear vínculos más constructivos en la relación entre poder Ejecutivo y Legislativo. El PRD, gran perdedor en la Cámara de Diputados El nuevo rol partidario del PRD, consiste en redefinir su línea política ante el traumatismo que ha sufrido por la pérdida de escaños en el Congreso Federal. Sus principios ideológicos que le dan origen, son democracia, justicia, igualdad, paz, soberanía, ética política (Vivero 2001, 449); y su principal demanda ha sido constituir una auténtica división de poderes, dada la larga trayectoria de influencia decisiva del Ejecutivo en el Legislativo. Ante su trayectoria de oponerse a proyectos de otros partidos y no negociar, debido a la exclusión de que fue objeto por el Ejecutivo (1988-1994), su primera posición en el gobierno del Distrito Federal le lleva a redefinir su estrategia política, rebasando el discurso de crítica radical al gobierno para pasar a acciones concretas en tanto que es gobierno. Otro factor que le obliga a cambiar su rol político en el nuevo escenario es su posición en el Congreso Federal. En la Cámara de Diputados, como ya se ha expresado, cuenta con un 10,4 por ciento de escaños; mientras en la Cámara de Senadores controla 15 escaños, representando un 11,7 por ciento; el partido no obstante ser la tercera fuerza política a nivel nacional, y en el recinto parlamentario, se reviste de una posición estratégica que le permite evaluar con qué fuerza política constituir alianzas para definir la orientación de su voto y dictaminar las iniciati- 12 \| European Review of Latin American and Caribbean Studies 74, April 2003 vas,14 novedoso rol que no es materia de este trabajo y que está abierto para ser investigado porque en el contexto de hegemonía priísta, difícilmente podían constituirse alianzas partidarias que eficientaran el trabajo parlamentario. Significa que el PRD está dejando atrás la postura de no cooperación en la que su estrategia fue la resistencia a colaborar con el gobierno, es decir, refrenda la necesidad de un nuevo sentido y credibilidad como partido opositor antes que reincidir en su antigua estrategia ‘de presentarse en términos de ruptura y cambio de régimen’ (Paramio 2001, 37). Un fenómeno distinto en el que cada fuerza política parlamentaria ha tenido que modificar sus patrones de comportamiento con relación a los otros partidos, potenciando sus propias propuestas y su lucha para que éstas se conviertan en ley, aunque fundamental es primero, que se supere la única coincidencia actual entre PRI y PRD, que es su sentido de la derrota. Ambos deben modificar sus estrategias políticas, asumiendo un rol de autocrítica, definiendo sus directrices como partido e innovarse para adaptarse a los nuevos tiempos. Con lo expuesto en este epígrafe, se resalta que los partidos políticos en México enfrentan actualmente cambios en su corpus doctrinario, con miras a mejorar su oferta ideológica para adecuarse a los nuevos tiempos, sin embargo, el dilema está en la fractura interna que agrava su unidad de partido, la cual se acentúa en el nuevo rol que asumen en un gobierno distinto al del PRI, es decir, buscan redefinir su identidad, su conciencia de pertenencia o valoración de ser parte de; la pertenencia a su partido que se da por una agregación de valores; pero que a su vez, experimenta una crisis de identidad en la constatación de su ‘transformación mediante la fusión o fisión de las fuerzas sociales que arman y desatan las alianzas que dominan dicha identidad’ (Fernández 1998). Actores políticos en el trabajo parlamentario El sistema presidencial mexicano es un gobierno dual, toda vez que existe una separación o independencia constitucional de poderes entre el Ejecutivo y el Legislativo.15 En la práctica, el problema en tiempos de hegemonía priísta, se derivó en la debilidad y mal funcionamiento del poder Legislativo frente a un amplio poder discrecional del Ejecutivo en donde las decisiones fueron tomadas por el Presidente sin mayores problemas, ahorrándose tener que buscar consensos con la oposición. Este esquema con rasgos autoritarios generó un poder Legislativo reactivo. Desde que el partido del Presidente no tiene mayoría absoluta en el Parlamento, se acabó el sometimiento del Legislativo respecto al Ejecutivo, sin embargo, el reto que enfrentan ahora estos actores, es crear condiciones de eficacia y funcionalidad para desahogar la agenda pública, pues de diciembre 2001 a marzo 2002, exceptuando la reforma fiscal, únicamente se han aprobado iniciativas ‘inevitables, como el presupuesto, o las que los propios legisladores hicieron suyas, como la de derechos indígenas, que no entrañan mayor controversia ni requieren acuerdos complejos y negociaciones profundas’ (Rubio 2001). Parte medular del desempeño de partidos, legisladores y Presidente de la República, han sido los conflictos suscitados para esos fines, los cuales han derivado en grandes tensiones como la imposibilidad del jefe del Ejecutivo para ver aprobados sus principales proyectos de ley por el Congreso. Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 13 Las relaciones de deterioro entre estas instituciones, se han expresado concretamente en las iniciativas de ley que el Presidente envió al Congreso para su análisis: a) Ley de Derechos y Culturas Indígenas; b) la Nueva Hacienda Pública Distributiva; c) Reforma Energética. Ninguno de sus proyectos que le darían un impacto tanto a nivel nacional como internacional, rindió los frutos esperados y no puede pasarse por alto este acontecimiento que da cuenta a casi año y medio de iniciado el gobierno, de que esta falta de logros puede ser recurrente a lo largo del sexenio. a) La Ley de Derechos y Culturas Indígenas, que había sido prometida por Fox, como uno de sus compromisos medulares de campaña política, guardó su relevancia al ser enviada al Senado como uno de sus primeros actos de gobierno, un proyecto que elaboró la Comisión de Concordia y Pacificación (Cocopa) en 1996 para reformar los artículos constitucionales 4, 18, 57, 73, 115 y 116. Esta iniciativa fue de gran controversia principalmente entre la cúpula del PAN y el Presidente. Representó un esfuerzo presidencial por darle contenido político a la Presidencia que actuaría dentro de los marcos de la legalidad, sin embargo terminó siendo símbolo de la incapacidad de un gobierno para tender y asegurar los puentes políticos necesarios para transformar el modo de hacer política (Delgado 2001). La dirigencia panista en la Cámara Alta, aliándose con los grupos más conservadores del PRI, modificó el proyecto de reforma, excluyendo los puntos de mayor interés para los pueblos indígenas,16 la aprobó el Senado, remitiéndola después a la Cámara de Diputados, que también la aprobó entre PAN, PRI y PVEM con 386 votos, por lo que la respuesta del zapatismo fue suspender negociaciones con el gobierno.17 En si, la iniciativa concluyó en un fracaso total, en el que los legisladores panistas rompieron con la tradicional disciplina del partido en el gobierno respecto a las disposiciones presidenciales, lo cual es positivo, pero no deja de ser una afrenta que los liderazgos panistas pusieron en marcha contra el Presidente, lo que en otros términos no es sino personalismos que muestran claras pugnas políticas entre sí. El PAN después de ver aprobada la Ley Indígena, que en nada favorece a los indígenas, – quizá con presiones – cambia su estrategia política respecto al Presidente, y comienza a manifestarle su respaldo, aunque éste está lejos de ser muestra de entreguismo como en los tiempos del autoritarismo con el PRI, quien sólo fuera el ejecutor de las políticas presidenciales. b) Sobre la Nueva Hacienda Pública Distributiva, se volvió a suscitar gran polémica, por la afectación directa a los ingresos familiares. La postura que todos los partidos asumieron, fue de rechazo a la iniciativa,18 en la que resalta el tema más polémico que es grabar con un impuesto del 15 por ciento, a los medicamentos y a los bienes de primera necesidad. El Ejecutivo, pretendió que fuera discutida en breve sin establecer los mecanismos de consenso entre las distintas fuerzas políticas, lo cual malogró el procedimiento. La respuesta de la Cámara de Diputados al Ejecutivo fue, que se aplazaría este debate para otro período ordinario porque los escasos días que restaban para finalizar el primero, no serían suficientes para dictaminar una ley de tal envergadura para el país.19 Es decir, se careció de un pacto político no entre las fuerzas parlamentarias, sino de éstas con el Ejecutivo. Ocho meses después de recibida en el Parlamento, fue aprobada pero sin gravar el IVA a ali- 14 \| European Review of Latin American and Caribbean Studies 74, April 2003 mentos y medicamentos; en cambio, los efectos resultaron muy controvertidos en otros renglones fiscales. Es decir, nuevamente el Ejecutivo ve frustrados sus proyectos de agenda pública. c) En cuanto a la apertura del sector eléctrico, en la que el Ejecutivo busca modificar las condiciones restrictivas a la inversión privada en áreas reservadas sólo para el Estado, también fue aplazado su dictamen. El tema de fondo en este ambiente de conflicto fue que la Comisión Permanente del Congreso de la Unión acordó interponer un juicio de controversia ante la Suprema Corte de Justicia de la Nación (SCJN) en contra del Presidente por considerar que invadió facultades del Legislativo al expedir un decreto que modifica el Reglamento de la Ley del Servicio Público de Energía Eléctrica.20 Senadores y Diputados de todos los partidos, unánimemente, aprobaron esta disposición, incluyendo al partido gobernante.21 Se trató de un asunto que no estaba en la agenda del día, que lo llevó a tribuna de la Permanente el PRI. La envergadura de este hecho es significativa porque se trata de un decreto que ya estaba en el Diario Oficial (24 de mayo de 2001) y que implicaba apertura del sector eléctrico al capital privado sin necesidad de modificar la constitución, es decir, pasando por alto la función del Poder Legislativo. Las relaciones del poder Ejecutivo con el poder Legislativo se han tornado en descalificaciones mutuas, y el equilibrio de poderes seguirá retrasándose, pues las tensiones han demostrado que los proyectos propuestos no han sido debatidos porque el cabildeo no ha sido una de sus cualidades. En definitiva, el rol individualista del Presidente le ha llevado a desdeñar las actividades del Congreso de la Unión; una actitud poco táctica pues no puede gobernar sin una mayoría parlamentaria, o al menos, requiere establecer acuerdos y sin la confianza de su partido en el Congreso, más difícil puede ser desahogar la agenda pública. Lo anterior, permite argumentar que, no obstante las tensiones respecto al comportamiento entre partidos, fuerzas parlamentarias y gobierno, éstos han asumido un rol razonablemente favorable; es decir, que ‘alimentan positivamente el conflicto político (Massari 1997, 126). Un conflicto que en la práctica, contiene una orientación democrática, aunque las tensiones se dan porque no parecen tener incentivos para cooperar fluidamente con el Ejecutivo. De ahí que la consecuencia esté finalmente en la dificultad de concretizar la consolidación democrática consensual en los términos de que hablaba Russo, como ya se ha expresado, es decir, con la cooperación como premisa más económica para dirimir las diferencias, esto es, el consenso en las grandes decisiones, que ha de provenir tanto del gobierno como de la oposición. Por todo ello urgente resulta, aprender a gobernar, negociando y generando compromisos; pero también aprender a ser oposición, con proyectos propositivos. Tal es la dinámica que tienen que explotar nuestros actores relevantes para consolidar la democrática en el país. Lo anterior da cuenta de que la Presidencia carece de un proyecto de gobierno consensuado por partidos políticos y actores relevantes, lo cual repercute en la imagen que a nivel nacional guarda el Presidente. Es un hecho que la tendencia ha sido una limitada eficacia que ha puesto en entredicho no sólo la imagen presidencial sino de los partidos y del Congreso. Los resultados de las encuestas así lo reflejan, como puede verse en el siguiente epígrafe. Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 15 La opinión ciudadana Una condición para el buen funcionamiento de la democracia, es la confianza en las instituciones; y puede ser medida a través de las percepciones de la ciudadanía respecto al Presidente, y la relación existente entre el poder Ejecutivo con el Legislativo. Encuestas aplicadas por el Grupo Reforma desde que inició su gestión Vicente Fox,22 muestran resultados interesantes en este ángulo. No obstante que se trata de estudios trimestrales, para fines operativos, se compara aquí el intervalo entre febrero 2001 y marzo 2002, período en el que se constata un descenso en casi todos los aspectos analizados. Al evaluar la popularidad del Presidente, se preguntó a los ciudadanos, ¿cómo calificaría la actuación de Vicente Fox, del 0 al 10?; la respuesta en febrero fue de 7,5, mientras que en marzo fue de 5,8; y si tomamos como referencia que el anterior Presidente, Ernesto Zedillo (1994-2000), nunca alcanzó ese 7,5 nivel de popularidad, podría sugerirse que Fox lo ha alcanzado por su carisma, o porque se trata de un liderazgo distinto al PRI, lo que alentó muchas expectativas; optimismo que parece haber sido de corta duración al descender la percepción ciudadana en la última encuesta hasta casi dos puntos porcentuales. Más aún, al asociar estos resultados con otras variables como el que un 53 por ciento de los que votaron por él en julio 2000, declararon en mayo 2001 que no lo hubieran hecho de haber conocido su proyecto de reforma fiscal, da cuenta de que existe una desmotivación social como amenaza para la consolidación democrática en el país. Este indicador es muy crudo porque significa que es una cifra muy alta de ciudadanos que estuvieron muy arrepentidos en ese momento, de haber elegido a Vicente Fox. La desaprobación de la reforma fiscal que implica necesariamente, aumento de impuestos a la ciudadanía, en marzo 2002 fue de un 62 por ciento. Las relaciones entre el poder Ejecutivo y el Legislativo se advierten desgastadas. El 51 por ciento de entrevistados calificó como buenas o muy buenas estas relaciones en febrero 2001, pero no en marzo 2002, donde descendió a 32 por ciento, es decir, en tan sólo un año, descienden 19 puntos porcentuales; y consecuentemente, del 9 por ciento que calificó como malas o muy malas esas relaciones en febrero, la cifra se eleva a un 29 por ciento que tienen esa opinión en marzo 2002. Esta evaluación obedece probablemente, a que fueron meses de tensión por el tratamiento que dieron estas instituciones, al tema de la Ley de Autonomía y Culturas Indígenas (caso de Chiapas), y en especial, a la afectación que vieron los ciudadanos a sus ingresos, si respaldaban la iniciativa de ley presidencial sobre la grabación de impuestos a los medicamentos y alimentos de primera necesidad. Por lo que se refiere a la evaluación de la ciudadanía a los tres principales partidos políticos, la tendencia también corrobora la crisis identitaria institucional. Se advierte una marcada desafección en términos de Paramio (1994, 17), es decir de desconfianza respecto a su acción política. El PAN como partido en el gobierno fue quien registró un decrecimiento de credibilidad ciudadana. La opinión favorable en febrero 2001 fue de 68 por ciento, 35 puntos porcentuales más que en marzo 2002 que fue de 33 por ciento, lo cual indica que ese partido en casi año y medio de gobierno no logró mantener una clientela estable desde que asumió el poder; el PRD pierdió once puntos porcentuales al pasar en febrero 2001 de 31 por ciento a marzo 2002 con 20 por ciento. De los tres partidos, el PRI fue quien menos credibilidad 16 \| European Review of Latin American and Caribbean Studies 74, April 2003 perdió en ese período, que registró un descenso de 31 a 24 por ciento, lo cual indica que en ese lapso apenas descendió 7 puntos porcentuales. Este panorama refleja por sí mismo que el PRI sigue siendo un fuerte competidor en el escenario político y que el reto de las tres fuerzas políticas para ampliar su representación ciudadana está en superar tan escasa identificación social. Consideraciones finales En México, se ha avanzado en la democracia porque existen condiciones claras como elecciones continuas, legítimas, y confiables; un sistema de partidos plural y un ambiente propicio que dio lugar a la alternancia en el poder tanto a nivel estatal como federal. Con el ascenso al poder de un partido diferente al PRI, y un parlamento sin mayoría absoluta, se despertaron expectativas desde el punto de vista cualitativo para que partidos, parlamento e institución presidencial asumieran la tarea inminente de transformar las estructuras adversas al proceso democrático, sin embargo, no obstante el corto tiempo de gobierno opositor, las primeras impresiones no han sido del todo alentadoras porque las acciones políticas han apuntado más a la pretericidad que invoca Aboy, cuando refiere que los actores relevantes corren el riesgo de reproducir las instituciones políticas sin cambios sustanciales. Si el cambio de gobierno no ha dado el paso paralelo de alentar la consolidación democrática, porque ésta depende de ‘profundizar las reformas políticas que garanticen su ejercicio [...] y del éxito que se tenga en superar o limitar la acción de los “enclaves autoritarios”, podría entonces identificarse como un obstáculo, los grupos que se resisten al cambio desde el poder o desde otros sectores; además de la permanencia de formas clientelares u otros hábitos que alientan comportamientos no democráticos en diversos grupos sociales’(Garretón 2000). En gran medida radican ahí los factores que impiden la concurrencia de los actores políticos para constituir un régimen liberado de mecanismos corporativos y controles caciquiles que han retardado por mucho tiempo el proceso democrático en el país. Este primer gobierno de oposición en México, se pronuncia por la consolidación democrática, pero sus acciones dificultan una pronta renovación institucional que apunte a dicho proceso. Se hace necesaria una definición de las relaciones entre actores políticos, asumir nuevos roles teniendo para sí, el reto de interactuar con un proyecto político de nación claramente definido, en el que todos los actores políticos concurran para ese fin, que se ve imposibilitado su logro ante la crisis interna que los componentes políticos experimentan, limitando las condiciones de cambio político. La presidencia depende a su vez de la capacidad de conformar mayorías legislativas. El actual Congreso de la Unión no ha optimizado sus recursos para construir coaliciones sólidas de entendimiento.23 Tanto el PRD como el PRI, han reconocido que en la LVIII Legislatura que inició en 2000 y concluirá su ejercicio legislativo en 2003, no se han logrado consolidar reformas de gran alcance en las que se de un debate de fondo, y sólo en cambio se han aprobado las reformas poco significativas. El partido en el gobierno (PAN) asume la misma posición, aunque dice avanzar poco a poco para actuar como Congreso de transición. Los conflictos entre los poderes Ejecutivo y Legislativo se han interpretado a nivel discursivo como un nuevo ‘equilibrio de poderes’, pero ha sido evidente una Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 17 falta de coordinación entre ambos, lo cual ha generado una relación de conflicto entre el Ejecutivo y el PAN, quien – dicho sea de paso – ha de resolver el dilema de si es partido en el gobierno o no lo es. Esta falta de articulación política se ha expresado en el trabajo legislativo con la iniciativa presidencial de reforma fiscal, la cual se pretendía fuera aprobada a escasos días de finalizado el período ordinario de sesiones. El tiempo limitado así como el desaire con el que se trató al Legislativo al pretender ratificar la iniciativa sin disponer de suficiente tiempo para analizarla, antepuso la erosión política entre instituciones, debilitando la gobernabilidad democrática. Por tanto, la agenda del gobierno se desahoga tardíamente a medias o no se desahoga. La gestión presidencial es un acierto que ha de provenir con una amplia legitimidad por el apoyo popular recibido en las elecciones y al comienzo de su gobierno, pero el problema que afronta es cómo mantener el poder, con un estilo de gobierno individualista en el que hay resistencias para arreglos institucionales; más aún que el PAN persiste sin una identidad definida con el Presidente y el PRD con dificultades para reagrupar su unidad y línea política; o el PRI que se torna defensor de las prácticas de antaño. Ante tal escenario, sugerente es preguntarse ¿cómo pueden instrumentarse mecanismos de largo plazo para consolidar la democracia en un régimen que no acaba de desterrar las viejas prácticas autoritarias? El Presidente Fox contó con una amplia legitimidad entre la ciudadanía al inicio de su gobierno, pero ese alto grado de aprobación se fue limitando ante las propuestas impopulares del gobierno, enfrentamientos entre poderes e ineficacia para concretizar los proyectos presidenciales, elementos que concurren en la limitada solución a los problemas que afectan a la población. En definitiva, México ya cuenta con instituciones democráticas, pero falta demostrar la eficacia de su desempeño, que se concretizará en la calidad de vida de la sociedad. Tal será la garantía de que el régimen actual avance en el orden democrático. * Margarita Jiménez Badillo es Maestra en Estudios Latinoamericanos por el Instituto Interuniversitario de Estudios de Iberoamérica y Portugal; candidata a Doctora en Ciencia Política y de la Administración por la Universidad de Salamanca en el Programa de Procesos Políticos Contemporáneos, orientando su línea de investigación a temas sobre el presidencialismo, relaciones entre poderes Ejecutivo y Legislativo y oposición parlamentaria en América Latina; recibió una beca de la Agencia Española de Cooperación Internacional para sus estudios de Doctorado. Este trabajo ha sido realizado en el marco del proyecto de investigación: ‘El Parlamento en América Latina. Rendimiento del Poder Legislativo: una clave interpretativa de los problemas de Gobernabilidad de la región’; dirigido por Manuel Alcántara Sáez en el Instituto Interuniversitario de Estudios de Iberoamérica y Portugal de la Universidad de Salamanca; cofinanciado con Fondos FEDER, Plan Nacional de I+D, Cortes de Castilla y León y Caja España. Ref. 1FD97-0906. <mirna@usal.es> 18 \| European Review of Latin American and Caribbean Studies 74, April 2003 Notas 1. Ante la crisis del sistema de partidos, manifestada en 1976 cuando no hay sino un único candidato presidencial que concurre a las urnas por el PRI, se evidencia la necesidad de crear normativas de competencia y representatividad para que concurra la oposición. Un signo de apertura del régimen, en el que se crean más tarde, mecanismos que le legitiman, tales como las reformas políticas de 1977, 1986, 1990-91, 1993, 1994 y 1996 que contribuyeron a democratizar al sistema político, con nuevas fórmulas de inclusión de los partidos políticos al Congreso, condiciones más equilibradas para participar en las campañas electorales, creación de un sistema electoral autónomo sin el control del gobierno y tener acceso a los medios de comunicación y financiamiento electoral. Así disminuyó el presidencialismo fuerte y correlativamente propició el desgaste del PRI que culminaría con su derrota en 2000. 2. Partido Revolucionario Institucional (PRI), creado en 1929 mediante un pacto fundacional de elites con el cual se institucionaliza el poder político, se mantiene en el poder hasta el año 2000, ha sido autodefinido como partido del centro al pretender aglutinar los distintos intereses sociales y políticos; el Partido Acción Nacional (PAN) surgido en 1939 para enfrentar el autoritarismo del partido en el gobierno, identificado como partido de derecha, representa fundamentalmente, los intereses conservadores de sectores eclesiásticos y empresariales y de clases medias; el Partido de la Revolución Democrática (PRD), originado de la fractura del PRI y constituido primero como Frente Democrático Nacional en donde se le unieron otros partidos y después identificado ya como PRD en 1989, representando los intereses de la izquierda. 3. Se define aquí al partido hegemónico en términos de Sartori, quien considera que es aquel que no permite la competencia por el poder, ni una competencia de facto; pueden existir otros partidos pero como partidos de segunda al no permitírseles competir con el partido hegemónico en términos antagónicos y en pie de igualdad. No sólo no se produce de hecho la alternación; no puede ocurrir, dado que ni siquiera se contempla la posibilidad de una rotación en el poder (1980, 278). 4. Un indicador que muestra esta debacle es su porcentaje de votación alcanzado en las últimas elecciones presidenciales: en 1976 obtuvo 92,3 por ciento; en 1982 el 68,4; en 1988 el 50,7; en 1994 el 50,2 y en 2000 el 42,5. Meyer, Lorenzo, en Reforma, 25 de mayo de 2001; y (Aguayo 2000). 5. Ernesto Zedillo en una entrevista, señaló que el excesivo poder fáctico del Presidente en México era también, ausencia de responsabilidad de los demás poderes y de otros interlocutores. Por ello, haciendo gala de la plena legitimidad popular con la que asumió la Presidencia, decidió demarcarse del ejercicio tradicional del poder y favorecer las condiciones para contar con un sistema de pesos y contrapesos entre poderes. De ahí la redefinición del Presidente con su partido, y de ahí también la participación del PRI en la construcción de nuevas reglas explícitas en las reformas electorales (Achard y Flores 1997, 147-152). 6. El primer día del año, – último del gobierno de Carlos Salinas – entraba en vigor el Tratado de Libre Comercio con América del Norte; mismo día en que surge públicamente el Ejercito Zapatista de Liberación Nacional, demandando el reconocimiento de los derechos de los pueblos indios. Otros factores relevantes fueron las fracturas internas del PRI, que en su lucha por el poder se hicieron manifiestas con los asesinatos del candidato priísta a la Presidencia y del que sería líder de la Cámara de Diputados. La crisis económica de 1994 es otro de los puntos endebles, heredados de la administración salinista y que son en los que cimenta su poder político. 7. Una legislatura reactiva es cuando el trabajo legislativo está dominado por el Ejecutivo, así como por las elites partidistas sobre mayorías sólidas y disciplinadas; mientras que una Legislatura activa, se refleja cuando el trabajo legislativo es más relevante en la proposición de iniciativas, examen, discusión y revisión de propuestas del Ejecutivo. (Mezey 1979). 8. De la vieja guardia tradicional, aquellos que se ampararon en los valores revolucionarios para expandir sus cotos de poder regionalista mediante mecanismos corporativos, clientelares y populistas que le permitieran mantener no sólo el control del proceso electoral sino de la vida política regional. 9. Desde la LVII Legislatura (1997-2000) diversas facciones, constituidas en buena parte por parlamentarios, que se han manifestado abiertamente en contra de la fracción más conservadora y renuente al cambio. Grupos autodenominados: Reflexión, Crítico, Galileo, todos ellos, expresión del no alineamiento a la tradición partidista. 10. Un mecanismo, resultado de la renovación del PRI en su Asamblea General en noviembre de 2001, Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 19 fue establecer que el Presidente de la República no puede ser ya su ‘jefe máximo’, es decir, el presidente del partido será electo mediante consulta directa a militantes del partido, y un requisito es que tenga cuando menos diez años de militancia y haya ocupado cuando menos un cargo de elección popular. 11. Meyer, Lorenzo, Reforma, 23 de agosto de 2001. 12. Las tensiones entre gobierno y PAN han llegado a tal grado que, la directiva panista representada por Luis Felipe Bravo, en ocasión de la inauguración del Primer Encuentro Nacional de Legisladores, tuvo que reafirmar que ‘el PAN es el partido en el gobierno y los primeros que deben entenderlo son los funcionarios y los legisladores de Acción Nacional [...] En la medida que se le niegue a Acción Nacional el carácter de partido en el gobierno, se corre el riesgo de equivocarnos y de que en el PAN se tienda a reproducir de manera inevitable los criterios, las acciones y los discursos que nos perfilaron como partido opositor durante 60 años’. Jornada, 24 de agosto de 2001. 13. A excepción del Secretario de Gobernación, que es de extracción panista y que cubre una cartera de primer nivel. 14. En declaración del coordinador de la bancada perredista de la Cámara de Diputados, Martí Batres, el PRD puede encontrar coincidencias con el PRI en materia de política exterior, algunos temas de la reforma del Estado como la redistribución del poder entre los diversos órganos y entre el centro y las regiones; en temas como la equidad de géneros. Con el PAN, las coincidencias se ubican en la transparencia de la administración pública, la limpieza electoral, la reforma democrática del Estado. Jornada, 19 de noviembre de 2000. 15. Linz (1997, 33-34) le identifica como legitimidad dual en tanto ambos poderes disfrutan de legitimidad democrática y no hay principio que marque o defina quién de los dos poderes representa la voluntad del pueblo. 16. Los acuerdos de San Andrés Larráinzar que desde 1996 se lograron entre gobierno y grupos indígenas fueron principalmente: libre determinación y la autonomía de los pueblos indígenas; desarrollar una política de verdadera sustentabilidad, que preserve las tierras, los territorios y los recursos naturales de los pueblos indígenas; proteger los derechos de los migrantes, indígenas y no indígenas, dentro y fuera de las fronteras nacionales; reconocer a los pueblos indígenas en la Constitución general; ampliar su participación y representación políticas; decidir su forma de gobierno interna y sus maneras de organizarse política, social, económica y culturalmente; participar en los órganos de representación nacional y estatal; promover los derechos y cultura de la mujer indígena. 17. El Congreso Nacional Indígena y el Ejercito Zapatista declararon que la ley aprobada en el Congreso no correspondía a la que fue aprobada por el gobierno mexicano en los Acuerdos de San Andrés y que es la que ellos firmaron con el gobierno, quien se retractó de ese acto y por ello, pasados cinco años, vuelve a estar en el centro del conflicto indígena. En consecuencia, el EZLN, decidió suspender todo contacto con el gobierno y regresar a la ‘resistencia y rebeldía’. 18. Los argumentos del PRD al negarse a aprobar el proyecto presidencial de reforma fiscal fueron que el gobierno de Fox buscó reducir más la inversión y el gasto social, transmitiendo el costo financiero del Estado a los sectores mayoritarios de la población, traducido en el aumento del IVA al consumo y protegiendo de gravámenes al capital. 19. El Senador del PAN, Carlos Medina declaró que ‘los funcionarios deben entender que no por ser legisladores panistas vamos a defender iniciativas o propuestas sólo porque vengan del Ejecutivo. Se equivocan. Desde que envió la iniciativa de reforma hacendaria [el Secretario de Hacienda], los senadores del PAN le pedimos que nos explicara sus alcances y su contenido. Seis meses después no lo había hecho. Si en el PAN no conocemos mínimamente si se trata de una reforma o de una miscelánea fiscal, y de sus alcances, esa iniciativa no pasa’. Jornada, 24 de agosto de 2001. 20. El decreto modifica los artículos 126 y 135 de la ley del servicio público de energía eléctrica para permitir que los particulares que ahora tienen capacidad de generación, en caso de tener excedentes, los pongan a disposición de la Comisión Federal de Electricidad, siempre y cuando tengan una capacidad instalada total hasta de 40 megavatios. Asimismo, por esa resolución, quienes tengan una capacidad instalada superior a 40 megavatios podrán vender hasta 50 por ciento de su capacidad total. Se precisa que ese porcentaje podrá ser modificado por la Secretaría de Energía, conforme a las necesidades que requiera la prestación del servicio público y el nivel de reserva de sistema eléctrico nacional. Jornada 28 de junio, 2001, México. 21. ‘En tribuna, el líder parlamentario del Senado, aclaró que fue “particularmente difícil” avalar la demanda contra el Presidente, pero que lo hacían porque son nuevos tiempos en los que hay realmente división de poderes’. La votación de los 35 miembros entre diputados y senadores en la Co- 20 \| European Review of Latin American and Caribbean Studies 74, April 2003 misión Permanente fue unánime aprobando la decisión, con ningún voto en contra. Se trata de la primera vez en su historia del Congreso de la Unión que se presenta una controversia constitucional en contra del Presidente de la República. Los coordinadores parlamentarios de PRI, PAN y PRD, argumentaron que la controversia ‘tiene como fin último preservar las facultades constitucionales del H. Congreso de la Unión’. No hay propósito alguno de limitar el ejercicio de las facultades presidenciales. 22. El Grupo Reforma, es un organismo de investigación y difusión periodística, que aplica encuestas trimestrales para conocer el pulso ciudadano respecto al desempeño de las instituciones políticas en el país. Su metodología en el último trimestre al 18 de febrero de 2002, fue un muestreo aleatorio sistemático con base en las secciones electorales del país previamente estratificadas por el criterio urbano, mixto y rural, y aplicadas a 1,200 mexicanos adultos en las 32 entidades del país. Seleccionaron 100 puntos de levantamiento en vivienda, que corresponden a 61 ciudades de más de 15 mil habitantes; margen de error +/- 2,5 por ciento nivel de confianza, 95 por ciento. Resultados de las encuestas publicados en Diario Reforma, 28 de febrero de 2002. 23. La diputada del PRI, Lorena Beauregard, sostiene que ‘no hemos asumido la posición de ser un Congreso de transición, y no sólo se debe hablar de democratizar la vida del país sino de consensuar en la Cámara de Diputados. Ese problema de falta de consenso se vive también en los demás grupos parlamentarios, lo que contribuye a que las Cámaras no asuman su papel histórico’. Proceso nº 1287, 1 de julio de 2001, México. Bibliografía Aboy, Carlés Gerardo (2001) Las dos fronteras de la democracia Argentina. La reformulación de las identidades políticas de Alfonsín a Menem. Ediciones Homo Sapiens, Colecc. Politeia, Argentina, pp. 333. Achard, Diego y Flores Manuel (1997) ‘Ernesto Zedillo: transición política con estabilidad’. Gobernabilidad: un reportaje de América Latina, Programa de las Naciones Unidas para el Desarrollo (PNUD) y Fondo de Cultura Económica, México, pp. 147-159. Aguayo, Sergio (2000) ‘Elecciones del 2000’. El Almanaque mexicano, Grijalbo, México, pp. 248-263. Alcántara, Manuel (1999) ‘Sistemas políticos de América Latina’. Vol. 1, América del Sur, Madrid, Tecnos, pp. 532. Banamex-Accival (2001) Panorama Social, ‘Se consolida el tripartidismo’, Informe mensual, diciembre de 2001, pp. 538-541. Cervantes, Jesusa y Alejandro Gutiérrez (2001) ‘Resiente la falta de un acuerdo político nacional. El Congreso, muy lejos de ser de transición’. Proceso nº 1287, 1 de julio, México. Crespo, José Antonio (2001) ‘El nuevo Congreso frente a Fox’. Revista Iniciativa nº 9-10, octubre 2000-marzo 20001, Instituto de Estudios Legislativos, Legislatura del Estado de México, pp. 53-56. Dahl, Robert A. (1989) La poliarquía. Participación y oposición. Tecnos, Madrid, pp. 228. Delgado, René (2001) ‘La indefinición’. Diario Reforma, 5 de mayo, México. Fernández U., Marco A. (1998) ‘Crisis de identidad. La derecha en los sesenta: La fundación del Partido Nacional’. Revista de Historia, año 8, Vol. 8, Universidad de Concepción, Chile. Garretón, M.A. (1997) ‘Revisando las transiciones en América Latina’. Nueva Sociedad nº 148, marzoabril, Caracas, pp. 20-29. ––– (2000) ‘El problema será la coalición de gobierno’. Suplemento Enfoque nº 314, Diario Reforma, 6 de febrero, México. Jiménez, Margarita (2001) ‘Partido Acción Nacional’. En: Alcántara, Manuel y Freidenberg, Flavia (Eds) Centroamérica, México y República Dominicana: partidos políticos en América Latina, Tomo nº19, Universidad de Salamanca, pp. 419-447. Linz, Juan (1997) ‘Democracia presidencial o parlamentaria: qué diferencia implica?’. En: Linz, Juan y Valenzuela, Arturo (Comps) La crisis del presidencialismo. Perspectivas comparativas, Tomo I, Alianza Universidad, Madrid, pp. 25-143. Lipset, Seymour Martin (2000) ‘Reinventar el ejercicio del poder’. Suplemento Enfoque nº 359, Diario Reforma, 17 de diciembre, México, pp. 4-7. Loaeza, Soledad (1996) Oposición y Democracia. Cuadernos de divulgación de la Cultura Democrática nº 11, Instituto Federal Electoral, México, pp. 77. –––(1997) ‘Gobierno y oposición en México. El Partido Acción Nacional’. Foro Internacional nº 1, Revista Europea de Estudios Latinoamericanos y del Caribe 74, abril de 2003 \| 21 enero-marzo, El Colegio de México, México, pp. 97-114. ––– (2001) ‘Dos oposiciones en busca de línea’. La Jornada, 19 de julio, México. Lujambio, Alonso (1996) ‘Federalismo y Congreso en el cambio político de México’, Instituto de Investigaciones Jurídicas, Serie E: Varios, nº 67. UNAM, México, pp. 236. Mainwaring, Scott y Timothy Scully (1997) ‘La institucionalización de los sistemas de partido en la América Latina’. América Latina Hoy nº 16, agosto, Salamanca, pp. 91-108. Mainwaring, Scott y Mathew Soberg Shugart (1998) ‘Presidencialismo y sistema de partidos en América Latina’. Post-Data nº 3 y 4, agosto, pp. 133-181. Mallo, Susana (2000) ‘El orden de lo diverso: las nuevas relaciones del gobierno y la oposición en Argentina y Uruguay’. Ponencia presentada en el Congreso de LASA, 2000, Miami, pp. 20. Massari, Oreste (1997) ‘Naturaleza y rol de las oposiciones político-parlamentarias’. En: Pasquino, Gianfranco, La oposición en las democracias contemporáneas. Eudeba, Argentina, pp. 71-127. Meyer, Lorenzo (1993) ‘El presidencialismo. Del populismo al neoliberalismo’. Revista Mexicana de Sociología nº 2, abril-junio, Instituto de Investigaciones Sociales/UNAM, México, pp. 57-81. ––– (2000) Editorial. Diario Reforma, 25 de mayo de 2001, México. Mezey, Michael (1979) Comparative Legislatives. Durham, NC, Duke University Press. Novaro, Marcos (1998) ‘El gobierno y la competencia entre los partidos argentinos en los ’90: una perspectiva comparada’, pp. 29. http://lasa.international.pitt.edu/LASA98/Novaro.pdf O’Donell, Guillermo (1996) ‘Otra institucionalización’. Agora nº 5, invierno, Argentina, pp. 5-28. Palermo, Vicente, y Marcos Novaro (1996) Política y poder en el gobierno de Menem. Grupo Editorial Norma, Facultad Latinoamericana de Ciencias Sociales (FLACSO), Buenos Aires, pp. 557. Paramio, Ludolfo (1994) ‘Consolidación democrática, desafección política y neoliberalismo’. Revista Internacional de Sociología, nº 7, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, pp. 11-30. ––– (2001) ‘Sin confianza no hay democracia: electores e identidades políticas’. En: Labastida, Julio; Del Campo, Martín y Camou, Antonio, Globalización, identidad y democracia. México y América Latina, UNAM, Siglo XXI, México, pp. 25-42. Pérez, H. Pedro (2001) ‘El sistema de partidos de México tras las elecciones del 2 de julio de 2000’. Revista Quórum nº 2, primavera, Universidad de Alcalá, España. Rubio, Luis (2001) ‘El desempate gubernamental’. Diario Reforma, 20 de mayo, México. Russo, Juan (1995) ‘Consolidación democrática y oposición en la Argentina. Una perspectiva comparada’. Agora, nº 3, invierno, Buenos Aires, pp. 115-140. Sartori, Giovanni (1980) Partidos y sistemas de partidos. Alianza, Madrid, pp. 450. S/A (2000) ‘El Congreso en México. Un retrato del cambio’. Este País nº 114, septiembre, México, pp. 54-59. Tuirán, Alejandro (2000) ‘Del monopartidismo a un país multicolor’. Suplemento Enfoque nº 359, Diario Reforma, 17 de diciembre, México, pp. 8-13. Venegas, Juan Manuel (2001) ‘A un año del cambio’. Jornada, 2 de julio, México. Vera, Rodrigo (2001) ‘Son seis grupos que ‘no generan políticas públicas’. El “gabinetazo” de Fox, su espada de Damocles’. Revista Proceso nº 1287, 1 de julio, México. Vivero, Arnulfo Igor (2001) ‘Partido de la Revolución Democrática’. En: Alcántara, Manuel y Freidenberg, Flavia (Eds) Centroamérica, México y República Dominicana: partidos políticos en América Latina, Tomo III, Universidad de Salamanca, pp. 447-477.
https://openalex.org/W2911342065	https://europepmc.org/articles/pmc6352939?pdf=render	English	null	Chemical composition and enzyme inhibition of <i>Phytolacca dioica</i> L. seeds extracts	Journal of enzyme inhibition and medicinal chemistry	2,019	cc-by	8,078	Introduction information is available on the chemical composition. Therefore, our aim was to investigate the potential bioactivity of P. dioica seeds extracts and relate them with the chemical profile. In gen- eral, aiming at the isolation of potential bioactive substances. The antioxidant capacity and the inhibitory activity on tyrosinase and xanthine oxidase were evaluated to understand the potential applications of these seeds in medicine. In the last years, the evaluation of medicinal plants and the isola- tion of their bioactive compounds have substantially increased due to their potential use as drugs in modern medicine. Different pharmacologically active compounds from medicinal plants, which can improve health, may act individually, additively or in synergy1,2. pp Indeed, enzyme inhibition is a promising strategy for drug development in several diseases15–17. Tyrosinase (E.C. 1.14.18.1) is an enzyme involved in melanogenesis in the skin, leading to the formation of melanin molecules, which act as a protective barrier against UV radiation from the sunlight. The skin is an organ quite vulnerable to oxidative stress and its continuous exposure to dir- ect UV can cause hyperpigmentation and pre-matured aging. Tyrosinase inhibitors are clinically useful for the treatment of some dermatological disorders associated with melanin hyperpig- mentation. It has been reported that melanogenesis produces hydrogen peroxide and other reactive oxygen species (ROS) that expose the human melanocytes to high levels of oxidative stress18. Thus, inhibitors of melanogenesis could also act as a nat- ural antioxidant in skin care products. The plant kingdom offers a considerable amount of plants with potential as drug candidates. Species of the genus Phytolacca, family Phytolaccaceae, are known for their use in popular medi- cine. Several ailments have been treated using these plants and different pharmacological actions have been reported, including antimicrobial, anti-inflammatory and anticancer3–6. Ethnopharmacological information also revealed that Phytolacca dioica is used to heal skin wounds7. Phytochemical screening of the extracts of P. dioica leaves and berries revealed the presence of alkaloids, tannins, saponins, phe- nols, lectins, and flavonoids, while triterpenoid aglycones and phlobatanins were absent8. The leaves and berries of P. dioica are rich sources of triterpene saponins, which have been described as displaying important biological actions such as molluscicidal, anti- inflammatory, antifungal, and antibacterial activities9–11. ABSTRACT ARTICLE HISTORY Received 19 October 2018 Revised 16 November 2018 Accepted 17 December 2018 Phytolacca, which belongs to the family of Phytolaccaceae, are known for their use in popular medicine. Bioactivity of five extracts from Phytolacca dioica seeds were evaluated in four bioassays. A selected group of compounds from the extract that displayed the best bioactivity was analysed. The ethyl acetate extract (EAE) possessed the highest content of phenolics, the highest inhibitory activity on the tyrosinase and xanthine oxidase enzymes and showed a high antioxidant activity. HPLC-DAD-MS was employed to iden- tify the phenolics profile of the most active one (EAE). HSCCC analysis of the EAE led to the isolation of phytolaccoside B and a mixture of 4 isomers, isoamericanol B1, B2, C1 and C2. These isoamericanol iso- mers presented activity against tyrosinase and xanthine oxidase. Our results revealed for the first time an interesting biological activity of the extract and isolated compounds from P. dioica seeds, which could be considered as a source of bioactive molecules. KEYWORDS Phytolacca dioica; tyrosinase; xanthine oxidase; enzyme inhibition; fatty acids CONTACT Antonella Fais fais@unica.it Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chemical composition and enzyme inhibition of Phytolacca dioica L. seeds extracts Amalia Di Petrilloa, Ana Marıa Gonzalez-Paramasb, Antonella Rosac, Valeria Ruggierod, Fabio Boylane, Amit Kumarf, Francesca Pintusa, Celestino Santos-Buelgab, Antonella Faisa and Benedetta Eraa aDepartment of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy; bPolyphenols Research Group. Unit of Nutrition and Bromatology, Faculty of Pharmacy, University of Salamanca, Salamanca, Spain; cDepartment of Biomedical Sciences, University of Cagliari, Monserrato, Italy; dDepartment of Medical Sciences and Public Health, University of Cagliari, Monserrato, Italy; eSchool of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; fDepartment of Mechanical, Chemical and Materials Engineering, University of Cagliari, Cagliari, Italy JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 2019, VOL. 34, NO. 1, 519–527 GC analysis Fatty acid methyl esters were analysed on a gas chromatograph Hewlett-Packard HP-6890 (Hewlett-Packard, Palo Alto, USA) with a flame ionisation detector (FID) and equipped with a cyanopropyl methyl-polysiloxane HP-23 FAME column (30 m 0.32 mm 0.25 lm) (Hewlett-Packard). Nitrogen was used as carrier gas at a flow rate of 2 ml/min. The oven temperature was set at 175 C, the injector temperature at 250 C, and the detector temperature at 300 C. The fatty acid methyl esters were identified by compari- son of the retention times to those of standard compounds. The composition of individual fatty acid was calculated as a percent- age of the total amount of fatty acids (g %), using the Hewlett- Packard A.05.02 software. Plant material The fruits of Phytolacca dioica L. were collected in Cagliari, Italy (coordinates were 39.224195 N, 9.105899 E). The plant was identi- fied by Dr. Cecilia Loi, Department of Life and Environmental Sciences, Section of Botany, University of Cagliari, Italy. A voucher specimen (1233/A Herbarium CAG) has been deposited in the Life and Environmental Sciences Department. Introduction Moreover, Phytolacca species also constitute one of the best sources of ribo- some-inactivating proteins (RIPs) that have been used both in the therapy against virus and tumours and in the construction of transgenic plants endowed with resistance to virus, bacteria, fungi, and insects12. An important biological source of oxygen-derived free radicals is xanthine oxidase (E.C. 1.2.3.2) (XO) that contributes to oxidative damage of living tissues that are involved in many pathological processes19,20. XO catalyses the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant production of hydrogen peroxide and superoxide anion. The generation of excess uric acid is harmful to the human body and may lead to gout, hyperuricaemia, and other symptoms of related diseases. However, limited research papers on medicinal and biological properties of P. dioica seeds have been published13,14, and scarce A. DI PETRILLO ET AL. 520 n-hexane and H2O, samples were centrifuged at 900 g. The hexane phase with fatty acid methyl esters was collected, the solvent was evaporated, the residue was dissolved in n-hexane and aliquots of the samples were injected into the GC system. All solvent evapor- ation was performed under vacuum. Several studies of plant extracts and synthetic compounds have been evaluated for their inhibitory and antioxidant activities in treatment of gout21,22. Therefore, inhibitors of XO may be poten- tially useful for the treatment of gout or other XO- induced diseases. Several studies of plant extracts and synthetic compounds have been evaluated for their inhibitory and antioxidant activities in treatment of gout21,22. Therefore, inhibitors of XO may be poten- tially useful for the treatment of gout or other XO- induced diseases. Therefore, the goal of our study was to report the chemical composition of the P. dioica seed extract to find bioactive mole- cules, which could be useful as potential drug candidates from natural origin. Chemical and reagents All chemicals were obtained as pure commercial products and used without further purification. Standards of fatty acids and fatty acid methyl esters, Desferal (deferoxamine mesylate salt), Trolox, Folin-Ciocalteau’s phenol reagent, 2,2’-azino-bis(3-ethylben- zothiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), kojic acid, allopurinol, XO from cow’s milk, xanthine, and all solvents used, of the highest available purity, were from Sigma-Aldrich (Milan, Italy). The methanolic HCl (3 N) was pur- chased from Supelco (Bellefonte, PA). Recording and integration of the chromatogram data was car- ried out through an Agilent OpenLAB Chromatography data sys- tem. The identification of fatty acids was made using standard compounds and conventional UV spectra. Calibration curves of all the compounds were constructed using standards and were found to be linear with correlation coefficients > 0.995. Determination of total phenolics and flavonoid contents The total phenolics and flavonoids content were evaluated accord- ing to the procedure previously reported25,26. For the total phe- nolics content, gallic acid was used as the standard, and the results were expressed as milligram of gallic acid equivalents per g of extract (mg GAE/g). Quantification of flavonoids was instead done based on standard curve of quercetin prepared in 80% etha- nol and results were expressed in milligram quercetin equivalent per gram of seeds extracts (mg QE/g). HPLC analysis Analyses of fatty acids were carried out with an Agilent Technologies 1100 liquid chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a diode array detector (DAD). Analyses of unsaturated free fatty acids (detected at 200 nm), obtained from oil saponification, were carried out with a XDB–C18 Eclipse (150 4.6 mm, 3.5 lm particle size) (Agilent Technologies) equipped with a Zorbax XDB-C18 Eclipse (12.5 4.6 mm, 5 lm par- ticle size) guard column (Agilent Technologies), with a mobile phase of CH3CN/H2O/CH3COOH (75/25/0.12, v/v/v), at a flow rate of 2.3 ml/min23. The temperature of the column was maintained at 37 C. Preparation of the extracts Freshly collected samples were washed with running water to remove glochids and impurities, air-dried and hand-peeled. The seeds were separated from the juicy pulp, washed abundantly with distilled water, then dried at room temperature for 24 h, weighed and reduced to a fine powder using a blender type A11 basic (IKA, Germany). The powdered seeds samples were stored at 20 C prior to analysis. The powdered seeds (20g) were extracted in 70% ethanol (etha- nol extract, EE). for 24h at room temperature under continuous stir- ring. The EE was filtered and centrifuged at 12,000g for 20min and then evaporated under reduced pressure to dryness. This extract was suspended in distilled water (water extract, WE) and sequentially frac- tionated by hexane (hexane extract, HE), ethyl acetate (ethyl acetate extract, EAE) and n-butanol (butanol extract, BE). After separation of the phases, the solvents were removed in a rotary evaporator at 45 C under vacuum. All extracts and fractions were submitted to biological assays. All analyses were performed in triplicate. HSCCC analysis High-speed counter-current chromatography (HSCCC) was carried out to isolate bioactive compounds from EAE. HSCCC was performed on Quattro Intro-Prep counter-current chromatograph (AECS, Bridgend, United Kingdom). The choice of solvent system was decided after comparing two-phase solvent systems containing n- hexane, ethyl acetate, methanol, and water (H:E:M:W) in different proportions (3:5:3:5 and 1:4:2:3 (v/v/v/v)). The partition coefficients (KD) of the compounds in the specific solvent system were evaluated by a UV detector at 254 nm in a Spectroline CX-20 UV Fluorescence Analysis Cabinet, followed by spraying TLC plates with vanillin (2% in methanol) and sulphuric acid (1% in methanol). The system HEMW 1:4:2:3 (v/v/v/v) was chosen because it showed a better distri- bution of the compounds in the two-phase solvent. Allopurinol was used as a standard inhibitor. Xanthine oxidase activity was expressed as percent inhibition of xanthine oxidase, calculated as (A B)/A 100, where A is the change in absorb- ance of the assay without the plant extract, and B is the change in absorbance of the assay with the plant extract. All assays were performed in triplicate. The IC50 value, a concentration giving 50% inhibition of XO activity, was determined by interpolation of dose-response curves. The mode of inhibition on the enzyme was performed using the Lineweaver–Burk plot. Different concentrations of substrate (20–70 mM) were used for the assay. The lower aqueous phase was used as the stationary phase, and the upper organic phase as the mobile phase. The sample solution was prepared by dissolving 500 mg of the dried EAE into 6ml of two-phase solvent, filtered through 0.45mm membrane filter prior to injection into the HSCCC system. The column was first filled with the lower stationary phase, subsequently the apparatus was rotated at 850 rpm while the upper phase was pumped into the inlet of the column as the mobile phase at a flow rate of 2mg/mL. After that equilibrium was established in the column, 6 ml of the extract were injected. After 100 min the rotation was stopped, and the lower phase was pumped into the column and separation was carried out for 100min more. The fractions were manually collected. The machine was used in the tail to head mode all the time. Determination of fatty acids The EAE of P. dioica seeds was analysed using a Hewlett-Packard 1200 chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a binary pump and a diode array detector (DAD) coupled to an HP Chem Station (rev. A.05.04) data-processing sta- tion. The HPLC system was connected via the DAD cell outlet to an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany) mass spectrometer (MS) consisting of an ESI source and a triple quadrupole-ion trap mass analyser, which was controlled by the Aliquots of dried HE extract (3 mg) were dissolved in ethanol and subjected to mild saponification at room temperature in the dark23. The saponifiable fraction with free fatty acids was collected and the solvent was evaporated. A portion of the dried residue was dissolved in CH3CN with 0.14% CH3COOH (v/v) and aliquots of the samples were injected into the HPLC system. An aliquot of dried fatty acids was methylated with 1 ml of methanolic HCl (3 N)23,24 for 30 min at room temperature. After addition of JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 521 521 Table 1. Fatty acid composition (%, g/100 g) by GC analysis of P. dioica HE. Fatty acid g/100 g 12:0 0.57 ± 0.14 14:0 0.93 ± 0.45 16:0 16.58 ± 0.63 16:1 n-7 0.20 ± 0.07 18:0 1.96 ± 0.18 18:1 n-7 0.20 ± 0.07 18:1 n-9 50.32 ± 3.43 18:2 n-6 15.33 ± 1.01 18:3 n-3 0.67 ± 0.08 20:0 0.77 ± 0.11 20:1 n-9 0.91 ± 0.19 SFA 20.82 ± 0.03 MUFA 51.71 ± 3.25 PUFA 16.00 ± 1.10 SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsat- urated fatty acids. Mean and standard deviation of 3 samples (n ¼ 3). Table 1. Fatty acid composition (%, g/100 g) by GC analysis of P. dioica HE. Fatty acid g/100 g 12:0 0.57 ± 0.14 14:0 0.93 ± 0.45 16:0 16.58 ± 0.63 16:1 n-7 0.20 ± 0.07 18:0 1.96 ± 0.18 18:1 n-7 0.20 ± 0.07 18:1 n-9 50.32 ± 3.43 18:2 n-6 15.33 ± 1.01 18:3 n-3 0.67 ± 0.08 20:0 0.77 ± 0.11 20:1 n-9 0.91 ± 0.19 SFA 20.82 ± 0.03 MUFA 51.71 ± 3.25 PUFA 16.00 ± 1.10 SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsat- urated fatty acids. Mean and standard deviation of 3 samples (n ¼ 3). concentration of the extract necessary to give a 50% reduction in the original absorbance (half maximal effective concentration, EC50). Tyrosinase assay The methodology of the spectrophotometric tyrosinase assay was described in detail in our previous works27. The percentage of inhibition of tyrosinase activity was calculated as inhibition (%)¼(A B)/A 100, where A represents the difference in the absorbance of control sample between an incubation time of 0.5 and 1.0 min, and B represents the difference in absorbance of the test sample between an incubation time of 0.5 and 1.0 min. p The IC50 value, a concentration giving 50% inhibition of tyro- sinase activity, was determined by interpolation of dose-response curves. Kojic acid was used as a standard inhibitor. The mode of inhibition on the enzyme was performed using the Lineweaver–Burk plot. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsat- urated fatty acids. y Mean and standard deviation of 3 samples (n ¼ 3). Analyst 5.1 software. An Aqua C18 125 Å column (250 4.6 mm, 5 lm; Phenomenex) thermostated at 35 C was used. The solvents were: (A) 0.1% formic acid and (B) acetonitrile. The elution gradi- ent was the same as previously described27. Compound identifica- tion was made based on their absorption and mass spectral characteristics (both positive and negative modes) and compari- son with previously published data. Xanthine oxidase assay XO activity was determined spectrophotometrically by measuring the formation of uric acid from xanthine. The xanthine solution was prepared by initially dissolving xanthine in a minimal volume of NaOH, adjusting pH to 7.5. The XO solution was prepared by diluting to a final concentration of 0.5 U/ml in cold 0.1 M phos- phate buffer (pH 7.5). The assay mixture consisted of 200 lL of plant extract solution, 690 lL 0.1 M phosphate buffer (pH 7.5), 60 lL of xanthine solution and 50 lL of XO. The change in absorb- ance was recorded at 295 nm for 3 min at room temperature. Statistical analysis Data are expressed as mean ± SD from three independent experi- ments. The analysis average of the treatment using multiple com- parisons was determined by using Duncan’s multiple range tests, and the data were compared using the p values: p < .05 was con- sidered statistically significant. The least significant difference (LSD) was used to determine the difference between the methods used to the investigation of the various antioxidant capacities. The statistical analysis of differences between various treatments was determined by the Student’s t-test. Values of p < .05 were consid- ered statistically significant. Statistical analysis was performed with SPSS v.18.0 (IBM Corp., Armonk, NY, USA). The purity of fractions obtained by HSCCC was determined by TLC analysis and HPLC-DAD-ESI/MS analyses and the compounds were identified by MS and 1H NMR analysis28. Antioxidant assay dioica extracts Table 2. Total phenolics and flavonoid content and free radical scavenging activity of P. dioica extracts Extract Total phenolics() Flavonoids() EC50 values (mg/mL) ABTS scavenging DPPH scavenging EE 102.07 ± 2.30c 18.43 ± 1.22bc 16.97 ± 0.32a 20.4 ± 1.23a HE 2.61 ± 0.21a 1.82 ± 0.42a >500 >500 EAE 396.41 ± 4.21e 43.57 ± 1.26d 3.1 ± 0.04a 4.85 ± 0.11a BE 143.64 ± 1.35d 15.19 ± 0.54b 5.64 ± 0.26a 6.28 ± 0.91a WE 46.34 ± 0.42b 19.66 ± 0.39c 127.48 ± 8.37b 137.79 ± 5.53b Mixture of isoamericanol B1, B2, C1 and C2 – – 7.1 ± 0.4 6.51 ± 0.74 Phytolaccoside B – – >500 >500 Trolox – – 3.4 ± 0.3 3.2 ± 0.4 () mg GAE/g of dry weight. () mg QE/g of dry weight. The data are given as mean ± standard deviation (SD) of triplicate experiments. The statistical comparison between values from the different plant extracts was performed using the post hoc Duncan test. Means followed by distinct letters in the same column were found to be significantly different (p < .05). ( ) mg QE/g of dry weight. The data are given as mean ± standard deviation (SD) of triplicate experiments. The statistical comparison between values from the different plant extracts was performed using the post hoc Duncan test. Means followed by distinct letters in the same column were found to be significantly different (p < .05). Figure 1. HPLC chromatogram of P. dioica seeds ethyl acetate extract recorded at 330 nm for phenolic compounds. Peak identification is given in Table 3. Figure 1. HPLC chromatogram of P. dioica seeds ethyl acetate extract recorded at 330 nm for phenolic compounds. Peak identification is given in Table 3. Figure 1. HPLC chromatogram of P. dioica seeds ethyl acetate extract recorded at 330 nm for phenolic compounds. Peak identification is given in Table 3. oil obtained by GC analysis are reported in Table 1. HE showed a concentration of approximately 20.82 ± 0.03% of saturated fatty acids (SFA, mainly palmitic acid 16:0, and stearic acid 18:0, being about 16.6 and 2% respectively), 51.71 ± 3.25% of monounsatu- rated (MUFA, mainly oleic acid 18:1 n-9, 50.3%), and 16.0 ± 1.1% of polyunsaturated (PUFA), mainly constituted by linoleic 18:2 n-6, 15.3%. highest phenolics content. In fact, it was respectively 2.8 and 3.9 fold higher than that determined in BE and EE extracts. Antioxidant assay To give some insight into the chemical composition of P. dioica seeds, quali-quantitative information on the individual fatty acids in HE extracts was obtained by GC and HPLC-DAD analyses. Fatty acid compositions (expressed as % of total fatty acids, g/100 g) of Radical scavenging activities were measured by using DPPH and ABTS radical scavenging assays as previously reported27. For both free radical methods, antioxidant activity was expressed as 522 A. DI PETRILLO ET AL. A. DI PETRILLO ET AL. oil obtained by GC analysis are reported in Table 1. HE showed a concentration of approximately 20.82 ± 0.03% of saturated fatty acids (SFA, mainly palmitic acid 16:0, and stearic acid 18:0, being about 16.6 and 2% respectively), 51.71 ± 3.25% of monounsatu- rated (MUFA, mainly oleic acid 18:1 n-9, 50.3%), and 16.0 ± 1.1% of highest phenolics content. In fact, it was respective fold higher than that determined in BE and EE ext also yielded the highest amount of flavonoids, fol EE, and BE. As it was expected, HE was the poorest olic and flavonoid compounds Previous studies have Table 2. Total phenolics and flavonoid content and free radical scavenging activity of P. dioica extracts Extract Total phenolics() Flavonoids() EC50 values (mg/mL) ABTS scavenging DPPH scavenging EE 102.07 ± 2.30c 18.43 ± 1.22bc 16.97 ± 0.32a 20.4 ± 1.23a HE 2.61 ± 0.21a 1.82 ± 0.42a >500 >500 EAE 396.41 ± 4.21e 43.57 ± 1.26d 3.1 ± 0.04a 4.85 ± 0.11a BE 143.64 ± 1.35d 15.19 ± 0.54b 5.64 ± 0.26a 6.28 ± 0.91a WE 46.34 ± 0.42b 19.66 ± 0.39c 127.48 ± 8.37b 137.79 ± 5.53b Mixture of isoamericanol B1, B2, C1 and C2 – – 7.1 ± 0.4 6.51 ± 0.74 Phytolaccoside B – – >500 >500 Trolox – – 3.4 ± 0.3 3.2 ± 0.4 () mg GAE/g of dry weight. () mg QE/g of dry weight. The data are given as mean ± standard deviation (SD) of triplicate experiments. The statistical comparison between values from the different plant extracts was performed using the post hoc Duncan test. Means followed by distinct letters in the same column were found to be significantly different (p < .05). Figure 1. HPLC chromatogram of P. dioica seeds ethyl acetate extract recorded at 330 nm for phenolic compounds. Peak identification is given in Tab Table 2. Total phenolics and flavonoid content and free radical scavenging activity of P. HPLC-DAD-ESI/MS analyses of phenolic compounds To identify the nature of phenolic compounds contained in the EAE of P. dioica seeds, HPLC-DAD-ESI/MS analyses were carried out. A representative HPLC chromatogram recorded at 330 nm is shown in Figure 1, and the phenolics’ composition is summarised in Table 3. Antioxidant assay The EAE also yielded the highest amount of flavonoids, followed by WE, EE, and BE. As it was expected, HE was the poorest in both phen- olic and flavonoid compounds. Previous studies have reported the total phenolic and flavonoid contents for other species of Phytolacca29,30. However, to best of our knowledge no studies have investigated the phenolic and flavonoid contents for the P. dioica seeds. It is important to highlight that the total phenolic and flavonoid contents reported in this study are appreciably higher than the previous studies29,30. Furthermore, the absolute content of the main unsaturated fatty acids in the P. dioica HE was determined by HPLC, as follows: approximately 488.7 mg/g of oil extract for 18:1 n-9, and minor amounts for 18:2 n-6 and 18:3 n-3 (148.4, and 8.1 mg/g of oil extract, respectively). MUFA formed a main part of fatty acid composition and the majority fatty acid identified in P. dioica oil extract was oleic acid (18:1 n-9). JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY NMR compound 1 – mixture of isoamericanol B1, B2, C1 and C2 Isoamericanol (B1, B2, C1, C2) 1H-NMR 13C-NM 1 – 129.2 2 6.90 (d, 2.5 Hz) 115.9 3 – 146.2 4 – 146.3 5 6.83 (d, 8.5 Hz) 115.7 6 6.76 (dd, 2.5, 8.5 Hz) 119.4 7 4.80 (d, 8 Hz) 76.2 8 3.97 (m) 78.7 9 3.46 (dd, 12.5, 5.5) 60.6 3.65 (dd, 12.5, 3) 1’ – 130.7 2’ 7.00 (m) 117.2 3’ – 145.7 4’ – 145.7 5’ 6.94 (m) 119.2 6’ 6.94 (m) 119.8 7’ – 76.1 8’ 3.97 (m) 78.6 9’ 3.46 (dd, 12.5, 5.5) 60.6 1” 3.65 (dd, 12.5, 3) 131.0 2” 6.94 (m) 115.9 3” – 144.0 4” – 145.6 5” 6.90 (d, 2.5) 117.5 6” 4.17 (dd, 6, 1.5 Hz) 119.2 7” 6.47 (d, 16 Hz) 129.8 8” 6.17 (dt, 16.6, 7.8 Hz) 128.5 9” – 62.0 JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 52 Table 3. Identification of polyphenol compounds in P. dioica seeds ethyl acetate extract by HPLC-DAD-ESI/MS analysis. Peak Rt (min) kmax (nm) Pseudomolecular ion [M-H] (m/z) MS2 (m/z), (%) Tentative identification 1 15.7 342 163 135(100) p-coumaric acid 121(42) 145(41) 119(20) 2 19 330 329 137(97) (iso)americanol A 3 21.7 324 297 135(41) caffeoyl-threonic acid (isomer I) 179(23) 4 23.1 320 687 179(100) Unknown 133(100) 269(28) 5 26.2 334 493 329(100) isoamericanol B1 or related isomers (B2 / C1 / C2 / (iso)princepin) 327(100) 165(64) 175(35) 137(32) 6 26.8 338 493 165(100) isoamericanol B1 or isomers (B2 / C1 / C2) 298(75) 329(40) 147(23) 7 27.1 320 297 135(36) caffeoyl-threonic acid (isomer II) 179(16) 8 27.6 320 462 289(93) Unknown lignan 165(100) 147(32) 9 30.3 334 657 493(100) Unknown lignan 327(41) 165(25) 10 31.1 324 329 211(32) trihydroxy-octadecenoic acid 229(31) 171(18) Relevant peaks in the extract were assigned as lignans. Peaks 5 and 6 were tentatively identified as different isomers of isoameri- canol (B1, B2, C1 or C2) with the molecular mass of 49431. This identification was further supported by the MS2 fragments at m/z 329 that might correspond to (iso)americanol A (also associated to peak 2) and at m/z 327 to (iso)americanin A31,32. Table 4. JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY NMR compound 1 – mixture of isoamericanol B1, B2, C1 and C2 Isoamericanol (B1, B2, C1, C2) 1H-NMR 13C-NMR 1 – 129.2 2 6.90 (d, 2.5 Hz) 115.9 3 – 146.2 4 – 146.3 5 6.83 (d, 8.5 Hz) 115.7 6 6.76 (dd, 2.5, 8.5 Hz) 119.4 7 4.80 (d, 8 Hz) 76.2 8 3.97 (m) 78.7 9 3.46 (dd, 12.5, 5.5) 60.6 3.65 (dd, 12.5, 3) 1’ – 130.7 2’ 7.00 (m) 117.2 3’ – 145.7 4’ – 145.7 5’ 6.94 (m) 119.2 6’ 6.94 (m) 119.8 7’ – 76.1 8’ 3.97 (m) 78.6 9’ 3.46 (dd, 12.5, 5.5) 60.6 1” 3.65 (dd, 12.5, 3) 131.0 2” 6.94 (m) 115.9 3” – 144.0 4” – 145.6 5” 6.90 (d, 2.5) 117.5 6” 4.17 (dd, 6, 1.5 Hz) 119.2 7” 6.47 (d, 16 Hz) 129.8 8” 6.17 (dt, 16.6, 7.8 Hz) 128.5 9” – 62.0 Table 4. NMR compound 1 – mixture of isoamericanol B1, B2, C1 and C2 HSCCC and structural identification of isolated compounds Two compounds were isolated from EAE extract using HSCCC. Structure of purified compounds was elucidated by interpretation of spectroscopic experiments (UV, MS, and 1H NMR) and by com- parison with literature. Compound 1: Mixture of Isoamericanol B1, B2, C1, and C2 UV (MeOH) kmax nm: 334; HRESI–MS m/z [M H]þ: 493 (related to iso- americanol C1 and C2); 1H NMR (Methanol-D4, 400 MHz)28 (Table 4). Compound 2: UV (MeOH)kmax(MeOH)nm: 210; HRESI–MSm/ z[M H]: 6 6 5 [M þ H] þ, 5 1 5 [M þ H-150]þ;1H NMR (Methanol-D4, 400 MHz): 0.78 (3H, s, H26), 0.91 (3H, s, H29), 1.12 (3H, s, H27), 1.15 (3H, s, H24), 1.26 (3H, s, H25), 2.68 (2H, brd, H- 18), 3.66 (3H, s, COOMe), 4.35 (1H, d, J ¼ 8 Hz, H3), 5.31 (1H, brs, H12), 5.32 (1H, d, 8 Hz, H1 Xyl). Compound 2 was identified as phytolaccoside B (Table 4). Total phenolics and flavonoids content The total phenolics content of the samples was quantified in each P. dioica seeds extract (Table 2). The EAE extract exhibited the JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 523 Relevant peaks in the extract were assigned as lignans. Peaks 5 and 6 were tentatively identified as different isomers of isoameri- canol (B1, B2, C1 or C2) with the molecular mass of 49431. This identification was further supported by the MS2 fragments at m/z 329 that might correspond to (iso)americanol A (also associated to peak 2) and at m/z 327 to (iso)americanin A31,32. HSCCC and structural identification of isolated compounds Two compounds were isolated from EAE extract using HSCCC. Structure of purified compounds was elucidated by interpretation of spectroscopic experiments (UV, MS, and 1H NMR) and by com- parison with literature. Compound 1: Mixture of Isoamericanol B1, B2, C1, and C2 UV (MeOH) kmax nm: 334; HRESI–MS m/z [M H]þ: 493 (related to iso- americanol C1 and C2); 1H NMR (Methanol-D4, 400 MHz)28 (Table 4). Compound 2: UV (MeOH)kmax(MeOH)nm: 210; HRESI–MSm/ z[M H]: 6 6 5 [M þ H] þ, 5 1 5 [M þ H-150]þ;1H NMR (Methanol-D4, 400 MHz): 0.78 (3H, s, H26), 0.91 (3H, s, H29), 1.12 (3H, s, H27), 1.15 (3H, s, H24), 1.26 (3H, s, H25), 2.68 (2H, brd, H- 18), 3.66 (3H, s, COOMe), 4.35 (1H, d, J ¼ 8 Hz, H3), 5.31 (1H, brs, H12), 5.32 (1H, d, 8 Hz, H1 Xyl). Compound 2 was identified as phytolaccoside B (Table 4). Table 3. Identification of polyphenol compounds in P. dioica seeds ethyl acetate extract by HPLC-DAD-ESI/MS analysis. Peak Rt (min) kmax (nm) Pseudomolecular ion [M-H] (m/z) MS2 (m/z), (%) Tentative identification 1 15.7 342 163 135(100) p-coumaric acid 121(42) 145(41) 119(20) 2 19 330 329 137(97) (iso)americanol A 3 21.7 324 297 135(41) caffeoyl-threonic acid (isomer I) 179(23) 4 23.1 320 687 179(100) Unknown 133(100) 269(28) 5 26.2 334 493 329(100) isoamericanol B1 or related isomers (B2 / C1 / C2 / (iso)princepin) 327(100) 165(64) 175(35) 137(32) 6 26.8 338 493 165(100) isoamericanol B1 or isomers (B2 / C1 / C2) 298(75) 329(40) 147(23) 7 27.1 320 297 135(36) caffeoyl-threonic acid (isomer II) 179(16) 8 27.6 320 462 289(93) Unknown lignan 165(100) 147(32) 9 30.3 334 657 493(100) Unknown lignan 327(41) 165(25) 10 31.1 324 329 211(32) trihydroxy-octadecenoic acid 229(31) 171(18) Table 4. Enzymes inhibitory activity All extracts were also evaluated for their inhibitory effect on tyro- sinase and xanthine oxidase enzymes (Table 5). The obtained results revealed that EAE exhibited the most potent inhibitory activity against mushroom tyrosinase. BE and EE were less potent, and no activity was detected for HE and WE. V / 1 EAE was also the most potent inhibitor of xanthine oxidase among all extracts; it exhibited 99.93 ± 1.94% inhibition at a con- centration of 200 mg/mL. EE and BE showed 50.5 ± 0.99% and 23.47 ± 1.66% inhibition, respectively, at same conditions, whereas the remaining extracts (HE and WE) showed no XO activity. Considering the complete inhibition of XO by EAE, further attention was focussed on its mode of inhibition of the enzyme, which was determined by Lineweaver–Burk plot analysis, as shown in Figure 2(A). The kinetic analysis revealed that this extract acts as a mixed-type inhibitor. In fact, increasing the concentration of the extract resulted in a family of lines, which intersected in the second quadrant. To calculate the equilibrium inhibition con- stants for the inhibitor binding with the free enzyme (KI) and the enzyme-substrate complex (KIS), two secondary graphs (data not shown) were obtained by plotting the Km/Vmax (slope) and 1/Vmax (intercept) versus the concentration of EAE. The values of KI and KIS were determined to be 0.68 and 0.007 mg/mL respectively. Figure 2. Lineawear-Burk plot for inhibition of EAE extract on xanthine oxidase (A) and tyrosinase (B). The inhibitor concentrations were 0 (), 0.04 (), 0.08 (w) and 0.1 () mg/mL, for xanthine oxidase assay. The inhibitor concentrations were 0 (), 0.05 (), 0.1 (w) and 0.2 () mg/mL, for tyrosinase assay. Figure 2. Lineawear-Burk plot for inhibition of EAE extract on xanthine oxidase (A) and tyrosinase (B). The inhibitor concentrations were 0 (), 0.04 (), 0.08 (w) and 0.1 () mg/mL, for xanthine oxidase assay. The inhibitor concentrations were 0 (), 0.05 (), 0.1 (w) and 0.2 () mg/mL, for tyrosinase assay. Table 6. Molecular properties of three classes of isoamericanol compounds. Radical scavenging activity B1, B2, C1 and C2) showed an EC50 for ABTS and DPPH of 7.1 and 6.5 mg/mL (Table 2) respectively, explaining in part the antioxidant activity determined in the EAE extract. B1, B2, C1 and C2) showed an EC50 for ABTS and DPPH of 7.1 and 6.5 mg/mL (Table 2) respectively, explaining in part the antioxidant activity determined in the EAE extract. P. dioica extracts were evaluated for their antioxidant activity using two different methods, leading to quite similar values for each extract analysed. In agreement with the phenolics content, the HE showed the poorest antioxidant activity, whereas the EAE and BE showed the highest antioxidant activity (Table 2). EC50 val- ues of EAE and BE are comparable to that of Trolox, used as refer- ence compound (Table 2). Compound 1 (Mixture of isoamericanol The bioassays experiments revealed antioxidant activity of the EAE and its constituent (compound 1) to be remarkably pro- nounced with respect to previous studies performed on Phytolacca americana29,30. Since, no data has been reported in the literature about the antioxidant activity of P. dioica seeds extracts. A. DI PETRILLO ET AL. 524 1/[L-DOPA], mM−1 -2 0 2 4 6 V / 1 0 2 4 6 8 10 12 14 1/[Xanthine], µM−1 -0.02 0 0.02 0.04 0.06 V / 1 0 2 4 6 8 10 12 14 16 18 20 Figure 2. Lineawear-Burk plot for inhibition of EAE extract on xanthine oxidas (A) and tyrosinase (B). The inhibitor concentrations were 0 (), 0.04 (), 0.08 (w and 0.1 () mg/mL, for xanthine oxidase assay. The inhibitor concentration were 0 (), 0.05 (), 0.1 (w) and 0.2 () mg/mL, for tyrosinase assay. Table 5. Inhibitory activity of the extracts of P. dioica seeds against tyrosinase and xanthine oxidase at 200 mg/mL. 1/[Xanthine], µM−1 -0.02 0 0.02 0.04 0.06 V / 1 0 2 4 6 8 10 12 14 16 18 20 and xanthine oxidase at 200 mg/mL. Extract Inhibitory activity (inhibition % ± SD) Tyrosinase Xanthine oxidase EE 22.35 ± 0.49a 50.5 ± 0.99b HE ND ND EAE 50.83 ± 0.84c 99.93 ± 1.94c BE 29.23 ± 0.90b 23.475 ± 1.66a WE ND ND Kojic acid 99.98 ± 0.02 Allopurinol 99.96 ± 0.003 ND: Not detected. V / 1 The results are expressed as Inhibition % (lg/mL). The data are given as mean- ± standard deviation (SD) of triplicate experiments. Enzymes inhibitory activity Physicochemical properties Isoamericanol AIsoamericanol BIsoamericanol C1 Molecular formula C18H18O6 C19H20O5 C27H26O9 Molecular Weight (g/mol) 330.33 328.36 494.49 Rotatable bonds 4 4 6 H-bond acceptor atoms 6 5 9 H-bond donor atoms 4 2 5 Molar refractivity 87.87 91.18 129.20 Polar Surface area (Å2) 99.38 68.15 138.07 Lipophilicity (consensus) 1.65 2.82 2.24 Water solubility Soluble Moderate Moderate Pharmacokinetics Isoamericanol AIsoamericanol BIsoamericanol C1 Gastrointestinal absoprtion High High High Blood brain barrier permeation No Yes No P-glycoprotein substrate Yes Yes No Cytochrome P450 2D6 inihibitor No Yes No Cytochrome P450 3A4 inihibitor No No Yes Druglikeness (lipinsky rule) Yes Yes Yes Table 6. Molecular properties of three classes of isoamericanol compounds. Physicochemical properties Isoamericanol AIsoamericanol BIsoamericanol C1 The mode of inhibition of this extract on tyrosinase was also determined using Lineweaver–Burk plot which displayed an uncompetitive inhibition of the enzyme, see Figure 2(B). The equi- librium constant for binding with enzyme–substrate complex (KIS) was calculated to be 0.113 mg/mL. The isolated mixture of isoamericanol B1, B2, C1, and C2 showed inhibitory activity on tyrosinase and xanthine oxidase enzymes, with IC50 of 0.110 ± 0.02 mg/mL and 0.145 ± 0.05 mg/mL respectively. No activity was detected for the phytolaccoside B. Radical scavenging activity The statistical comparison between values from the different seeds extracts was performed using the post hoc Duncan test. Means followed by distinct letters in the same column were found to be significantly different (p < .05). -2 0 2 4 6 V / 1 0 2 4 6 8 10 12 14 Conclusions Furthermore, the chemical composition of the hexane extract from the P. dioica seeds showed that major fatty acid identified was oleic acid (18:1 n-9), which is very important for the nutri- tional value of oils. It has been documented that MUFA may reduce LDL cholesterol, while it might possibly increase HDL chol- esterol39. Oleic acid may promote insulin resistance and it has been reported as anti-apoptotic and anti-inflammatory agent via down regulation of cyclooxygenase-2 and inducible nitric oxide synthase through the activation of nuclear factor-kappa B (NF-jB)39. The development of new inhibitors, synthetic or from natural sources, of tyrosinase and xanthine oxidase activity is a very prom- ising field of research34–37. The results of this study showed that the ethyl acetate extract of P. dioica seeds possessed a good XO and tyrosinase inhibi- tory activity. Xanthine oxidase catalyses the oxidation of hypoxanthine to xanthine and subsequently to uric acid. This enzyme plays a vital role in the onset of hyperuricaemia and gout. Furthermore, its re- oxidation involves molecular oxygen and during this reaction reactive oxygen species are produced. The obtained results indi- cate that the ethyl acetate extract of P. dioica seeds presents con- stituents with medicinal properties that could be exploited to treat diseases associated with oxidative stress and xanthine oxi- dase enzyme activity. This extract can also be considered a good candidate for further investigations to evaluate its effect on the inhibition of skin pigmentation as deduced from its effects on tyrosinase. All in all, for the first time the chemical composition and inhibi- tory enzyme activity of P. dioica seeds extracts is here reported. It seems that this plant could be a source of bioactive molecules. The results achieved open promising perspectives for further stud- ies of the biological effect and medicinal use of these nat- ural products. Acknowledgements The authors are grateful to Dr. Cecilia Loi (University of Cagliari) for plant authentication. Moreover, the isolated mixture of isoamericanol (B1, B2, C1, and C2) showed inhibitory activity on tyrosinase and xanthine oxi- dase enzymes. This is the first time that these isoamericanols have been described as inhibitors of the above mentioned enzymes and the presence of these compounds in the ethyl acetate extract may contribute to the activity observed for the plant. Indeed, we Molecular and physicochemical properties of compounds Physicochemical properties of three classes of isoamericanol were calculated using Swiss-ADME web tool33 (Table 6). Due to critical importance for pharmacokinetics drug discovery, we report, in addiction to simple molecular attribute, the lipophilicity, water solubility, and drug likeliness characteristics for the three classes. Among the three classes of isoamericanol, A and B display similar molecular flexibility reflected from their same number on rotatable bonds, while C1 exhibiting highest flexibility. Further, isoamerica- nol C1 possess the highest number of hydrogen (H-bond) acceptor and donor atoms, molecular refractivity and polar surface area (PSA). On the other hand, isoamericanol B showed highest lipophilic behaviour, while best water solubility characteristics was noted for isoamericanol A. lipophilic behaviour, while best water solubility characteristics was noted for isoamericanol A. lipophilic behaviour, while best water solubility characteristics was noted for isoamericanol A. Concerning pharmacokinetics, all the three classes displayed high gastrointestinal absorption, while only isoamericanol B showed promising blood brain barrier (BBB) permeation character- istics. We used a novel Egg-boiled model, which is simple and intuitive graph prediction of passive intestinal absorption and brain penetration, as a function of lipophilicity and apparent JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 525 Figure 3. Novel BOILED-Egg construction using two physicochemical descriptors, lipophilicity (LogP) and polar surface area (PSA) for the three isoamericanol com- pounds. The white region represents the physicochemical space of gastrointestinal absorption and the yellow region brain penetration. Figure 3. Novel BOILED-Egg construction using two physicochemical descriptors, lipophilicity (LogP) and polar surface area (PSA) for the three isoamericanol com- pounds. The white region represents the physicochemical space of gastrointestinal absorption and the yellow region brain penetration. polarity (Figure 3). If plotted molecule falls inside the white ellipse, the probability of a good intestinal absorption is high, while inside the yellow ellipse (i.e. the yolk), the probability of a good BBB crossing is high. Interestingly, all the three classes displayed drug likeness calculated according to Lipsinky’s rule. found promising physicochemical and pharmacokinetics properties for the three classes of isoamericanol. The ethyl acetate extract also showed highest antioxidant cap- acity if compared with other extracts. The EC50 values were strongly correlated with total phenolic contents, suggesting the contribution of these compounds to the antioxidant properties of the extracts. Moreover, most of the polyphenolics identified were lignans with well-known antioxidant activity38. References 19. Chiang HC, Lo YJ, Lu FJ. 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https://openalex.org/W2009865231	https://zenodo.org/records/1812667/files/article.pdf	English	null	A school outbreak of diphtheria due to a carrier case	Public health	1,910	public-domain	988	By ANDREW SMITH, M.D., Medical Officer of Health, Whickham. T T HERE was a small epidemic of eleven cases in June and July at Whickham which was of a peculiar if not unique character. The school was closed for the usual summer holidays in the same week as this child was again isolated, and no more cases occurred ; indeed, there were no cases at all throughout the whole district during the next month, when all the schools were closed. After that isolated cases occurred as usual, but without any obvious connection with any of the schools. p q At the end of June one case occurred, and at the beginning of July a series of others in rapid succession, living widely apart in the village. It was noted that they were all of the same age, and on inquiry I found they were all attending the same class in the infant school. I visited the school and found that they were all even in this room usually close together; as others came in the area widened, but still they were all from the same class-room. It was obvious that they were all coming from some source of infection in the school. At this time the school was within a week of closure for the summer holidays. It was too great a task to get swabs taken from all the children, because we have no power to do so without consent of parents, and the time was too short to get this and carry out the work. I, how- ever, suspected a probable origin in a child that I had attended five months previously for laryngeal diphtheria, and who had had a chronic discharge from the ear since it was two years old. This child was isolated until throat swabs were negative about the sixth week, and it did not return to school till several weeks later, as it continued under treatment for ear discharge. Treatment failing, it was sent to the Newcastle Infirmary with a view to operation, but it was decided to continue local treatment--antiseptic syringing and eardrops. This had been persistently carried out, but still discharge continued. My suspicion fell on this discharge, so I sent a swab from the ear for bacteriological examination and it proved positive ; diphtheria bacilli were present, and that in spite of the long continuance of antiseptic treatment. By ANDREW SMITH, M.D., Medical Officer of Health, Whickham. SCARLET FEVER CARRIERS.---I am convinced that scarlet fever is to a very large extent spread by "carriers," viz., persons who have had such a very mild dose of the poison as to produce no observable rash or other symptoms except perhaps a slight sore throat ; and it may often happen that they simply carry the germs in their throats, if one may judge by the analogy of diphtheria where it is easily possible to show the germs under the microscope. In these diphtheritic cases the patient may have no clinical symptoms whatever, but he may be able to transmit infection to others. A somewhat wide experience of scarlet-fever has made me believe that disinfection of the mouth and throat of scarlet-fever contacts with a really active bactericide such as a spray of I in ~ooo perchloride of mercury applied every two or three hours is a really useflll means of destroying or in- hibiting the growth of the scarlatinal micro- organism, and thus limiting the spread of the disease. The surgeon uses similar means to destroy micro-organisms on his hands previous to an operation, and prima facie there is reason to suppose that in certain cases it might be effective in the destruction of micro-organisms in the throat, if used before they have had time to enter the circulation in large numbers.--Anmtal Report of Dr. Bertra~n 77zomton, Medical Ofwer of Health, Margate. Another swab taken in lhe same way after all antiseptic treatment was stopped for three days was again returned positive, as was also a swab from the throat. A consultation with a throat and ear specialist was then held, and on his advice the case was admitted to the isolation hospital and there operated on for radical cure of chronic otitis media, enlarged tonsils and adenoids being re- moved at the same time. The removed tonsils, adenoids, a swab from the antrum and a swab from the tympanum were all submitted to bac- teriological examination. All gave a negative result except that from the tympanum, which contained the discharge as it was coming from the middle ear. MIDWIVES IN RURAL DISTRICTS.--If rural dis- tricts are in any way to retain the valuable services of a qualified midwife, the law must be so amended as to allow such women to do all such work as they can possibly attend to. PUBLIC HEALTH. PUBLIC HEALTH. MAY, a26 A SCHOOL OUTBREAK OF DIPH- THERIA DUE TO A CARRIER CASE.* By ANDREW SMITH, M.D., Medical Officer of Health, Whickham. A SCHOOL OUTBREAK OF DIPH- THERIA DUE TO A CARRIER CASE.* been acting as an incubating chamber for the diphtheria bacilli, and that these were being const~antly passed with the discharge from the ear and occasionally also down the eustachian tube to the throat, and this had apparently been going on for six months, which was the interval between the attack of diphtheria and the operation. By ANDREW SMITH, M.D., Medical Officer of Health, Whickham. I therefore suggest that in certain sparsely populated areas, women be appointed who shall act as midwife, sick nurse, school nurse, and health visitor, and if the various authorities and societies who are engaged in push- ing their own particular pet theories would com- bine, the necessary funds for the maintenance of such women could easily be found.--A nnual Report of Dr. S. Noy Scott, Medical O~cer of Health, Plymptou St. Ma~y. It was clear, therefore, that the middle ear had Ammal Report, ~9o9 , Ammal Report, ~9o9 ,
https://openalex.org/W4313179768	https://periodicos.ufmg.br/index.php/edrevista/article/download/36551/31584	Portuguese	null	DISABILITY, DIVERSITY, AND DIFFERENCE: IDIOSYNCRASIES AND CONCEPTUAL DIFFERENCES	Educação em Revista	2,022	cc-by	9,603	EDUR • Educação em Revista. 2022; 38:e36551 DOI: http://dx.doi.org/10.1590/0102-4698368536551 Preprint DOI: https://doi.org/10.1590/SciELOPreprints.3012 EDUR • Educação em Revista. 2022; 38:e36551 DOI: http://dx.doi.org/10.1590/0102-4698368536551 Preprint DOI: https://doi.org/10.1590/SciELOPreprints.3012 https://creativecommons.org/licenses/by/4.0/ JACKELINE SUSANN SOUZA DA SILVA1;2 https://orcid.org/0000-0002-9779-7663 JACKELINE SUSANN SOUZA DA SILVA1;2 https://orcid.org/0000-0002-9779-7663 JACKELINE SUSANN SOUZA DA SILVA1;2 https://orcid.org/0000-0002-9779-7663 1 Professora Substituta no Departamento de Habilitação Pedagógica da Universidade Federal da Paraíba (UFPB). João Pessoa, PB, Brasil. 2 <jackelinesusann@usal.es>. 1 Professora Substituta no Departamento de Habilitação Pedagógica da Universidade Federal da Paraíba (UFPB). João Pessoa, PB, Brasil. 2 <jackelinesusann@usal.es>. RESUMO: Desde 1980, a Organização das Nações Unidas (ONU) assume um papel preponderante na difusão de diretrizes contra exclusão, violência, discriminação e privação de direitos humanos e liberdades fundamentais. Diante disso, o marco político e legal brasileiro para a inclusão expandiu-se consideravelmente nas últimas décadas, trazendo regulamentações transversais em favor de grupos identitários específicos como pessoas com deficiências, mulheres e coletivos etnicorraciais. Assim, há o consenso público de que a inclusão é um princípio regulador das políticas nacionais. Embora sejam partes da pauta emergente, diante do complexo quadro de barreiras e exclusões sociais, os discursos oficiais em defesa da inclusão e diversidade apresentam idiossincrasias e contradições que, inclusive, camuflam intencionalidades políticas, muitas vezes, desconectadas das lutas dos coletivos que são focos das políticas de inclusão. Problematizando este contexto, o presente artigo tem como objetivo caracterizar diferenças conceituais entre deficiência, diversidade e diferença humana. Conclui-se que as definições, objetos de análise, ora alinham-se às perceptivas críticas do discurso, ora reproduzem a visão normativa de sujeitos- corpos-mentes, camuflando, de forma muitas vezes sutil, na retórica oficial sobre inclusão e diversidade, a construção social assimétrica das diferenças humanas. alavras-chave: Deficiência, Diversidade, Diferença Humana, Política de Inclusão. Palavras-chave: Deficiência, Diversidade, Diferença Humana, Política de Inclusão. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 DISABILITY, DIVERSITY, AND DIFFERENCE: IDIOSYNCRASIES AND CONCEPTUAL DIFFERENCES ABSTRACT: Since 1980, the United Nations (UN) has played a leading role in disseminating international guidelines against violence, discrimination, and deprivation of human rights and fundamental freedoms. Considering this, the Brazilian political and legal framework for inclusion has expanded considerably in recent decades, bringing transversal regulations in favor of specific identity groups, such as people with disabilities, women, and ethnic-racial collectives. Although they are part of the emerging agenda, when confronted by the complex framework of barriers and social exclusions, official inclusion and diversity discourses present idiosyncrasies and contradictions that can even camouflage political intentions, often disconnected from the struggles of the collectives focused by inclusion policies. In this context, this article aims to characterize conceptual differences between Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 disability, diversity, and human differences. We conclude that the definitions and objects of analysis sometimes align with the critical perceptions of the discourse, while at other times reproduce the normative view of subjects-body-minds, camouflaging, often subtly, the asymmetric social construction of human differences in official rhetoric about inclusion and diversity. Keywords: Disability, Diversity, Human Difference, Inclusion Policy. Palabras clave: Discapacidad, Diversidad, Diferencia humana, Política de inclusión. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 INTRODUÇÃO O objetivo do presente artigo é caracterizar diferenças conceituais entre deficiência, diversidade e diferença humana. Tais termos começam a ser incorporados nas agendas internacionais a partir da década de 1980 com a publicação de uma série de documentos pela ONU. Acompanhando a discussão sobre deficiência, diversidade e diferença, a inclusão social surge como tema transversal nas metas internacionais com o propósito de estabelecer acordo com Estados-Partes para o investimento em ações contra todas as formas de discriminação e exclusão social, incluindo a eliminação de barreiras no processo de escolarização de crianças e jovens em condição de deficiência e outras vulnerabilidades (ONU, 1990; 1994; 2006; 2015). Mais recentemente, a Agenda ONU 2030 reconhece a inclusão social nos Objetivos para o Desenvolvimento Sustentável-ODS (ONU, 2015-2030). Em específico, o princípio da inclusão compõe o ODS 10 para a redução da desigualdade em âmbito local e no contexto internacional: Objetivo 10. Redução das Desigualdades: [...] até 2030, empoderar e promover a inclusão social, econômica e política de todos, independentemente da idade, gênero, deficiência, raça, etnia, origem, religião, condição econômica ou outra. Garantir a igualdade de oportunidades e reduzir as desigualdades de resultados, inclusive por meio da eliminação de leis, políticas e práticas discriminatórias e da promoção de legislação, políticas e ações adequadas a este respeito (ONU, 2015, art. 10). Esta meta da ONU, para até 2030, reforça a necessidade dos países investirem em um projeto amplo de inclusão, focando a igualdade, sobretudo, em favor de grupos em condição de vulnerabilidade como crianças com deficiência, grupos étnicos-raciais e imigrantes (ONU, 2015). No âmbito da educação, a Agenda da ONU sinaliza para o investimento na melhoria das instituições educacionais para que sejam “apropriadas para crianças e sensíveis às deficiências e ao gênero, e que proporcionem ambientes de aprendizagem seguros e não violentos, inclusivos e eficazes para todos” (ONU, 2015, art. 4). Todos é abertura para que os outros, à margem, sejam unificados na mesma categoria com o intuito de atingir um propósito comum, neste caso, o acesso à educação. g p p O trecho da ONU “inclusivos e eficazes para todos” é ponto crucial para reflexão acerca do sentido da diversidade no discurso internacional sobre inclusão. Nas diretrizes internacionais, a inclusão é um conceito otimista que ganha sentido quando relacionado à diversidade humana (ONU, 2015; UNESCO, 2002; 2009). DISCAPACIDAD, DIVERSIDAD Y DIFERENCIA: IDIOSINCRASIAS Y DIVERGENCIAS CONCEPTUALES RESUMEN: Desde 1980, la Organización de las Naciones Unidas (ONU) ha desempeñado un papel central en la difusión de directrices contra la violencia, la discriminación y la privación de derechos humanos y libertades fundamentales. Frente a eso, el marco político y legal brasileño para la inclusión se ha ampliado considerablemente en las últimas décadas, y ha traído regulaciones transversales a favor de grupos identitários específicos como las personas con discapacidad, las mujeres y los colectivos étnico- raciales. Así, hay el consenso público de que la inclusión es un principio regulador de las políticas nacionales. Aunque sean partes de la pauta emergente frente a la compleja situación de barreras y exclusiones sociales, los discursos oficiales en defensa de la inclusión y de la diversidad presentan idiosincrasias y contradicciones que, incluso, camuflan intenciones políticas, muchas veces desconectadas de las luchas de los colectivos que son focos de las políticas de inclusión. En este contexto, este artículo tiene como objetivo caracterizar diferencias conceptuales entre discapacidad, diversidad y diferencia humana. Se concluye que las definiciones, objetos de análisis, a veces se alinean con las percepciones críticas del discurso, a veces reproducen la mirada normativa de sujetos-cuerpos-mentes, camuflando, de manera sutil, en la retórica oficial sobre inclusión y diversidad, la construcción social asimétrica de las diferencias humanas. Palabras clave: Discapacidad, Diversidad, Diferencia humana, Política de inclusión. Palabras clave: Discapacidad, Diversidad, Diferencia humana, Política de inclusión. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 INTRODUÇÃO Segundo Lopes (2004), a narrativa de diversidade é constituinte da ordem reguladora das relações humanas que exclui para incluir e inclui excluindo. Em outras palavras, mais do que naturalizar a diversidade humana como referência para as políticas de inclusão, é necessário pensá-la como parte da ordem social de organização e funcionamento das relações humanas frente à materialização da inclusão/exclusão, uma vez que [...] Inclusão e exclusão estão articuladas dentro de uma mesma matriz epistemológica, política, cultural e ideológica. Todo o espaço determinado por uma determinada ordem é delimitado e governado pela norma. Norma esta que classifica, compara, avalia, inclui e exclui. Toda a lei mantém aqueles que denominamos de excluídos fora de seu controle, pois não cabe a ela pensar o excluído, mas cabe prever o incluído (LOPES, 2004, p. 11). Com isso, é a pertença naturalizada de sujeito/grupo da cultura dominante no espaço social que cria o ponto de referência para a exclusão de características humanas não ou pouco aceitas na estrutura social. Neste caso, a subjetividade que molda o lugar da inclusão – ou mesmo aquele que já está incluído - é, inevitavelmente, o que forma a fronteira da não-pertença. Na análise de Foucault (1979), pode-se pensar a inclusão da diversidade pelo prisma do poder em que ninguém é propriamente o titular do poder, mas este é legitimado por uma direção, criando oposições, isto é, a dualidade de quem é o incluído e o excluído: não se sabe ao certo quem detém o poder, mas se sabe quem não o possui. Para Sawaia (2001), a exclusão é um processo complexo e multifacetado, com dimensões materiais, políticas, relacionais e subjetivas, no entanto, a exclusão não é uma oposição da inclusão, mas sim uma parte dela, constituindo-se assim em um: [...] processo sutil e dialético, pois só existe em relação à inclusão como parte constitutiva dela. Não é uma coisa ou um estado, é processo que envolve o indivíduo por inteiro e suas relações com os outros. Não tem única forma e não é uma falha no sistema, devendo ser combatida como algo que perturba a ordem social, ao contrário, ela é produto do funcionamento do sistema (SAWAIA, 2001, p. 9). Nas palavras de Sawaia (2001), a exclusão é parte do sistema, não sendo uma reação externa, mas uma das faces de determinada ordem social. INTRODUÇÃO Nesta lógica, uma sociedade somente torna-se inclusiva quando é tolerante e valoriza a diversidade humana, sendo, com isso, a inclusão e o respeito à diversidade propósitos interdependentes das nações e, em particular, das instituições escolares. No texto da Declaração Universal sobre Diversidade Cultural (UNESCO, 2002, Art. 1), o conceito de diversidade cultural é entendido como “patrimônio comum da humanidade” e caracteriza- se como “originalidade e pluralidade de identidades de grupos que compõem a humanidade”. Há, neste sentido, por parte dos órgãos das Nações Unidas a percepção horizontal das relações humanas, presentes, por exemplo, na definição de todos e humanidade (ONU, 2015-2030; UNESCO, 2002). O ponto de tensão desta retórica está na ideia de que a originalidade dos povos parte do pressuposto do essencialismo plural das identidades, isto é, o que é naturalmente original ou próprio das diversas identidades faz com que o todo da diversidade torne-se um dado fixo, natural e equiparado não, sendo portanto, questionado na sua construção social (WOODWARD, 2000). No entanto, quem, de fato, está circunscrito na noção de todos? A palavra todos, na retórica sobre diversidade induz, muitas vezes, ao simplismo discursivo de que somos todos humanos. Essa lógica opera, por exemplo, na contestação corriqueira de que é suficiente uma consciência humana em oposição ao Dia da Consciência Negra ou, igualmente, que o direito constitucional à educação de todos é suficiente para a garantia do acesso e da permanência de grupos específicos, sem que com isso haja a necessidade de criação de políticas afirmativas. O discursivo homogeneizador da diversidade acaba por reduzir a inclusão do outro nos mesmos sistemas normativos e, com isso, apaga os significados políticos das diferenças culturais (SKILIAR; SOUZA, 2000). Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 A noção de diversidade humana nas diretrizes internacionais para inclusão é mais do que um texto oficial, pois é contraponto para entender a subjetividade dos arranjos culturais e fronteiras humanas - ainda que essas últimas não sejam mencionadas diretamente na categoria todos. Segundo Lopes (2004), a narrativa de diversidade é constituinte da ordem reguladora das relações humanas que exclui para incluir e inclui excluindo. Em outras palavras, mais do que naturalizar a diversidade humana como referência para as políticas de inclusão, é necessário pensá-la como parte da ordem social de organização e funcionamento das relações humanas frente à materialização da inclusão/exclusão, uma vez que - ainda que essas últimas não sejam mencionadas diretamente na categoria todos. INTRODUÇÃO Portanto, a reivindicação por inclusão da diversidade é necessariamente indicativo de que há uma ordem normativa estabelecida. Especificamente, a dicotomia “inclusão versus exclusão” é o marco de referência para refletir sobre a experiência da deficiência. Neste caso, a supervalorização dos atributos da não-deficiência contribui para a noção de incapacidade relacionada às pessoas com deficiência e, consequentemente, para a exclusão desse grupo. Com isso, é o processo de legitimidade da não-deficiência (SILVA, 2019) que estabelece padrões aceitos de corponormatividade (MELLO; FERNANDES, 2017) e isso, forçosamente, designa a outra face da inclusão: os critérios subjetivos para a exclusão daquilo que enquadra-se em deficiência. j p q q q Prevendo isso, Hughes (1999) assume que é preciso desestabilizar a cultura dominante (lê- se cultura da não-deficiência), pois ela não é neutra e está, na verdade, relacionada a “uma mitologia da normalidade, da verdade, da beleza e da perfeição” (HUGHES, 1999, p.164). Pinto (2014) acrescenta que é fundamental perceber como a cultura vem construindo pressupostos que se assumem como naturais na forma do direito, mas que mantêm a distinção entre as pessoas com e sem deficiência (eles/as e nós). Assim, se por um lado, a diversidade é compreendida como abordagem positiva para o diálogo horizontal e apaziguador no discurso oficial em defesa da inclusão de todos (ONU, 1990; 1994; 2006; 2015; UNESCO, 2002; 2008); por outro, este mesmo conceito é analisado, na teoria crítica, como uma categoria que camufla o processo o embate entre culturas e as assimetrias entre diferenças humanas (SKILIAR; SOUZA, 2000; WOODWARD, 2000; SILVA, 2019). A respeito desse último aspecto, o campo dos Estudos da Deficiência constitui-se como vertente crítica para contestação das estruturas de desigualdades próprias das relações entre diferenças humanas, especificamente, quando tratam-se de marcadores sociais da deficiência e não-deficiência - muito além de parâmetros fixados na diversidade biológica (SILVA, 2019). Segundo Tom Shakespeare (1996), a teoria social coloca em evidência o dualismo entre biologia e sociedade para contestar o determinismo biológico das interpretações acerca do corpo-mente. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 Nos Estudos da Deficiência, a deficiência não é tomada como uma categoria fixa da diversidade humana em que os aspectos biológicos e naturais são as principais referências – tal como categoriza-se a diversidade entre diferentes espécies de animais ou plantas. Ao invés disso, a deficiência é compreendida no “mundo social; na vida […]. DEFINIÇÃO DA DEFICIÊNCIA NO MARCO POLÍTICO-LEGAL Esta seção tem como objetivo conceituar a deficiência nas diretrizes nacionais e internacionais. O critério de escolha dos documentos delimitados aqui foi o recorte do período pós Declaração de Salamanca (ONU, 1994), no qual, em virtude da influência dessa Declaração, houve uma ampla adoção de orientações em favor da inclusão educacional no Brasil, especificamente, focando o coletivo de pessoas com deficiência. Neste marco, a educação inclusiva fundamenta-se no Capítulo V da Lei de Diretrizes e Bases da Educação Nacional (LDB) n° 9394/1996; na Convenção Interamericana para a Eliminação de Todas as Formas de Discriminação, promulgada pelo Decreto brasileiro n° 3956/2001; no Decreto de Acessibilidade n° 5296/2004; na Convenção sobre os Direitos das Pessoas com Deficiência (ONU, 2006); na Política Nacional de Educação Especial na Perspectiva da Educação Inclusiva (MEC, 2008); no Relatório Mundial da Deficiência (OMS, 2001) e, mais recentemente, no Estatuto da Pessoa com Deficiência, regido pela Lei n° 13146/2015. Os documentos delimitados contemplam abordagens diferentes da deficiência que, em alguns momentos, aproximam-se do modelo médico e, em outros do modelo social (OLIVER, 1983). No entanto, há também definições que tentam equilibrar ambos modelos, tal como aparece no Relatório Mundial da Deficiência (OMS, 2010). Neste ponto, Carol Thomas (2008) destaca que, embora sejam inegáveis as contribuições do modelo social da deficiência, este modelo não pode anular o que ela denomina de “impairment effects”, isto é, os aspectos empíricos da vivência da deficiência que não se resumem às opressões próprias das barreiras sociais. Dessa maneira, nota-se que o conceito de deficiência não é consensual, existindo abordagens divergentes, assim como aproximadas. g g p Apesar disso, o modelo social da deficiência tem grande reconhecimento por parte de representantes do movimento internacional das pessoas com deficiência (ONU, 2006). Este modelo é estabelecido em oposição ao modelo médico. A principal crítica ao modelo médico da deficiência está no fato de que essa abordagem focaliza o problema na própria pessoa e, com isso, compreende a deficiência isoladamente (OLIVER, 1983), não considerando barreiras, discriminações e impedimentos sociais, sendo esses reais incapacitantes na experiência da deficiência (PINTO, 2014). No modelo médico, a segregação, a exclusão e as opressões que historicamente afetam as pessoas com deficiência acabam sendo justificadas pela condição individual e biológica deste grupo, não sendo, portanto, questionadas como produção social e cultural. INTRODUÇÃO A deficiência é um fenômeno sociocultural e pessoal corporificado de forma fisiológica ou psicológica” (GOODLEY, 2017, p. 1). Essa percepção da deficiência contextualiza a experiência social e evidencia outras marcas de identidades associadas às dimensões culturais, econômicas, geográficas, de gênero, de raça/etnia, de nacionalidade (BISH, 2015). É justamente para refletir acerca deste amplo debate que este artigo tem o intuito caracterizar variações conceituais entre deficiência, diversidade e diferença humana nas seções a seguir. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 DEFINIÇÃO DA DEFICIÊNCIA NO MARCO POLÍTICO-LEGAL ( ) No início do presente século, com a publicação das leis de acessibilidade – Lei n° 10.048/2000 que dá prioridade de atendimento às pessoas que específica e Lei n° 10.098/2000 que estabelece normas gerais e critérios básicos para a promoção da acessibilidade – o Brasil avança acerca da inclusão ao determinar modificações estruturais, focando a construção de ambientes acessíveis para as demandas de pessoas com deficiência e outros coletivos denominados de pessoas com mobilidade reduzida, sendo essas últimas qualquer indivíduo “que, não se enquadra no conceito de pessoa com deficiência, mas que tem, por qualquer motivo, dificuldade de movimentar-se, permanente ou temporariamente, gerando redução efetiva da mobilidade, flexibilidade, coordenação motora e percepção” (BRASIL, 2004, art. 5°). O avanço na legislação está no fato de que antes era a pessoa com deficiência que deveria adaptar-se à sociedade ou este grupo era excluído totalmente da experiência social. Entretanto, com publicação das leis de acessibilidade (BRASIL, 2000; 2004), é a estrutura social que deve ser modificada para incluir pessoas com deficiência e com mobilidade reduzida. p p O Decreto n° 5296/2004 traz definições detalhadas sobre o significado da deficiência, caracterizando esse grupo como aquelas pessoas que possuem limitação ou incapacidade para o desempenho de atividade e enquadram-se nas seguintes categorias: [...] Deficiência física: alteração completa ou parcial de um ou mais segmentos do corpo humano, acarretando o comprometimento da função física, apresentando-se sob a forma de paraplegia, paraparesia, monoplegia, monoparesia, tetraplegia, tetraparesia, triplegia, triparesia, hemiplegia, hemiparesia, ostomia, amputação ou ausência de membro, paralisia cerebral, nanismo, membros com deformidade congênita ou adquirida, exceto as deformidades estéticas e as que não produzam dificuldades para o desempenho de funções. Deficiência auditiva: perda bilateral, parcial ou total, de quarenta e um decibéis (dB) ou mais, aferida por audiograma nas frequências de 500Hz, 1.000Hz, 2.000Hz e 3.000Hz. Deficiência visual: cegueira, na qual a acuidade visual é igual ou menor que 0,05 no melhor olho, com a melhor correção óptica; a baixa visão, que significa acuidade visual entre 0,3 e 0,05 no melhor olho, com a melhor correção óptica; os casos nos quais a somatória da medida do campo visual em ambos os olhos for igual ou menor que 60o; ou a ocorrência simultânea de quaisquer das condições anteriores. Deficiência mental: Funcionamento intelectual significativamente inferior à média, com manifestação antes dos dezoito anos e limitações associadas a duas ou mais áreas de habilidades adaptativas, tais como: 1. DEFINIÇÃO DA DEFICIÊNCIA NO MARCO POLÍTICO-LEGAL Em oposição, o modelo social rejeita a perspectiva de culpabilização do indivíduo com deficiência em virtude da sua condição físico-cognitiva e a justificativa do recorte social deste grupo em razão da discriminação das suas mentes-corpos. Este modelo transfere a responsabilidade de supressão das barreiras para a sociedade: [...] As dificuldades e a marginalização experimentadas por tantas pessoas com deficiência não são mais explicadas com base nas suas incapacidades individuais ou em patologias do foro biológico, mas pela incapacidade da sociedade responder adequadamente às suas necessidades e características diversas. Por outras palavras, tal como a classe social, o gênero, ou a pertença étnica, a experiência da deficiência é reinterpretada como sendo mais pela incapacidade da sociedade responder adequadamente às suas necessidades e características diversas (PINTO, 2014, p. 12). Como afirma Pinto (2014), a deficiência é uma questão social, não sendo uma característica restritamente biológica. É importante destacar que, no âmbito dos Direitos Humanos – embora seja reconhecida as diferenças identitárias entre próprias pessoas com deficiência (ONU, 2006) –, a conceituação da deficiência, como categoria unitária, representa a demarcação da história de luta social e de (auto)afirmação dos direitos, das liberdades fundamentais e das contribuições coletivas deste grupo no percurso da humanidade (SILVA, 2014). A (auto)afirmação da identidade da deficiência representa, portanto, um processo de reconhecimento coletivo e visibilidade social (FERREIRA, 2004; SOARES, 2010). ) Com ênfase ao reconhecimento dos direitos das pessoas com deficiência, a LDB (BRASIL, 1996) determina que é dever do Estado garantir a matrícula das pessoas com deficiência no sistema de ensino regular, bem como facilitar a oferta do Atendimento Educacional Especializado (AEE) - serviço transversal a todos os níveis e as modalidades de ensino (BRASIL, 1996, art. 4°). Os critérios para a definição do perfil de estudantes-públicos da inclusão, nas determinações legais brasileiras, alinham-se às orientações internacionais, como a Convenção de Guatemala (OEA, 1999). Esta Convenção reafirma os direitos humanos e liberdades das pessoas com deficiência e, principalmente, assume o compromisso de eliminar e prevenir todas as formas de discriminação em razão de deficiência. O artigo primeiro deste documento define deficiência como uma condição individual influenciada por fatores externos, sendo, portanto, uma “restrição física, mental ou sensorial, de natureza permanente ou transitória, que limita a capacidade de exercer uma ou mais atividades essenciais da vida diária, causada ou agravada pelo ambiente econômico e social” (OEA, 1999, art. 1°). Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 DEFINIÇÃO DA DEFICIÊNCIA NO MARCO POLÍTICO-LEGAL Comunicação; 2. Cuidado pessoal; 3. Habilidades sociais; 4. Utilização dos recursos da comunidade; 5. Saúde e segurança; 6. Habilidades acadêmicas; 7. Lazer; e 8. Trabalho. Deficiência múltipla: associação de duas ou mais deficiências (BRASIL, Decreto n° 5296/2004, art. 5°). Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 Embora haja a mudança de foco, com a publicação da legislação de acessibilidade, que passa do ajuste/exclusão/segregação da pessoa com deficiência (visibilidade da condição individual da deficiência) à transformação do ambiente de modo acessível (visibilidade das barreiras externas), o detalhamento das categorias da deficiência extraídas, anteriormente, do Decreto de Acessibilidade (BRASIL, 2004) remete ao modelo médico. No mencionado decreto há o enquadramento físico, cognitivo, comportamental e comunicacional aos padrões de percepção corporal, visual, auditiva e intelectual (FERREIRA, 2004). Esse modelo é útil para a conceituação no campo biológico, no entanto, traz forte influência para a naturalização de estereótipos limitantes e essencialistas sobre as pessoas com deficiência, principalmente, aquelas pessoas definidas pela categoria de deficiência mental, particularmente, porque o texto do decreto afirma que tais pessoas são caracterizadas “pelo funcionamento intelectual significativamente inferior à média” (Decreto n° 5296/2004, art. 5°), sendo a ideia de sujeito médio o suposto padrão de normalidade. Justamente, em oposição às formas diretas e sutis de discriminação contra as pessoas com deficiência que a Convenção sobre os Direitos das Pessoas com Deficiência (ONU, 2006, p. 1) reconhece o conceito deficiência como uma definição “em evolução”, uma vez que tal condição é parte da diversidade humana e resulta “da interação entre pessoas com deficiência e as barreiras devidas às atitudes e ao ambiente que impedem a plena e efetiva participação dessas pessoas na sociedade em igualdade de oportunidades com as demais pessoas” (ONU, 2006, p. 22). A Convenção fundamenta seu conteúdo nos direitos humanos e por isso agrega princípios importantes para a definição de deficiência como barreiras atitudinais, participação social e igualdade de oportunidade (ONU, 2006). No texto da Convenção (ONU, 2006), pessoas com deficiência são aquelas com impedimento “físico, mental, intelectual ou sensorial, os quais em interação com diversas barreiras, obstruem a sua participação plena e efetiva na sociedade em igualdades de condições com as demais pessoas” (ONU, 2006, art. 1). Desta forma, a condição de deficiência caracteriza-se na relação da pessoa com os ambientes e os demais; interação esta que pode favorecer ou não o usufruto pleno dos direitos humanos e liberdades fundamentais. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 DEFINIÇÃO DA DEFICIÊNCIA NO MARCO POLÍTICO-LEGAL A OMS (2011) reconhece as barreiras nos ambientes, nos contextos e nas relações como fatores agravantes na experiência de interação da pessoa com deficiência. Nesse marco de referência, a ideia de deficiência é construída a partir de três fatores principais: “a) alterações das estruturas/funções corporais, por exemplo, paralisia ou cegueira; b) dificuldades para executar atividades, por exemplo, caminhar ou comer e c) restrições à participação em atividades, por exemplo, enfrentar discriminação no emprego ou nos transportes” (OMS, 2011, p. 5). Nesse marco de referência, a ideia de deficiência é construída a partir de três fatores principais: “a) alterações das estruturas/funções corporais, por exemplo, paralisia ou cegueira; b) dificuldades para executar atividades, por exemplo, caminhar ou comer e c) restrições à participação em atividades, por exemplo, enfrentar discriminação no emprego ou nos transportes” (OMS, 2011, p. 5). O esquema, a seguir, mostra como o conceito de deficiência é definido pela OMS (2011): O esquema, a seguir, mostra como o conceito de deficiência é definido pela O Figura 1. Representação da Deficiência na CIFS (OMS, 2011) Figura 1. Representação da Deficiência na CIFS (OMS, 2011) Chama atenção que a “atividade” está no centro da representação e não a deficiência ou condição cognitivo-corporal. O esquema demonstra que para medir a execução da atividade examina-se, de forma conjunta, as funções corporais e as condições de participação. Esse processo de mensuração envolve aspectos de saúde, fatores ambientais (barreiras) e pessoais (família, renda, acesso à tecnologia assistiva, entre outros). As variações de dados coletados são a base para classificação da deficiência, tornando-se indispensável a análise interdisciplinar e contextual da experiência da deficiência. p p p Vale destacar que a OMS cita a CIFS como parâmetro para mensurar elementos positivos da experiência no uso das funcionalidades humanas para, assim, criar novos marcos de referências acerca das “funções corporais, das atividades, da participação e facilitação ambiental” (OMS, 2011, p. 5). O Relatório Mundial da Deficiência (OMS, 2011) reconhece os diversos ensejos que estão por trás dos discursos sobre deficiência que os aproximam da área de Saúde e, ao mesmo tempo, de âmbitos variados da vida humana, incluindo os fatores individuais, familiares e sociais. O mesmo relatório faz alusão ao termo “diminuição de capacidade” como resultado das condições pessoais e das limitações de saúde, aliadas aos fatores externos: “diminuições de capacidade são diminuições específicas das funções e estruturas corporais, geralmente identificadas como sintomas ou sinais de problemas de saúde. DEFINIÇÃO DA DEFICIÊNCIA NO MARCO POLÍTICO-LEGAL Quase dois anos após a publicação da Convenção da ONU (2006), o Ministério da Educação lançou a Política Nacional de Educação Especial na Perspectiva da Educação Inclusiva (PNEEpEI) com o objetivo assegurar a inclusão escolar de alunos com deficiência, transtornos globais do desenvolvimento e altas habilidades/superdotação, orientando os sistemas de ensino para garantia do acesso ao ensino regular, bem como a participação e a aprendizagem (BRASIL, 2008). Com a implementação desta política, as escolas receberam do MEC e das Secretarias Municipais e Estaduais uma série de orientações acerca da oferta do AEE e da abertura das Salas de Recursos Multifuncionais no sistema de ensino regular. A Política de Inclusão (BRASIL, 2008) apresenta definições acerca do público alvo do AEE, acrescentando parâmetros para as deficiências, os transtornos globais do desenvolvimento e as altas habilidades e superdotação: [...] consideram-se alunos com deficiência àqueles que têm impedimentos de longo prazo, de natureza física, mental, intelectual ou sensorial, que em interação com diversas barreiras podem ter restringida sua participação plena e efetiva na escola e na sociedade. Os estudantes com transtornos globais do desenvolvimento são aqueles que apresentam alterações qualitativas das interações sociais recíprocas e na comunicação, um repertório de interesses e atividades restrito, estereotipado e repetitivo. Incluem-se nesse grupo estudantes com autismo, síndromes do espectro do autismo e psicose infantil. Estudantes com altas habilidades/superdotação demonstram potencial elevado em qualquer uma das seguintes áreas, isoladas ou combinadas: intelectual, acadêmica, liderança, psicomotricidade e artes, além de apresentar grande criatividade, envolvimento na aprendizagem e realização de tarefas em áreas de seu interesse (BRASIL, 2008, p. 11). A definição de deficiência da PNEEpEI (BRASIL, 2008) retoma o texto da Convenção da ONU (2006) ao reconhecer as características corporais e cognitivas da deficiência no contexto da interação com barreiras sociais. Além disso, o texto desta política incorpora outros coletivos como público-foco da política de inclusão, como os estudantes com altas habilidades e superdotação, que não necessariamente, são pertencentes à categoria deficiência. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 No marco das diretrizes internacionais, o Relatório Mundial da Deficiência (OMS, 2011), documento elaborado sob coordenação da Organização Mundial da Saúde (OMS), tornou-se referência nacional para categorização das deficiências na Classificação Internacional de Funcionalidade, Incapacidade e Saúde (CIFS). A CIFS foi desenvolvida por profissionais de diversas áreas entre eles, acadêmicos, médicos e técnicos com deficiência. A conceituação de deficiência parte da junção do modelo médico e o modelo social. A RETÓRICA DA DIVERSIDADE NA PAUTA SOBRE INCLUSÃO Como apresentado na seção introdutória deste artigo, o tema da diversidade humana é parte das diretrizes internacionais (ONU, 1948; 1990; 1994; OEA, 1999). A partir das mudanças globais com o pós Segunda Guerra, o discurso sobre tolerância à diversidade e respeito às diferenças ganha visibilidade, tal como aparece na Declaração Universal dos Direitos Humanos (ONU, 1948): [...] Todos os seres humanos podem invocar os direitos e as liberdades proclamados na presente Declaração, sem distinção alguma, nomeadamente de raça, de cor, de sexo, de língua, de religião, de opinião política ou outra, de origem nacional ou social, de fortuna, de nascimento ou de qualquer outra situação. Além disso, não será feita nenhuma distinção fundada no estatuto político, jurídico ou internacional do país ou do território da naturalidade da pessoa, seja esse país ou território independente, sob tutela, autônomo ou sujeito a alguma limitação de soberania (ONU, 1948, art. 2°). A garantia dos direitos humanos e das liberdades fundamentais (ONU, 1948) estão na base da discussão sobre diversidade. Reivindicações coletivas por direitos e inclusão revelam tensões entre diferentes grupos seja, por exemplo, em razão étnica, geracional, de gênero, de deficiência e de nacionalidade. O acirramento entre grupos identitários indica a segmentação plural das relações sociais, antes caracterizada, na teoria moderna, apenas em dois grandes grupos: burguesia e povo. No contexto atual, existe a percepção de um quadro muito mais complexo, de multiplicidades humanas e de fronteiras socioculturais, nas quais as diferenças são interseccionais e tocam em outros fatores além da questão econômica como o gênero, a deficiência e a cor da pele (HALL, 1997). A expectativa acerca da redução de conflitos entre diferentes grupos sociais é fundamento para a celebração da diversidade humana na busca por uma cultura de paz mundial (ONU, 1948). Essa positividade na afirmação da diversidade está presente, sistematicamente, nos discursos oficiais e por isso tornou-se objeto de análise da teoria crítica. Tais teóricos desmontam o simplismo por trás do discurso de tolerância à diversidade, pois na verdade, essa retórica camufla o real projeto para os grupos excluídos e discriminados na sua inclusão em demandas geradas pela era da globalização (FERREIRA, 2015). DEFINIÇÃO DA DEFICIÊNCIA NO MARCO POLÍTICO-LEGAL A deficiência surge da interação entre problemas de saúde e fatores contextuais” (OMS, 2011, p. 5). Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 Por fim, o Estatuto da Pessoa com Deficiência (BRASIL, 2015), em vigor no Brasil desde 2015, reforça o modelo social da deficiência ao enfatizar as necessidade de eliminação de barreiras e de promoção da participação plena das pessoas com deficiência em igualdade de condições com as demais (BRASIL, 2015, art. 2°). O artigo dois, da mesma lei, destaca que a avaliação da deficiência, quando necessária, “será biopsicossocial realizada por equipe multiprofissional e interdisciplinar, considerando: os impedimentos nas funções e nas estruturas do corpo; os fatores socioambientais, psicológicos e pessoais; a limitação no desempenho de atividades e a restrição de participação” (BRASIL, 2015, art. 2°). Este trecho vai de encontro com o Relatório da Deficiência da OMS (2011) ao direcionar o modelo biopsicossocial como parâmetro para medir a deficiência. A conciliação entre modelo médico e modelo social no modelo biopsicossocial é uma tentativa de trazer a dimensão sociológica da experiência da deficiência sem esquecer dos aspectos que estão implicados no corpo ou “lesão” (impairment) (THOMAS, 2007). Mas, será que essa conciliação entre os modelos da deficiência é suficiente para resolver o problema da normalidade? Com propósito de aproximação com este debate, as próximas seções abordam a deficiência nas noções de diversidade humana e diferenças culturais, presentes na retórica internacional e nas teorias críticas. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 A RETÓRICA DA DIVERSIDADE NA PAUTA SOBRE INCLUSÃO Segundo Ferreira (2015), o conceito de diversidade solidificou-se no contexto da globalização e sobressai a própria luta dos movimentos sociais, pois não é apenas uma retórica de interesse dos grupos excluídos, mas sim dos grandes representantes do poder econômico mundial: O processo da globalização da economia, impulsionado pela revolução tecnológica que se iniciou no século passado, ganha força no século XXI com a realização de grandes eventos mundiais, cujos encontros as comunidades política, econômica e acadêmica internacionais com poder se reúnem para definir movimentos de incorporação de grandes massas humanas [...] em suas agendas econômicas, políticas e também educacionais, porque as massas devem ser preparadas para responder às demandas geradas pela era da informação, da globalização e da sociedade do conhecimento (FERREIRA, 2015, p. 301). No sentido expresso pela autora, percebe-se que o conceito de diversidade alicerça-se nos interesses globais do neoliberalismo em um movimento de apropriação das pautas reivindicadas por determinados coletivos. Para Ferreira (2015, p. 305), o emergente uso do termo diversidade nas políticas e leis formuladas tanto Brasil como em âmbito internacional assume a retórica do compromisso com as Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 “massas”, mas há a tendência do esvaziamento do termo diversidade e ainda o processo de homogeneização das diferenças humanas para responder aos interesses econômicos. A autora chama atenção a sintonia conceitual entre diversidade e outros termos como inclusão, tolerância, aceitação, igualdade e liberdade; palavras que tornaram-se princípios norteadores para elaboração de materiais didáticos, bases curriculares e planos institucionais (FERREIRA, 2015, p. 305). Deste modo, o tema da diversidade é compulsoriamente introduzido na política e práticas educacionais. Nesta linha, a Declaração de Salamanca (ONU, 1994) foi um dos documentos importantes na consolidação de políticas para a diversidade porque trouxe o princípio da inclusão associado aos sistemas de ensino comum a partir da ênfase no direito à educação dos estudantes que apresentam algum tipo de necessidade educacional que são [...] deficientes e superdotados, de rua e que trabalham, de origem remota ou de população nômade, pertencentes a minorias linguísticas, étnicas ou culturais, e de outros grupos desavantajados ou marginalizados (...). Muitas crianças experimentam dificuldades de aprendizagem e portanto possuem necessidades educacionais especiais em algum ponto durante a sua escolarização (ONU, 1994, p. 3). A RETÓRICA DA DIVERSIDADE NA PAUTA SOBRE INCLUSÃO Ao abranger as diversidades de características e condições linguísticas, étnicas, culturais, socioeconômicas e habilidades cognitivas, a Declaração de Salamanca (ONU, 1994) chama atenção às diferenças humanas e às desigualdades que são geradas pelos sistemas educacionais, principalmente, devido à adoção de um modelo de pedagogia instrumental. No que se refere às características humanas, este documento menciona, como necessidade educacional, as características físicas, sociais ou culturais que marcam os estudantes e os colocam em condição de vulnerabilidade (ONU, 1994, p. 3). O conceito de diversidade humana é compreendido na Declaração, portanto, por dimensões biológicas e na sua construção cultural. Gomes (2007) diferencia essas dimensões como: [...] a diversidade biológica, no caso dos seres humanos, caracteriza-se pela igualdade em nossa condição de gênero humano que possui diferenças, a diversidade cultural está representada exatamente nessas diferenças que são modeladas no processo histórico e cultural e no contexto das relações de poder [e que] recebem leituras estereotipadas e preconceituosas, passando a ser tratadas desigualmente e de forma discriminatória (GOMES, 2007, p. 17). Diferentemente da dimensão do poder expressa por Gomes (2007), ao analisar a diversidade cultural, o termo diversidade biológica sempre “insinua” o reconhecimento da multicultura e o respeito ao pluralismo. No entanto, este mesmo termo é direcionado para aqueles que são vulneráveis à experiência de exclusão e discriminação e não para os que não os são. No caso, o coletivo da diversidade é aquele que é identificado com uma desvantajosa diferença. As teorias críticas (SKLIAR, 2000; SHAKESPEARE, 1996; WOODWARD, 2000) – no debate acerca do processo de construção de identidades, coletividades e diferenças – analisam o processo de naturalização e despolitização do conceito de diversidade. Nesta abordagem teórica, desloca-se tanto os sujeitos taxados como diferentes (os coletivos que compõem a diversidade e as chamadas “minorias”) como também aqueles que permanecem intocáveis, ou seja, os sujeitos que atendem à norma. A pergunta é: “Como refletir sobre surdos, indígenas, mulheres, negros, mestiços, desempregados, crianças [em situação] de rua, cegos etc. sem se esconder atrás da máscara da terminologia de natural pluralidade e da natural diversidade dos outros?” (SKLIAR, 2000, p. 1). No âmbito da Educação Especial, crianças, jovens e adultos com deficiência, tradicionalmente, foram rotulados por nomenclaturas específicas, como ‘especiais’, ‘excepcionais’ e ‘deficientes’. Agora, essas terminologias foram substituídas pelo termo diversidade ou estudantes públicos do AEE. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 As normas e os valores sobre corpos e mentes completos, autossuficientes, disciplinados e belos, constituem a base dos discursos, das práticas e da organização das instituições especiais. Em geral, a norma tende a ser implícita, invisível e é esse caráter de invisibilidade que a torna inquestionada. Nos documentos oficiais e nos discursos das instituições da educação especial, é frequente encontrar a utilização do termo diversidade. Diversidade neste e em outros contextos A TEORIZAÇÃO SOBRE DIFERENÇA HUMANA PARA DESLOCAR A NORMALIDADE Três abordagens são predominantes na conceituação da deficiência: o modelo médico que foca a deficiência em características físico-cognitivas (FERREIRA, 2004); o modelo social, no qual a deficiência é entendida como experiência associada às barreiras e aos impedimentos impostos pela sociedade (OLIVER, 1983) e o modelo biopsicossocial que tenta equilibrar informações no âmbito da Saúde e da funcionalidade humana e aspectos da experiência empírica da deficiência (OMS, 2011). Seguindo o fluxo das diretrizes internacionais, o marco político-legal nacional situa esses modelos, mas não apresenta retórica única para definir a categoria deficiência, pois em alguns textos a conceitua dentro de especificações médicas, como no Decreto de Acessibilidade n° 5296/2004 e, em outros momentos, busca-se interpretação no modelo social ou no modelo biopsicossocial, tal como aparece na Lei Brasileira de Inclusão (BRASIL, 2015). ( ) Além desses modelos, as diretrizes para inclusão trazem a tolerância e o respeito à diversidade como princípios-chaves para a formulação de políticas nacionais (ONU, 2015-2030), sendo as pessoas com deficiência grupo-foco da denominada diversidade humana. No âmbito global, o termo diversidade surge como alternativa para os Estados resolverem o problema social da exclusão e da intolerância contra grupos específicos e, ao mesmo tempo, unificarem as massas para responderem às exigências neoliberais, sem que isso altere os sistemas de dominância (FERREIRA, 2015; SKLIAR; SOUZA, 2000). Nesta conjuntura, uma terceira via de análise surge para a teorização sobre deficiência: a teoria crítica da diferença presente, por exemplo, nos Estudos Culturais e nos Estudos de Gênero (SILVA, 2000; WOODWARD, 2000; PINTO 2014; FARIAS, 2011; SOARES, 2010; MELLO, FERNANDES, 2013; SILVA, 2014). Nesse viés, a desconstrução da normalidade é o ponto de partida para a reflexão sobre diferenças humanas e identidades. As análises críticas do discurso contestam até que ponto as características biológicas servem de base para justificar valores culturais e reforçar desigualdades humanas (WOODWARD, 2000; SKLIAR, 2000). Segundo Skliar e Souza (2000), avalia- se, no discurso crítico, sobre a deficiência que [...] não é a pessoa que está em uma cadeira de roda ou que usa um aparelho auditivo ou que não aprende segundo o ritmo e a forma como a norma espera, se não os processos sociais, históricos, econômicos e culturais que regulam e controlam a forma acerca de como são pensados e inventados os corpos e mentes dos outros. Para explicá-lo mais detalhadamente: a deficiência não é uma questão biológica e sim uma retórica social, histórica e cultural. A RETÓRICA DA DIVERSIDADE NA PAUTA SOBRE INCLUSÃO O que parece um avanço ainda esconde os pressupostos que fundamentam a antiga ideologia da normalidade, conforme dito nas palavras de Skliar e Souza (2000): mais amplos, retrata uma estratégia conservadora que contêm, obscurece, o significado político das diferenças culturais. A ambiguidade com que se pensa e se constrói a diversidade, gera consequência, no melhor dos casos, a aceitação de um certo pluralismo que se refere sempre a uma norma ideal (SKLIAR; SOUZA, 2000, p. 7). mais amplos, retrata uma estratégia conservadora que contêm, obscurece, o significado político das diferenças culturais. A ambiguidade com que se pensa e se constrói a diversidade, gera consequência, no melhor dos casos, a aceitação de um certo pluralismo que se refere sempre a uma norma ideal (SKLIAR; SOUZA, 2000, p. 7). Desta forma, o conceito de diversidade é construído para os desajustados, para aquelas pessoas e grupos que são “considerados “diferentes” daquilo que a classe dominante define como “padrão”: uma referência “certa” que passa a ser naturalizada nas relações sociais” (FERREIRA, 2015, p. 307). Nesta lógica, resta para os que não são diferentes a perpetuação dos valores que compõem seu domínio e privilégio. Skliar e Souza (2000) argumentam que o discurso sobre a tolerância à diversidade tende a dissimular o que é político na diferença e não resolve o problema gerador da exclusão e discriminação, ao contrário, mantêm a norma em seu lugar. Neste sentido, a diferença passa a ser definida “como diversidade que é entendida quase sempre como as variantes aceitáveis e respeitáveis do projeto hegemônico da normalidade” (SKLIAR; SOUZA, 2000, p. 6-7). Segundo Ferreira (2015), a maneira como o termo diversidade é exaltado nos discursos oficiais não impulsiona mudanças necessárias para a qualidade da educação brasileira e ainda mantêm “aqueles que são chamados de “diferentes” em um vácuo teórico-conceitual” (FERREIRA, 2015, p. 306). Por essa razão, o conceito de diferença humana é indispensável na análise sobre a narrativa da inclusão e diversidade. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 A TEORIZAÇÃO SOBRE DIFERENÇA HUMANA PARA DESLOCAR A NORMALIDADE A deficiência não é um problema dos deficientes ou de suas famílias ou dos especialistas. A deficiência está relacionada a própria ideia de normalidade e com sua historicidade (SKLIAR; SOUZA, 2000 p. 5). deficiência está relacionada a própria ideia de normalidade e com sua historicidade (SKLIAR; SOUZA, 2000 p. 5). Portanto, a deficiência não é uma questão reduzida ao âmbito da medicina. Muito mais do que isso, é uma narrativa de ordem coletiva que tem mais relação com o que é estabelecido como normal do que com o que pressupõe-se como anormalidade. Analisando esses aspectos, Pinto (2014) afirma que a demarcação do que é deficiência existe dentro de sistemas de representação que valorizam determinados atributos físicos, classificando, definindo e separando pessoas e grupos. Do mesmo modo, Woodward (2000) argumenta que o corpo é um dos locais em que são estabelecidas fronteiras ou aproximações a partir do processo de (auto)identificação de diferenças e semelhanças humanas. Tais diferenças são o que constituem as identidades. Em outras palavras, a identidade só existe porque há uma diferença contrastante. Por isso, recorre-se a possível essencialismo biológico ou histórico para justificar e agrupar os sujeitos considerados semelhantes e distinguir aqueles julgados na diferença. Na abordagem crítica pós-estrutural do discurso, o conceito de diferença parte da noção de que é preciso desnaturalizar qualquer característica humana dita como biológica e essencialista para encontrar as normas sociais que as governam, uma vez que “as diferenças empíricas – como a cor da pele ou a surdez – não possuem, em si mesmas, nenhum valor natural. Não carregam qualquer sentido intrínseco”, no entanto, “recebem significados como efeito das relações históricas e políticas que se articulam nas sociedades onde estas marcas de diferenças estão presentes” (CANGUILHEM, 1995, p. 186). Deste modo, são as representações sociais formadas por discursos, símbolos e práticas de legitimação e significação que fazem com que o indivíduo, por meio de um processo psicossocial, atribua significado a experiência e posicione-se, sendo possível, portanto, encontrar respostas para questões como: Quem sou? De onde sou? Quem é diferente? Quem é igual? (WOODWARD, 2000; SILVA, 2000). ) Assim, é a partir do significado das representações de ser diferente e ser igual que a identidade vai constituindo-se na arena cultural. Neste caso, aprendemos, ao longo da vida, a ser quem somos e, nesse sentido, a diferença do outro é base da nossa identidade (SILVA, 2000). Conclui-se, assim, que não existe identidade sem diferença. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 CONSIDERAÇÕES FINAIS Este artigo teve como objetivo caracterizar diferenças conceituais entre deficiência, diversidade e diferença humana. Nota-se que as definições, objetos de análise, ora alinham-se às perceptivas críticas do discurso, ora reproduzem a visão normativa de sujeitos-corpos-mentes, camuflando, de forma muitas vezes sutil, na retórica oficial sobre inclusão e diversidade, a construção social assimétrica das diferenças humanas. Em resumo, examinou-se que a definição de deficiência não é unitária tanto nas diretrizes nacionais e internacionais como na abordagem da teoria crítica social. Entre os modelos da deficiência disseminados está o modelo médico em que a deficiência é tomada como objeto das Ciências da Saúde e as características físico-cognitivas do indivíduo são deslocadas do contexto social, sendo interpretadas como um dado “natural”. Em oposição, o modelo social tornou-se uma segunda referência para conceituação da deficiência, muito além da marca biológica, sendo um fenômeno coletivo (FERREIRA, 2004). O modelo social denuncia as diferentes barreiras que impedem o usufruto, das pessoas com deficiência, dos direitos humanos e das liberdades fundamentais (ONU, 2006). Mais recentemente, com a publicação da Lei Brasileira de Inclusão (BRASIL, 2015), a retórica oficial brasileira alinha-se a tentativa de conciliação entre a perspectiva médica e sociológica da deficiência na adoção do modelo biopsicossocial. Essa última abordagem aproxima-se daquilo que Carol Thomas (2008) reconhece como aspectos empíricos da vivência da deficiência - que não são diretamente barreiras sociais, mas revelam as dimensões pragmáticas e particulares da experiência da deficiência. Com a disseminação de documentos, por órgãos das Nações Unidas, em defesa da tolerância e do respeito à diversidade houve o crescimento expressivo do marco político-legal para a educação inclusiva no Brasil (BRASIL, 1996; 2000; 2004; 2008; 2015). Para a UNESCO (2002), o conceito de diversidade é entendido como patrimônio da humanidade e compõe o que é original e plural nas identidades humanas. Embora o reconhecimento do valor da diversidade humana induza a positividade no discurso oficial, esta narrativa transmite a ideia ilusória de que há simetria entre grupos sociais e que, supostamente, a ampla tolerância é suficiente para romper as tensões culturais e promover a paz mundial. Além disso, a defesa da diversidade na sua forma essencialista e naturalizante mantêm o status quo e o projeto de normalidade que tanto imagina-se combater (SKLIAR; SOUZA, 2000). A teoria crítica da diferença contraria a noção simplista da diversidade para desestabilizar a normalidade. A TEORIZAÇÃO SOBRE DIFERENÇA HUMANA PARA DESLOCAR A NORMALIDADE Somos quem somos porque nos vemos pelo reflexo de quem não nos representa e de quem julgamos ser parecidos com nós. Este jogo de aproximação e exclusão do outro é marcado pela relação de alteridade: [...] a alteridade permanece reabsorvida em nossa identidade e a reforça ainda mais; a faz possível, mais arrogante, mais segura e mais satisfeita de si mesma. A partir do ponto de vista, o louco confirma e reforça nossa razão; a criança nossa maturidade; o selvagem, nossa civilização; o marginal, nossa integração; o estrangeiro, nosso país e o deficiente, nossa normalidade (LARROSA; PEREZ, 1998, p. 3). A questão é que no processo de alteridade para construção de identidades criam-se normas, estigmas e desigualdades (WOODWARD, 2000). Por exemplo, no caso das pessoas com deficiência, a sociedade considera “determinados corpos como inferiores, incompletos ou passíveis de reparação/reabilitação quando comparados aos padrões hegemônicos funcionais/corporais” (MELLO; FERNANDES, 2013). Na sociedade, valorizam-se determinadas formas físicas em detrimento de outras a partir de padrões sociais de funcionalidade, comunicação, estética, gênero, atividade e cognição (SILVA, 2014). Consequentemente, isso faz com que a pessoa com um corpo marcado por algum tipo de deficiência seja estigmatizada e colocada em posição de desvantagem social. Tal experiência coloca a pessoa estigmatizada como sujeito desviante da norma(lidade) que ao se sentir “estranho a si mesmo” abre margem à “dominação que se articula ideológica e politicamente através do enfraquecimento das coletividades, de inferiorização, da repressão dirigida, do divisionismo para que deixem de questionar a ordem hegemônica e até passando a identificar-se com ela” (SKLIAR; SOUZA, 2000, p. 3). Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 g p ( p ) Portanto, as representações de identidade e diferenças humanas criam fronteiras entre as ditas pessoas com deficiência e sem deficiência. No processo de socialização, as pessoas apropriam-se do lugar a elas direcionado e aprendem a falar sobre si, a defender ou resistir a sua identidade. Qualquer discurso que constitua os modelos da deficiência estão embasados em sistemas de representação (WOODWARD, 2000). Assim, tanto o modelo médico como o modelo social – bem como o modelo biopsicossocial – são operantes nas dinâmicas da normalidade que só ganham sentido nos arranjos culturais em que são construídos. A TEORIZAÇÃO SOBRE DIFERENÇA HUMANA PARA DESLOCAR A NORMALIDADE A depender do que é legitimado na coletividade, os modelos da deficiência podem impor estereótipos limitantes, intensificar fronteiras ou, de outra forma, reconstruir significados sociais e novas representações que sejam mais benéficas às pessoas com deficiência no processo involuntário de diferenciação e aproximação na constituição das identidades. Educação em Revista\|Belo Horizonte\|v.38\|e36551\|2022 CONSIDERAÇÕES FINAIS Neste caso, não são apenas os sujeitos marcados por diferenças estigmatizadas que ganham visibilidade no debate, mas também aquele que é intocável pelo privilégio de ser o sujeito normal. No âmbito da educação, a discussão sobre diferença e identidade busca desnaturalizar qualquer valor que estigmatiza um grupo ou pessoa, bem como questiona a posição dominante. Esse processo inicia-se pela desconstrução dos sentidos implicados nas relações escolares e nas normas pedagógicas, nos procedimentos didáticos e nas relações de poder-saber. O normativo na educação escolar beneficia os atributos da não-deficiência e o perfil de aluno-médio – isto é, o estudante previsto pela norma, excluindo aqueles que não se encaixam no padrão estético, de aprendizagem, de comunicação, de comportamento e de interação (SILVA, 2019). Desta forma, ao colocar em evidência a norma que conduz os corpos e mentes (des)ajustados no lugar comum, o reconhecimento da diferença, como constituinte das identidades, torna-se um marco de referência potente para refletir sobre a experiência da deficiência. Quando refere-se ao tema da deficiência, diversidade e diferenças humanas, a análise crítica é o caminho para compreender quais são as bases conceituais que fundamentam os discursos oficiais. A narrativa da inclusão, de fato, responde aos interesses dos diferentes grupos sociais ou está alinhada ao projeto de normalidade? Cabem às teorias contemporâneas desnaturalizarem e politizarem as categorias deficiência e diferença para, assim, deslocarem a normalidade legitimada – e intocada no discurso de diversidade –, uma vez que é a norma a raiz do fenômeno denominado de (in)exclusão. deslocarem a normalidade legitimada – e intocada no discurso de diversidade –, uma vez que é a norma a raiz do fenômeno denominado de (in)exclusão. REFERÊNCIAS BRASIL. Lei nº 9.394 de 20 de dezembro de 1996. Estabelece as Diretrizes e Bases da Educação Nacional. Brasília, 1996. Brasília, 1996. BRASIL. Lei nº 10.048, de 8 de novembro de 2000. 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Informe mundial de la UNESCO: Investir en la diversidad cultural y el diálogo intercultural, 2009. Disponível em: < https://oibc.oei.es/uploads/attachments/73/Invertir_en_la_diversidad_cultural_y_el_di%C3%A1logo _intercultural_2009.pdf>. Acesso em: 18/10/2021. UNESCO. Declaração Universal sobre a Diversidade Cultural, 2002. Disponível em: <https://www.oas.org/dil/port/2001%20Declara%C3%A7%C3%A3o%20Universal%20sobre%20a %20Diversidade%20Cultural%20da%20UNESCO.pdf>. Acesso em: 22/04/2022. WOODWARD, Kathryn. Identidade e diferença: uma introdução teórica e conceitual. In: SILVA, Tomaz Tadeu. Identidade e Diferença: A perspectiva dos estudos culturais. Petrópolis: Vozes, 2000. p. 1-48. 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https://openalex.org/W2620911639	https://globalizationandhealth.biomedcentral.com/track/pdf/10.1186/s12992-017-0252-6	English	null	A health partnership to reduce neonatal mortality in four hospitals in Rwanda	Globalization and health	2,017	cc-by	5,811	* Correspondence: t.lissauer@imperial.ac.uk 8Imperial College Healthcare Trust, London, UK 9Centre for International Child Health, Imperial College London, Norfolk Place, London W2 1PG, UK Full list of author information is available at the end of the article A health partnership to reduce neonatal mortality in four hospitals in Rwanda Placide Ntigurirwa1, Kathy Mellor2, Daniel Langer3, Mari Evans4, Emily Robertson5, Lisine Tuyisenge6, Alan Groves7 and Tom Lissauer8,9* Abstract Background: A health partnership to improve hospital based neonatal care in Rwanda to reduce neonatal mortality was requested by the Rwandan Ministry of Health. Although many health system improvements have been made, there is a severe shortage of health professionals with neonatal training. Methods: Following a needs assessment, a health partnership grant for 2 years was obtained. A team of volunteer neonatologists and paediatricians, neonatal nurses, lactation consultants and technicians with experience in Rwanda or low-income countries was assembled. A neonatal training program was provided in four hospitals (the 2 University hospitals and 2 district hospitals), which focused on nutrition, provision of basic respiratory support with nasal CPAP (Continuous Positive Airway Pressure), enhanced record keeping, thermoregulation, vital signs monitoring and infection control. To identify if care delivery improved, audits of nutritional support, CPAP use and its complications, and documentation in newly developed neonatal medical records were conducted. Mortality data of neonatal admissions was obtained. Results: Intensive neonatal training was provided on 27 short-term visits by 10 specialist health professionals. In addition, a paediatric doctor spent 3 months and two spent 6 months each providing training. A total of 472 training days was conducted in the neonatal units. For nutritional support, significant improvements were demonstrated in reduction in time to initiation of enteral feeds and to achieve full milk feeds, in reduction in maximum postnatal weight loss, but not in days for regaining birth weight. Respiratory support with bubble CPAP was applied to 365 infants in the first 18 months. There were no significant technical problems, but tissue damage, usually transient, to the nose and face was recorded in 13%. New medical records improved documentation by doctors, but nursing staff were reluctant to use them. Mortality for University teaching hospital admissions was reduced from 23.6% in the 18 months before the project to 21.7%. For the two district hospitals, mortality reduced from 10% to 8.1%. A major barrier to training and improved care was low number of nurses working on neonatal units and staff turnover. Conclusion: This health partnership delivered an intensive program of capacity building by volunteer specialists. Improved care and documentation were demonstrated. CPAP was successfully introduced. Mortality was reduced. This format can be adapted for further training and improvement programs to improve the quality of facility-based care. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ntigurirwa et al. Globalization and Health (2017) 13:28 DOI 10.1186/s12992-017-0252-6 Ntigurirwa et al. Globalization and Health (2017) 13:28 DOI 10.1186/s12992-017-0252-6 Open Access Background by visiting the neonatal units, attending medical wards rounds and direct observation of medical and nursing care. The units varied in size. The University hospitals each had 12 incubators, 3 radiant warmers, 9 cots and 5 beds for 24-h Kangaroo Mother Care (KMC). Although relatively well equipped with incubators and radiant warmers, there was a shortage of beds for mothers to stay and conduct KMC. The district hospitals had 3–10 incubators, 2–8 radiant warmers and 15 cots. One hos- pital had 8 beds for KMC, the other initially had none. Only 3 of the units had a functioning oxygen saturation monitor. Each unit was supervised by a paediatrician, but most medical care in the University hospitals was provided by recently qualified doctors or postgraduate trainees; in the district hospitals by junior doctors also covering other specialties. Nurse staffing was very limited, ranging from 10 to 16 for each unit, with 2 to a maximum of 4 nurses per shift. There was no formal neonatal training programme for either doctors or nurses. The major needs identified were: by visiting the neonatal units, attending medical wards rounds and direct observation of medical and nursing care. The units varied in size. The University hospitals each had 12 incubators, 3 radiant warmers, 9 cots and 5 beds for 24-h Kangaroo Mother Care (KMC). Although relatively well equipped with incubators and radiant warmers, there was a shortage of beds for mothers to stay and conduct KMC. The district hospitals had 3–10 incubators, 2–8 radiant warmers and 15 cots. One hos- pital had 8 beds for KMC, the other initially had none. Only 3 of the units had a functioning oxygen saturation monitor. Each unit was supervised by a paediatrician, but most medical care in the University hospitals was provided by recently qualified doctors or postgraduate trainees; in the district hospitals by junior doctors also covering other specialties. Nurse staffing was very limited, ranging from 10 to 16 for each unit, with 2 to a maximum of 4 nurses per shift. There was no formal neonatal training programme for either doctors or nurses. The major needs identified were: g In September 2000, global political leaders adopted the United Nations Millennium Declaration, committing to a series of Millennium Development targets including improving health. Millennium Development Goal 4 (MDG 4) focused on reducing <5 years mortality. Background In Rwanda, a target was set to reduce child mortality from 152/1000 live births in 1990 to 50/1000 live births by 2015 [1]. By 2010 under 5 years mortality was still 76/ 1000 live births, with neonatal mortality (< 28 days old) comprising 27/1000 live births, and the reduction in neo- natal mortality was slower than that in older children. From 2010, a partnership in child health was in place in Rwanda to provide ETAT+ (Emergency Triage, Assessment and Treatment plus Admission) courses for the recognition and initial management of sick children (Imperial College London, subsequently Royal College of Paediatrics and Child Health, UK and Rwanda Pediatric Association, Rwandan Ministry of Health and Kenya Paediatric Association health partnerships). This well- established, evidence-based, 5-day course covers the 10 commonest causes of paediatric admissions in East Africa [2]. Although the ETAT+ course has a neonatal component, a more extensive neonatal training program was requested by Dr. Agnes Binagwaho, former Rwandan Minister of Health (personal communication, TL). i) Nutrition – to overcome long delays in the early introduction of breast milk feeds to preterm infants. This was because of physical separation of mother and infant (as the mother was in the maternity unit) and lack of advice and help in initiating expression of breast milk, together with reluctance by staff to begin and advance enteral milk feeds in preterm infants due to concerns that the babies were too small to tolerate feeds and fear of risk of serious gastrointestinal complications (necrotising enterocolitis). In Rwanda, there are just over 400,000 births/year [3]. Over 90% of births occur in health centres or district hospitals. Rwanda has under 0.45 healthcare workers per thousand population [4], far below the minimum level recommended by WHO of 2.3 per 1000. There were only about 26 paediatricians and no neonatologists or specialist neonatal nurses in the country. ii) Provision of basic respiratory support - no unit was able to provide this for infants with respiratory distress, although this is standard care in high-income countries. Bubble CPAP (Continuous Positive Airway Pressure) was considered to be the most pragmatic way to provide this. ii) Provision of basic respiratory support - no unit was able to provide this for infants with respiratory distress, although this is standard care in high-income countries. Bubble CPAP (Continuous Positive Airway Pressure) was considered to be the most pragmatic way to provide this. Background The aim of this partnership was to improve neonatal care and reduce neonatal mortality in 4 major neonatal units, including the 2 University hospitals and 2 District hospitals, one of which was the largest maternity unit in the country and the other a medium-sized district hospital. After identifying their major training require- ments, we aimed to provide a program of intensive capacity building by visiting volunteer health profes- sionals. In order to determine if this resulted in changes in service delivery, several audits were conducted. Our experience would determine if this partnership model could be adopted for future partnerships. iii)Enhanced Record Keeping - hospital medical records were unsuitable for neonatal care and data was often missing or difficult to retrieve. Introduction of focused neonatal-specific records was considered a priority to support structured clinical assessments and to allow monitoring of improvements in care [5]. iv) Thermoregulation - to improve temperature regulation of preterm and sick infants with appropriate use of available incubators and increase use of KMC. Abstract Keywords: Global health partnership, Low-income country, Newborn, Infants, Capacity building, Quality improvement, Nutrition, Continuous positive airways pressure, Medical records, Rwanda © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ntigurirwa et al. Globalization and Health (2017) 13:28 Page 2 of 7 Health partnership grant countries, circuits were washed and re-used to reduce cost and help overcome supply difficulties. As CPAP was new to all the units, and machine availability limited, guidelines were developed to maximise their potential benefit by targeting preterm infants with difficulty breathing and a birth weight 1.0–2.5 kg, and to encour- age rapid weaning when breathing improved. In conjunction with the Rwanda Pediatric Association and Rwandan Ministry of Health, a grant was obtained, funded by the United Kingdom Department for International Development (DfID) and managed by the Tropical Health & Education Trust (THET). The primary aim was reduc- tion in neonatal mortality in the 4 hospitals. The grant was for US$45,000 over 2 years, which clearly constrained the size of the project. g p g g p To overcome the difficulty in using the standard hos- pital medical records, a new neonatal medical record booklet was designed, in consultation with local health professionals and hospital directors. The record consists of structured collection of initial data on antenatal and perinatal health, clinical assessment and management followed by multiple, specially designed, daily summary pages for medical and nursing records and for recording of vital signs as well as fluid input/output charts. Growth charts are also included. After piloting, booklets for the University hospitals were printed. Development of the notes, staff induction and a 3-week training period with feedback was conducted at the University hospitals. Newborn care training and service improvement program Core issues identified in the needs assessment were addressed with a newborn care training program. A team of neonatologists, neonatal nurses, lactation con- sultants and technicians with experience of teaching and training, and willing to participate in the program on a voluntary basis was assembled. Multiple short visits of intensive training were organized, as well as three long- term placements, one 3-months and two 6-months, by paediatric doctors with neonatal experience. A local lead clinician was appointed at each hospital to lead and co- ordinate the program; in 3 hospitals it was the paediatri- cian, in one district hospital it was a nurse on the unit. Temperature regulation was problematic as many different makes of incubator were in use, and their efficacy was variable. Demonstrations were provided to optimise their use, and increased use of KMC encouraged. Needs assessment v) Vital Sign Monitoring – to increase access to monitoring of oxygen saturation and vital signs and improve staff’s appreciation of their interpretation. A detailed needs assessment of neonatal care in the four hospitals was performed. In addition to a detailed inven- tory of facilities, equipment, investigations and drugs, the training needs were determined by a team of UK neonatologists and neonatal nurses. This was achieved vi) Infection Control – improve hand hygiene, which was compounded by lack of hand basins with running water and hand gel. Page 3 of 7 Page 3 of 7 Ntigurirwa et al. Globalization and Health (2017) 13:28 Health partnership grant To enhance nutritional support, training was provided emphasising the importance of supporting mothers to provide early and frequent breast milk expression and not to wait until mothers were well enough after delivery to come to the neonatal unit. Training by lactation consultants and neonatal nurses was mainly conducted directly with staff on the maternity and neonatal units, supplemented by lectures and demonstrations. Posters, mainly diagrammatic, but with any text translated in English, French or Kinyarwanda, for mothers and staff were produced to demonstrate techniques and import- ance of expressing breast milk for preterm infants. Post- ers were distributed on maternity and neonatal units. Systems for cleaning and sterilising milk expressing equipment, and safe storage of expressed breast milk were introduced. Vital signs monitoring was improved by the provision of extra oxygen saturation monitors and guidance on their interpretation. Infection control was improved by practising hand washing technique. Audits and data collection To determine if the training program resulted in changes in clinical practice, 3 detailed audits were con- ducted. The nutrition audit was led by visiting long-term paediatric doctors (ER and ME), in conjunction with local doctors and nurses. Data from a random sample of records of babies of birthweight <2.5 kg on 3 of the units before and after training were extracted: time to initiation of enteral feeds, time to full milk feeds, max- imum postnatal weight loss (percentage of birthweight) and number of days for birthweight to be regained. Respiratory support was provided by bubble CPAP, using a simple system of nasal CPAP in widespread use in Europe and North America (Fisher Paykel), except that air was provided by a small compressor which was part of the system instead of from a wall supply. The system was driven by air, with additional oxygen pro- vided as required. It included an oxygen analyser to allow the oxygen concentration to be monitored. The circuit generated heated humidified gases that were delivered to the infant by nasal cannulae or face mask. The CPAP pressure was regulated by bubbling the gases through a bottle of water. An oxygen saturation monitor suitable for continuous monitoring in newborn infants was provided with each machine. Instead of each patient receiving a new CPAP circuit, as in high-income The audit of respiratory support with CPAP was led by a Rwandan paediatrician (PN). CPAP use and complica- tions were recorded using a checklist which staff com- pleted on a daily basis. The audit of completeness of documentation of med- ical records was led by the long-term visiting paediatric doctors (DL, ME, ER). Medical records were selected at random before and after introduction of the new version and completeness assessed according to a check-list. Mortality data of infants admitted to the neonatal units, both before and during the project, is collected by the hospital every month and was checked with entries in the unit admission books. Ntigurirwa et al. Globalization and Health (2017) 13:28 Page 4 of 7 Ethical approval Five CPAP machines were provided (2 financed by this grant and 3 from a separate donation) and installed in the 4 hospitals. Altogether, CPAP was used on 365 infants during the first 18 months, and was rapidly adopted by all 4 units. The most frequent complication was transient nasal or facial trauma (13%) attributed to excessive pressure applied by the cannulae in the nose or by the face mask to the nasal bridge. Significant abdominal distension was noted in 2% of infants. Pneumothorax (air adjacent to the lung), an uncommon but potentially serious complication, was not identified in any patient. As this can be difficult to identify clinic- ally, and chest X-rays were often unavailable, this com- plication may have occurred but not identified. Technical problems were minor and occurred in only 2% of infants. Ethical approval was not required as audits were conducted for quality assurance and data was anon- ymised. Mortality data is collected routinely for the Ministry of Health. Impact of revised medical records Completeness of documentation in 30 sets of medical records after new records were introduced were com- pared with 30 sets before implementation. It improved in multiple areas e.g. maternal history was completed in 75% compared with 35% in previous records, all birth details in 75% compared with 44%, admission assess- ment of the baby in 78% compared with 53% and an admission plan in 71% compared with 51%. Daily status updates were recorded in 57%% vs 23% previously and complete prescriptions were provided in 82% of charts vs 57% previously. Growth charts, not previously avail- able, were completed for 50% of infants at admission. Whilst all the medical staff adopted the new notes, some of the nurses continued to use their separate forms. Impact of nutritional intervention Data was collected from 58 babies with birth weight < 2.5 kg before and 38 babies after training in 3 of the hospitals. There was a significant reduction in time to initiation of enteral feeds, mean 2.4 days (Standard Deviation 1.5 days) Impact on thermoregulation and kangaroo mother care The large district hospital provided KMC to 42 mothers in the year before this program, which increased to 71/year followed by 157/year during our program. In the other district hospital, following discussions with the hospital director, a room adjoining the neonatal unit was converted into a KMC room for 4 mothers. Table 1 Number of health professionals. visits and training days Health professionals No. people No. visits Training days provided Neonatologists 3 6 63 Neonatal nurses 4 16 154 Medical technician 1 3 17 Lactation consultants 2 2 18 Paediatric doctors (long-term placements) 3 4 220 Table 1 Number of health professionals. visits and training days Health professionals No. people No. visits Training days provided Statistical methods compared to 1.8 days (Standard Deviation 1.0 days), (p = 0.02); for time to full milk feeds (> 150 ml/kg/day of feeds or demand feeding) 10.5 days (IQR 5–14) vs 6 days (IQR 3–12) (p = 0.02); and for a reduction in maximum postnatal weight loss, 15% (IQR 8–20) vs 9.0% of birth- weight (IQR 4.2–15), (p = 0.026). There was no significant difference in the number of days for birth weight to be regained (p = 0.22). Analysis of data from the nutrition audit was with Graphpad Prism statistical software version 7.0. Two- tailed Mann-Whitney statistical analysis was used to compare before and after training results except for data for the day of life enteral feeds were started as this had a normal distribution and so unpaired two-tailed t- test was used. Training provided The frequency and duration of training provided over the two years of the project is shown in Table 1. Between February 2012 and January 2014, 27 short-term visits by a team of 10 specialist health professionals from the UK were made, most making multiple visits. They provided a total of 252 training days. The medical technicians assisted in setting up the CPAP machines and with initial training. Visits were mostly short and intensive, lasting 1–2 weeks. This was because of difficulty in staff obtaining leave from their hospitals in the UK, with visits generally taken as annual rather than professional leave. Visits were frequent to reinforce the training. Training was provided to 84 Rwandan health profes- sionals on CPAP. The one paediatric doctor who each spent three months and the two who spent six months in Rwanda provided an additional 220 days of training. A major limitation was the small number of nursing staff available for training, with only 2–4 nurses working each shift. In addition, medical and nursing staff often rotated to other departments and to other hospitals. Discussion Although this was a small program, the considerable effort and intensity of training provided is shown by the many visits made by experienced doctors, neonatal nurses, lactation consultants and medical technicians from the UK. Most of the training was conducted in the neonatal unit itself and concentrated on practical aspects of neonatal care. The advantage of this training model is that through regular, short visits, intensive training can be delivered and problems dealt with without undue delay, but avoids the potential risk of trainers taking over the clinical care of the babies from local staff. The addition of some longer-term clinicians to the program allowed new initiatives to be undertaken, such as the introduction of CPAP, development of new medical records and assisting with conducting the audits, as well as reinforcing the training. However, a major issue in the implementation and adoption of improvements in care was the very small number of nurses working on each neonatal unit. This limited both nursing staff availability for training and their ability to provide additional aspects of care recommended by the partnership. Fre- quent staff turnover was another issue, affecting both nurses and doctors, as it necessitated repeated training of new staff and severely impeded institutional memory within the unit of new working practices. This is par- ticularly problematic in neonatal care as new nurses have received minimal training in the care of new- born infants as part of their general nurse training. Local medical and nursing leadership and staff motiv- ation were also major issues, aggravated by frequent moves to other departments. pp y g p Our experience with the introduction of CPAP high- lights many of the issues in implementing new technol- ogy. Once introduced into the country, demand for CPAP rapidly increased. An additional 4 similar CPAP machines were subsequently purchased by another donor, but other new programs have resulted in different machines being introduced. Each requires its own set of guidelines and training, and different circuits and main- tenance. Some, for example, do not heat or humidify the gases, making them cheaper to purchase and simpler to use; evidence to guide the choice of system according to outcomes is not available [6]. Impact on neonatal mortality For the University teaching hospitals, overall mortality was reduced from 23.6% (389 deaths, 1649 admissions) in Ntigurirwa et al. Globalization and Health (2017) 13:28 Page 5 of 7 Page 5 of 7 the 18 months before the project to 21.7% (463 deaths, 2135 admissions) during the 2 years of the project. For the district hospitals, overall mortality was reduced from 10% (141 deaths, 1415 admissions) to 8.1% (179 deaths, 2206 admissions). The higher mortality of the University teach- ing hospitals was because they were referred many high- risk mothers and preterm or sick newborn infants from other hospitals. the time it took to achieve full milk feeds and to reduce maximum weight loss. It was disappointing that there was no significant difference in the number of days for birth weight to be regained, but this is a more complex measure and may have needed a larger number of babies to show a significant difference; however, the reduction in maximum weight loss suggests that nutrition was improved. Posters demonstrating technique and advising on importance of expressing breast milk for preterm in- fants continued to be displayed on maternity and neo- natal units after the end of the project, but regular reinforcement of training was required to maintain lac- tation support and early feeding of preterm infants. Consent for publication Not applicable. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Abbreviations Abbreviations CPAP: Continuous Positive Airway Pressure; ETAT +: Emergency Triage, Assessment and Treatment plus Admission.; IQR: Interquartile Range; KMC: Kangaroo Mother Care Acknowledgements ld l k h We would like to thank all the paediatricians, neonatal nurses, lactation consultants and technicians for the training they provided on a voluntary basis. Their enthusiastic support was crucial to the success of this program. Mortality rates declined during this program, which is reassuring. However, as with most quality improvement initiatives, there were many other changes occurring sim- ultaneously to this project. In particular, towards the end of the program, the University paediatric departments had some additional paediatricians, nurses and technical sup- port, mostly from the United States, providing long-term training as part of the HRH (Human Resources for Health) program [9]. Identifying the specific contribution made by our program was not possible, although ours was specifically neonatal and did not cover the care of older children, the main focus of other programs. Funding The grant for this project (Imperial College London – Rwanda Paediatric Association, Rwanda Ministry of Health, Grant Holder Dr. Tom Lissauer) was funded by DfID (Department for International Development, UK) and managed by THET. Four of the CPAP systems were funded by the Belgian Technical Cooperation (BTC). Availability of data and materials Not applicable. Availability of data and materials Not applicable. Conclusion This program provides an example of how health partner- ships can evolve and expand from small beginnings. Some initial informal training evolved into a major program of ETAT+ courses for training in the recognition and initial management of sick children for all medical students over a period of 6 years and for health professionals in 18 hospitals. This neonatal program developed as an offshoot of that large program to address the specific needs of hospital care of newborn infants and the determination of the Ministry of Health in Rwanda to achieve its Millen- nium Development Goal 4 target, which was achieved [10]. It also resulted in an invitation for us and one of the University hospitals to join the WHO African Partnership for Patient Safety and the development of a further partnership to improve infection control. Authors’ contributions This paper was conceived and written by TL, with contributions by other authors who all approved the final version. DL and ER spent 6 months, ME spent 3 months in Rwanda, and conducted 2 of the audits and collected data. KM organized the visits by the neonatal nurses, lactation consultants and technicians. Discussion CPAP allows the oxygen concentration deliv- ered to be adjusted according to need, and usually markedly reduces the oxygen concentration required, with many managed just with air. Screening for and treatment of retinopathy of prematurity is standard of care in preterm infants in high-income countries, but was unfortunately not available during this program. It is reassuring that technical problems were uncommon, being seen in only 2% of infants in this cohort. Efforts to standardise the CPAP machines and guidelines nation- ally and to organise purchase of replacement circuits and machine maintenance are now underway, but will require new investment and agreement between all parties involved. The main immediate problems were the small numbers of staff available locally for training and high staff turn- over. For long-term sustainability of the changes intro- duced, the main issues are local leadership, both medical and nursing, by trained staff. The number of doctors undergoing postgraduate paediatric training is slowly in- creasing and a small cohort of nurses are receiving some specialist neonatal training, which should help address this problem in the future. This program’s intensive training helped improve facility-based neonatal care, as documented by the audits showing better nutrition, successful introduction of respiratory support with bub- ble CPAP and more structured and complete medical documentation. Further improvements in neonatal and other branches of medical care could be achieved by developing similar partnership programs. The design and implementation of a new dedicated neonatal medical record produced significant improve- ments in documentation by doctors. However, use by nurses was inconsistent; on questioning nursing staff about this, many expressed reluctance to make entries in the same notes as the doctors. Further training and amendments to fit local agendas may be required. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Discussion Availability of only a lim- ited number of machines in a unit can create difficult ethical problems for staff in choosing which babies should be given CPAP, and if a machine should be trans- ferred from one infant to another with greater need. This project was not designed to demonstrate improved outcomes from CPAP as it is standard care in high- income countries. Some authors have though argued that more evidence is needed for its use in low-income countries [7], but others disagree and consider it unnecessary [8]. Monitoring and preventing complica- tions are clearly important, particularly tissue trauma to the nose and nasal bridge. Although this can largely be prevented by regular re-assessment and adjustment of the nasal cannula or face mask, this is much more diffi- cult when the nurse to patient ratio is so much lower than in high-income countries. Rates of infection related to CPAP, and in particular assessment of risks of re-use of CPAP circuits and water in the humidifier could not be determined since the investigations required were not usually available. Permanent visual impairment from ret- inopathy of prematurity is another potential complica- tion from delivering excessively high oxygen to preterm infants, and all infants on CPAP should have continuous monitoring of their oxygen saturation, and their inspired oxygen concentration adjusted accordingly. However, prior to the introduction of CPAP in this partnership, it was not uncommon for preterm infants with respiratory distress to be managed with high flow rates of 100% oxy- gen, as this was the only respiratory support available, This project markedly raised the profile of newborn care in the country. A Rwandan Neonatal Network was formed and meetings and workshops held for doctors and nurses from all hospitals, with contributions by vis- iting specialists from our program. Two of the local lead paediatricians were awarded a Royal College of Paediat- rics and Child Health visiting fellowship to spend a month in a neonatal unit in the UK. However, most of the local paediatricians who provided leadership for this program are no longer working in neonatology. For improved nutrition, we were able to introduce new feeding practices and showed significant improve- ment in the day of life that enteral feeds were started, Ntigurirwa et al. Globalization and Health (2017) 13:28 Page 6 of 7 but placed them at increased risk of retinopathy of pre- maturity. Competing interests TL is Honorary Adviser in Child Health at THET. No other competing interests declared. Ntigurirwa et al. Globalization and Health (2017) 13:28 Ntigurirwa et al. Globalization and Health (2017) 13:28 References References 1. Countdown to 2015. http://countdown2030.org/documents/2014Report/ Rwanda_Country_Profile_2014.pdf (Accessed Feb 2017). 2. ETAT+ (Emergency triage, Assessment and Treatment plus Admission) guidelines. http://www.idoc-africa.org (Accessed Feb 2017). 3. UN Statistics. https://unstats.un.org/unsd/demographic/products/vitstats/ serATab3.pdf (Accessed Feb 2017). 4. National Human Resources for Health Policy, Ministry of Health, Rwanda 2014. http://www.moh.gov.rw/fileadmin/templates/policies/Human_ Ressource_for_Health_Policy.pdf (Accessed Feb 2017). 5. Gathara D, Nyamai R, Were F, et al. Moving towards routine evaluation of quality of inpatient Pediatric Care in Kenya. Choonara I, ed. PLoS One. 2015; 10(3):e0117048. doi:10.1371/journal.pone.0117048. 6. Lissauer T, Duke T, Mellor K, et al. Nasal CPAP for neonatal respiratory support in low and middle-income countries. Arch Dis Child Fetal Neonatal Ed Published Online First. 2017;(0):F1–3. doi:10.1136/archdischild-2016-311653. 7. McAdams RM. Bubble CPAP may be safe and efficacious for neonates in low and middle income countries, but more evidence is needed. Evid Based med. 2015;20(2):62. doi:10.1136/ebmed-2014-110135. 8. Jensen EA, DeMauro SB, Kirpalani H. Has enough evidence accumulated to consider CPAP a first-line standard of care in developing countries? Arch Dis Child Fetal Neonatal Ed. 2014;99:F443–4. 9. Binagwaho A, Kyamanywa P, Farmer PE, et al. The human resources for health program in Rwanda–new partnership. N Engl J Med. 2013;369(21): 2054–9. doi:10.1056/NEJMsr1302176. 10. Rwanda Demographic and Health Survey 2014–15. Final report. Republic of Rwanda. http://www.statistics.gov.rw/publication/demographic-and-health- survey-20142015-final-report (Accessed Feb 2017). 1. Countdown to 2015. http://countdown2030.org/documents/2014Report/ Rwanda_Country_Profile_2014.pdf (Accessed Feb 2017). 2. ETAT+ (Emergency triage, Assessment and Treatment plus Admission) guidelines. http://www.idoc-africa.org (Accessed Feb 2017). 3. UN Statistics. https://unstats.un.org/unsd/demographic/products/vitstats/ serATab3.pdf (Accessed Feb 2017). 5. Gathara D, Nyamai R, Were F, et al. Moving towards routine evaluation of quality of inpatient Pediatric Care in Kenya. Choonara I, ed. PLoS One. 2015; 10(3):e0117048. doi:10.1371/journal.pone.0117048. 6. Lissauer T, Duke T, Mellor K, et al. Nasal CPAP for neonatal respiratory support in low and middle-income countries. Arch Dis Child Fetal Neonatal Ed Published Online First. 2017;(0):F1–3. doi:10.1136/archdischild-2016-311653. 7. McAdams RM. Bubble CPAP may be safe and efficacious for neonates in low and middle income countries, but more evidence is needed. Evid Based med. 2015;20(2):62. doi:10.1136/ebmed-2014-110135. 7. McAdams RM. Bubble CPAP may be safe and efficacious for neonates in low and middle income countries, but more evidence is needed. Evid Based med. 2015;20(2):62. doi:10.1136/ebmed-2014-110135. 8. Jensen EA, DeMauro SB, Kirpalani H. Has enough evidence accumulated to consider CPAP a first-line standard of care in developing countries? Arch Dis Child Fetal Neonatal Ed. 2014;99:F443–4. 9. Binagwaho A, Kyamanywa P, Farmer PE, et al. Author details 1 1Muhima Hospital, Kigali, Rwanda. 2BirthLink, Ickenham UB10 8HN, UK. 3Epsom & St Helier University Hospitals NHS Trust, Surrey, UK. 4Great Ormond Street Hospital, London, UK. 5Royal Devon and Exeter Hospital, Exeter, UK. 6University Teaching Hospital of Kigali, Kigali, Rwanda. 7Weill Cornell Medicine, New York, USA. 8Imperial College Healthcare Trust, London, UK. 9Centre for International Child Health, Imperial College London, Norfolk Place, London W2 1PG, UK. This program provided a considerable amount of training from a range of neonatal health professionals. As they were all volunteers, this was done at low cost. Page 7 of 7 References The human resources for health program in Rwanda–new partnership. N Engl J Med. 2013;369(21): 2054–9. doi:10.1056/NEJMsr1302176. 10. Rwanda Demographic and Health Survey 2014–15. Final report. Republic of Rwanda. http://www.statistics.gov.rw/publication/demographic-and-health- survey-20142015-final-report (Accessed Feb 2017). 10. Rwanda Demographic and Health Survey 2014–15. Final report. Republic of Rwanda. http://www.statistics.gov.rw/publication/demographic-and-health- survey-20142015-final-report (Accessed Feb 2017). • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W4391270420	https://journal.ypidathu.or.id/index.php/lingeduca/article/download/586/414	English	null	Learning Arabic with Animaker-Based Learning Videos for Elementary School Students	Journal of Language and Education Studies	2,023	cc-by-sa	7,353	Rahayu Mustika Rafi 1 Rahayu Mustika Rafi 1 Rahayu Mustika Rafi 1 1 Universitas Islam Negeri Mahmud Yunus Batusangkar, Indonesia Corresponding Author: Name, Rahayu Mustika Rafi E-mail; rahayumustikarafi@gmail.com Article Information: Received November 10, 2023 Revised November 19, 2023 Accepted December 1, 2023 ABSTRACT The development of technology in this era means that teachers are increasingly required to create a pleasant learning atmosphere, no longer monotonous by using thick textbooks. The pleasure of learning can certainly make students more interested in the lesson and easily digest the lesson. The aim of this research is to prove that animaker-based learning videos can make learning Arabic fun and also make it easier for teachers in the teaching and learning process. The method used in this research is descriptive qualitative by referring to reliable research. This research found that by using the Animaker application, learning Arabic for elementary school students will not feel difficult and will be more fun because of the interesting videos. Keywords: Animaker application, Development of technology, Monotonous Journal Homepage https://journal.ypidathu.or.id/index.php/lingeduca This is an open access article under the CC BY SA license https://creativecommons.org/licenses/by-sa/4.0/ How to cite: Rafi, R, M. (2023). Learning Arabic with Animaker-Based Learning Videos for Elementary School Students. Lingeduca: Journal of Language and Education Studies, 2(3). https://doi.org/10.55849/lingeduca.v2i3.586 Published by: Yayasan Pendidikan Islam Daarut Thufulah Corresponding Author: Name, Rahayu Mustika Rafi E-mail; rahayumustikarafi@gmail.com Article Information: ABSTRACT Corresponding Author: Name, Rahayu Mustika Rafi E-mail; rahayumustikarafi@gmail.com Article Information: R i d N b 10 2023 ABSTRACT Received November 10, 2023 Revised November 19, 2023 Accepted December 1, 2023 The development of technology in this era means that teachers are increasingly required to create a pleasant learning atmosphere, no longer monotonous by using thick textbooks. The pleasure of learning can certainly make students more interested in the lesson and easily digest the lesson. The aim of this research is to prove that animaker-based learning videos can make learning Arabic fun and also make it easier for teachers in the teaching and learning process. The method used in this research is descriptive qualitative by referring to reliable research. This research found that by using the Animaker application, learning Arabic for elementary school students will not feel difficult and will be more fun because of the interesting videos. Keywords: Animaker application, Development of technology, Monotonous Keywords: Animaker application, Development of technology, Monotonous Journal Homepage https://journal.ypidathu.or.id/index.php/lingeduca This is an open access article under the CC BY SA license https://creativecommons.org/licenses/by-sa/4.0/ How to cite: Rafi, R, M. (2023). Learning Arabic with Animaker-Based Learning Videos for Elementary School Students. Lingeduca: Journal of Language and Education Studies, 2(3). https://doi.org/10.55849/lingeduca.v2i3.586 Published by: Yayasan Pendidikan Islam Daarut Thufulah Lingeduca: Journal of Language and Education Studies, 2(3) - December 2023 266- 282 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Lingeduca: Journal of Language and Education Studies, 2(3) - December 2023 266- 282 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students ingeduca: Journal of Language and Education Studies, 2(3) - December 2023 266- 282 Lingeduca: Journal of Language and Education Studies Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Rahayu Mustika Rafi 1 1 Universitas Islam Negeri Mahmud Yunus Batusangkar, Indonesia INTRODUCTION The world is currently experiencing various changes and developments, both in terms of offices, social, community, and even in terms of education which has led to progress in line with technological developments (Mustafiyanti dkk., 2023). These changes and developments not only produce negative influences, but also have many positive impacts on the current state of the world. Especially in terms of education nowadays, progress is very visible (Auliani dkk., 2023). Education is an activity that is really needed in today's world (Wanti dkk., 2023). In the Ministry of National Education (2003) it is explained that education is a human effort that is carried out consciously and planned to create an atmosphere and learning process so that students Lingeduca: Journal of Language and Education Studies Lingeduca: Journal of Language and Education Studies Learning Arabic with Animaker-Based Learning Videos for Elementary School Students can actively participate in and develop the potential and skills that exist within them (Mulyasari dkk., 2023). Studying and learning are two things that are closely related to education, because the success of an education is determined by the success of the learning process carried out. In essence, learning is a regulatory process, so that these rules can encourage students to carry out the teaching and learning process (Noer dkk., 2023). Learning is a process or activity with one's own experience whose aim is to change behavior or attitudes (Al Maarif dkk., 2023). Learning is a process of development within humans where this development will be realized in the form of behavior in order to improve human qualities, such as skills, habits, understanding, thinking power and other abilities (Utami dkk., 2023). According to Slameto, learning is an effort that a person makes to achieve a complete change in actions and behavior, as a result of his own observations in interacting with his environment (Emda, 2018). Then, learning is any or all activities that discuss ways to help an individual get to know and understand new things or new values. As Muslims, it is of course highly recommended for us as educators to teach Arabic to students. Because remembering that Arabic is the language of the Koran and is the holy book for Muslims. To understand the holy book in Arabic, it is fitting for us as someone who adheres to Islam to learn Arabic (Pamuji & Limei, 2023). Arabic is an important subject for students. INTRODUCTION So, according to BharatatTechnology is the science of ways of working in the industrial sector the science which deals with industrial arts (Peres dkk., 2020). Heavy industrial work for humans will be greatly facilitated by With technology, production in an industry becomes faster, easier and more efficient without draining human energy. There is also according to Paul B. Wesz, he said that technology is the application of scientific discoveries purely for practical purposes, a product that is ready to be sold or ready for public consumption (Spiegel dkk., 2018). And not much different from what Paul B. Wesz expressed, Harahap Felino believes that technology is practical industrial knowledge, systematic knowledge about industrial capabilities (Torres & De-la-Torre, 2021). Humans will not be able to improve their welfare without using technology (Hermansyah dkk., 2023). According toPhilip SpurtTechnology is a body of knowledge that is structured in an organized way systematic based on scientific discovery through experimentation, or simply based on years of practice successful ones, which enable the practical production of a particular object or service (Nicholas dkk., 2023). With With technology, work that was previously heavy will become lighter, difficult work will also become easier. So, according to BharatatTechnology is the science of ways of working in the industrial sector the science which deals with industrial arts (Peres dkk., 2020). Heavy industrial work for humans will be greatly facilitated by With technology, production in an industry becomes faster, easier and more efficient without draining human energy. There is also according to Paul B. Wesz, he said that technology is the application of scientific discoveries purely for practical purposes, a product that is ready to be sold or ready for public consumption (Spiegel dkk., 2018). And not much different from what Paul B. Wesz expressed, Harahap Felino believes that technology is practical industrial knowledge, systematic knowledge about industrial capabilities (Torres & De-la-Torre, 2021). From the things above, it can be concluded that technology is really needed in every aspect of humans and It has even developed and influenced the smooth running of life itself. In the world of education, for example, technology is like a new light that is getting brighter. Because technology provides convenience and ease to study. To keep up with this increasingly developing technology, teacher skills are needed adequate so that technology can be utilized properly (Azizah dkk., 2022). INTRODUCTION Most students today tend to be weak in remembering the mufrodats that have been memorized, in other words, they often forget the mufrodats. Not only that, Arabic lessons also seem to be an enemy for students, because they are known for their difficulties. Therefore, the students have deeply invested itmind setThey think Arabic lessons are difficult and are lazy to learn them, and even tend to take them for granted because they were not interested from the start. The most likely cause of this is that the books used by students tend to be monotonous and have no variation. So the book becomes uninteresting and difficult for students to remember. That is why it is very necessary for teachers to be able to utilize technology to make it easier for teachers to teach Arabic. Technological progress will be felt if it is accompanied by adequate capabilities to manage, utilize and use it (Fiqih dkk., 2023). If not, then technological development and progress will only be in vain. Educational progress will also be felt if accompanied by technology. It cannot be denied that technological developments have had a major influence on the world of education. What is meant by technological development is the increasing sophistication of technology from time to time (Alharizeh & Khasawneh, 2022). This development is of course influenced by human creativity and innovation. So, it can be said that education and technology are very closely related.Technological progress produces modernity characterized by economic growth, education, social mobility, cultural expansion or expansion (Marginson, 2018). Where through advances in communication technology it is also becoming more sophisticated and cheaper. Regardless of the benefits for human life, apparently there are still many who do not understand the meaning of technological development itself. Technology itself is an extension of humans, which means technology really helps humans in living their lives. 267 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Humans will not be able to improve their welfare without using technology (Hermansyah dkk., 2023). According toPhilip SpurtTechnology is a body of knowledge that is structured in an organized way systematic based on scientific discovery through experimentation, or simply based on years of practice successful ones, which enable the practical production of a particular object or service (Nicholas dkk., 2023). With With technology, work that was previously heavy will become lighter, difficult work will also become easier. INTRODUCTION Especially for teachers who teaches Arabic, remembering that Arabic is seen as a difficult subject for students. Because Therefore, it is necessary to update and look for things that make students happier and more interesting in learning Arabic. Therefore, as a manifestation of technological progress, so that students have interest and enjoy learning Arabic, the researcher offers or explains here a learning media that can be used by teachers in teaching Arabic to learners or students. Learning media is a tool used to assist in the learning process which can be used by a teacher to improve student learning achievement (Putri dkk., 2023). The mufrad form of the word media is medium, which means intermediary or introduction as something used to communicate (Vicky dkk., 2023). Media is a facility for distributing a message or learning information that will be conveyed by the sender of the message to the recipient or destination of the message (Winarto dkk., 2020). According to Santyasa, learning media is a tool that aims to stimulate an individual's interest or desires by channeling messages in the form of learning materials (Holly dkk., 2023). The learning media here is very useful for stimulating students' thoughts, feelings and skills or abilities so that they are encouraged to follow the existing learning (Levan’s dkk., 2022). According to Oemar Hamalik, learning media is a method and technique used to make interaction and communication between educators and students more effective in carrying out educational and teaching activities in the school environment (Saputra dkk., 2022). Learning media is an important component in determining whether the 268 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students learning is successful or not (Liam dkk., 2023). Therefore, it occupies a quite important position in the world of education. The learning media that can be used in teaching Arabic is in the form of learning videos. What is meant by learning video is a media that uses audio and visuals as its main features and contains learning material by combining various concepts, principles, theories and procedures (Saskia dkk., 2023). The aim of this learning video is not only to help students understand the subject matter being taught, but it will also make it easier for teachers to convey the subject matter (Rasmitadila dkk., 2020). Moreover, education in Indonesia currently uses the 2013 curriculum. INTRODUCTION This curriculum requires students' independence to learn, formulate and think for themselves about existing materials, no longer with long teacher explanations (Fathia dkk., 2022). In other words, the teacher explains all the material from the beginning to the end of the learning process. The development of the 2013 curriculum is based on thinking about future challenges, people's understanding of knowledge and pedagogy, future abilities and emerging negative phenomena (Maryati dkk., 2022). The curriculum is one element that can make a significant contribution to the process of developing the potential quality of students among the many elements of educational resources (Madani, 2019). Soedijarto believes that the curriculum is a series of planned activities with the aim of achieving the learning objectives of an educational institution (Madani, 2019). With a curriculum, the learning process in an educational institution can be more focused and conceptualized. The function of this curriculum is as a tool to achieve educational goals (Azzahra dkk., 2023). However, teachers should not be careless in making these learning videos. There are also several aspects that teachers must pay attention to when making these videos, such as appropriate delivery techniques, presentation of material must be correct, making videos with optimal quality, andskill a teacher in making videos must also be appropriate to current developments (Andriyani & Suniasih, 2021). Apart from that, there are also several aspects that teachers must pay attention to regarding the teaching in the video, such as stating learning objectives, presenting interesting, stimulating material, providing feedback and outside of class hours, teachers should provide tutoring services for their students (Halim dkk., 2023). Teachers as providers of knowledge must be able to act as facilitators in learning activities so that learning activities become more effective and meaningful (Rapanta dkk., 2020). For this reason, a teacher should be able to create a pleasant and interesting teaching and learning atmosphere for students (Roberts dkk., 2018). As has been explained, learning that is fun for students can be done by learning using videos (Andriyani & Suniasih, 2021). Learning videos that are needed to support Arabic learning so that elementary school students are interested and enjoy learning Arabic can be created using the Animaker application. Various studies on the benefits and effectiveness of Animaker on the quality of learning processes and outcomes in schools the basics have been done a lot. INTRODUCTION The use of Animaker as a learning medium in elementary schools has been empirically proven has 269 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students a positive influence on students' learning motivation and students' interest in reading, increasing learning activities, students' scientific attitudes and critical thinking skills (Lin & Wu, 2021). The positive influence of using Animaker is deep learning process which will then lead to improved student learning outcomes in elementary schools several subjects, as explained in various research results (Fitria dkk., 2023). Syarifuddin (2014) stated that the Animaker development phenomenon has indeed spread to all corners community life in a fast time (Krismanto, 2018). As the name suggests, the Animaker application is free software that is useful for creating various videos attractive animation (Niswatin dkk., 2023). In this application there are various features that will support it making animated videos. This animaker application was first launched in Open Beta version 2014 (Dorathy & Chandrasekaran, 2018). In February 2015, this Animaker application was officially launched based ona freemium model which allows users to enjoy the application for free. Since its launch in 2014, this Animaker application is equipped with attractive characters and templates so that users can use this application easily. Animaker itself is a combination of words(portmanteau) from “animation” and“maker” (Winarto dkk., 2020). So it can be exported to Facebook, YouTube or downloaded as a video mp4, animaker uses HTML5 and Adobe Flash to create the animation. This animaker application developed at a company founded by RS Raghavan by SaaS video-based Animaker, Inc (Schwendicke dkk., 2020). This software is also available in the Chrome Web store as a Chrome Extension File (Guess dkk., 2020). By using this application, it can make students interested in taking lessons, because it exists interesting videos. The teacher's job is also of course easier, because the teacher no longer explains clearly overall, but the teacher only needs to show the video and explain a little to the students based on it the video. Animaker has a product namedanimaker whiteboard,background and characters what is needed also already exists (Sallam, 2023). Based on the explanation above, it can be concluded that the aim of this research is to see effectiveness Animaker application to make it easier for teachers to present lessons and help students understand the material lessons taught. INTRODUCTION The benefits of this research are that it makes it easier for students to understand the material, because videos will provide a fun experience for students so they are interested and like them in learning Arabic, then this research will also improve teachers' abilities with videos interesting animated learning. For schools, this research will provide a new breakthrough increasing students' Arabic learning outcomes. And finally, for the researchers themselves, this research will add insight and knowledge by practicing the theory obtained. RESEARCH METHODOLOGY In this research, researchers used qualitative descriptive methods. Which in this method is human is the main instrument and the results of this research are conveyed in words or statements according to existing circumstances, in other words what actually happened (Seixas dkk., 2017).The qualitative descriptive method is a research method 270 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students based on the philosophy of postpositivism which is used to research the conditions of natural objects, and not experiments (Crawford & Serhal, 2020). In this research, the researcher acts as the key instrument, then the data collection technique is carried out using triangulation (combination), and the data analysis is inductive/qualitative, and the results of this research place more emphasis on meaning. In other words, research Qualitative descriptive is the use of a method that utilizes qualitative data and then explains it descriptively later. This research was conducted at SD IT Mutiara Hati Payakumbuh on 23 May – 1 July 2022. Researchers collected data by means of interviews, observation and practice or also called triangulation (combination). Interviews were carried out by asking questions to teachers in order to obtain a lot of information about learning Arabic and what methods and learning outcomes using the methods used in elementary schools are (Jailani dkk., 2021). Then the researcher continued the research using observation, namely by witnessing directly how the teaching and learning process occurred using this method. Then later the researcher will practice the learning method that the researcher proposes, namely by using animaker- based learning videos. Finally, the researcher will analyze the data and results that have been obtained to obtain conclusions for this research. RESULT AND DISCUSSION In this sophisticated era, where technology has improved and all sectors of life in general have used and utilized technological sophistication, the education sector must not be inferior to other sectors (Jones-Jang dkk., 2021). In fact, it should be the education sector that should be at the forefront by relying on existing technology. A situation that allows students to gain knowledge, intelligence and attitudes is a learning medium, including teachers, books and the environment (Diani dkk., 2018). An educator today must be creative and be able to master existing technology so that the learning process runs smoothly (Frederix dkk., 2019). Especially in using applications that can be used as learning media. Learning media is a very important instrument because it plays a role in determining the success or failure of a teaching and learning process (Deuflhard dkk., 2019). In this modern era, students often use something related to technology, both for learning and playing, the most important thing for them is that it is interesting. So, technology can now be used to attract students' interest in learning so that the teaching and learning process occurs effectively and can achieve the desired results (Simamora, 2020). To get maximum learning results, especially in learning Arabic, educators must try to find learning methods or media that can make students more enthusiastic and interested in learning Arabic (Tangcharoensathien dkk., 2020). Researchers have proposed here a learning medium that can be used by educators, namely the Animaker application. This application is believed to be able to make students more interested and happy in learning Arabic because it will make it easier for students to understand the material (Falloon, 2020). In essence, students as young as children who are still in elementary school will like various animated videos (Priyono dkk., 2020). That is why 271 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students researchers strongly recommend using animaker for learning Arabic at the elementary school level. However, this does not exclude that the competence of educators must also be in accordance with current developments in making animated videos. In other words, educators must strive to create interesting videos and learning objectives can also be achieved using this method. As explained, this research will produce video media for learning Arabic. RESULT AND DISCUSSION The use of learning videos using the Animaker application in Arabic language learning for fifth grade SD IT Mutiara Hati students through several procedures such as defining, designing, developing and disseminating. The first stage, namely the definition stage, is carried out by analyzing needs. Based on interviews with Arabic teachers at SD IT Mutiara Hati, it was found that the source of students' learning was only through the books provided at school. Teachers do not provide media to support learning so that students are less enthusiastic and unmotivated in the learning process. That is a problem that needs to be addressed. The next step is to determine competency standards and learning indicators that need to be better understood by students based on a learning needs analysis. The aim is that the media developed can help teachers teach students according to the demands of competency in learning (Syakur dkk., 2020). Then proceed to practice, first the researcher designs the design plan and components that will be used to learn with the Animaker application. The first thing the researcher does is collect learning material or understand the material that will be taught to students and that way it will be easy for the researcher to design what kind of video to make. After that, the researcher will continue by making Arabic learning videos using the Animaker application. This video will later become a result that can be utilized by SD IT Mutiara Hati to become a benchmark and reference for better videos in the future. Introduction to Various Features in Animaker Figure 1.Animaker Utama Display Figure 1.Animaker Utama Display 272 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students On the main display of Animaker, users will be presented with various menus and interesting features. First, users must log in first before using this application. Researchers use a website to open this application to make it easier. In the top menu, users are presented with a search feature, so that users can search for the model or type of video they want without having to search by looking at them one by one. RESULT AND DISCUSSION Then, under the search feature, users will be given a classification of what kind of editing the user wants to make it easier, such as videos for social media,my tools to cut orcrop video, voice over to edit sound,presentation for editing videos in the form of presentations and there are alsonew apps which includes various menus for editing videos, photos, sound and so on. Then, in partcreate project The form of video that will be used is further classified, such as horizontal videos, vertical videos, square videos, Instagram videos and Facebook videos. Figure 2.Video model selection Animaker also provides featurespreview. This feature aims to see the details in each video before the user decides to choose the video model they like or want (Blake dkk., 2020) Besides featurespreview there are also featuresuse which can be selected if the user has decided to choose the video model. Figure 2.Video model selection Animaker also provides featurespreview. This feature aims to see the details in each video before the user decides to choose the video model they like or want (Blake dkk., 2020) Besides featurespreview there are also featuresuse which can be selected if the user has decided to choose the video model. 273 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Figure 3.Display the video to be edited After deciding on the video model, users just click on the menuuse which is on the right sidepreview. Then, the user will be directed to the display for editing the video, as in Figure 3. There are several menus that can be used, on the left side there is a menucharacter that the user can use, it dependscharacter like what will be used. Then, on the right side there isscene-scene which can be used as a shortcut for users to viewscene-scene certain. Then, at the bottom there is a video duration table, through this the user can cut or lengthen it according to the desired video duration. Finally, to edit the writings you want to convey, users just click on the part of the writing, then replace it by typing according to the writing or purpose of the material you want to convey. RESULT AND DISCUSSION Figure 3.Display the video to be edited Figure 3.Display the video to be edited Figure 3.Display the video to be edited After deciding on the video model, users just click on the menuuse which is on the right sidepreview. Then, the user will be directed to the display for editing the video, as in Figure 3. There are several menus that can be used, on the left side there is a menucharacter that the user can use, it dependscharacter like what will be used. Then, on the right side there isscene-scene which can be used as a shortcut for users to viewscene-scene certain. Then, at the bottom there is a video duration table, through this the user can cut or lengthen it according to the desired video duration. Finally, to edit the writings you want to convey, users just click on the part of the writing, then replace it by typing according to the writing or purpose of the material you want to convey. Figure 4. Writing Changes Figure 4. Writing Changes Figure 4. Writing Changes 274 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students Writing changes can be made by clicking the menu on the right, then the user can select the desired writing form. Changing the size of the letters can also be done by changing the numbers in the text menu. changing the numbers in the text menu. Figure 5.Expression Change Expressioncharacter What the user chooses can also be changed according to the context of the word or video being edited. This feature is located on the right side with the nameexpressions, then there is also a menuactions which will show various states or movements that the user wants. Figure 6.Continue Editing to the Next Stage Users can continue editing forscene-scene further according to one's own wishes. With the many menus available, users can more easily edit videos according to their own wishes (Smith & Magnani, 2019). Figure 5.Expression Change Figure 5.Expression Change Expressioncharacter What the user chooses can also be changed according to the context of the word or video being edited. This feature is located on the right side with the nameexpressions, then there is also a menuactions which will show various states or movements that the user wants. Expressioncharacter What the user chooses can also be changed according to the context of the word or video being edited. CONCLUSION Based on the data analysis that the researchers have carried out, it can be concluded that learning Arabic using animaker is quite effective because it makes it easier for educators and students to learn. For educators, this web-based animaker application can facilitate the learning process because educators can explain the material just by using edited videos, then educators can also increase their competency due to increasedskill to edit various videos (Blake dkk., 2020). For students, learning Arabic using the Animaker application can increase the level of enthusiasm for studying (Alharizeh & Khasawneh, 2022). It is proven that videos have a big role in today's developments, considering that many children prefer watching rather than reading (Saurabh & Ranjan, 2020). Therefore, learning Arabic using videos is fun while eliminating the negative stigma of children who think learning Arabic is difficult and difficult to understand (Hulaihel dkk., 2023). This stigma has existed since ancient times, even though Arabic is the language of Muslims and the Muslim holy books were revealed in Arabic. RESULT AND DISCUSSION This feature is located on the right side with the nameexpressions, then there is also a menuactions which will show various states or movements that the user wants. Figure 6.Continue Editing to the Next Stage Users can continue editing forscene-scene further according to one's own wishes. With the many menus available, users can more easily edit videos according to their own wishes (Smith & Magnani, 2019). Figure 6.Continue Editing to the Next Stage Users can continue editing forscene-scene further according to one's own wishes. With the many menus available, users can more easily edit videos according to their own wishes (Smith & Magnani, 2019). Figure 6.Continue Editing to the Next Stage Figure 6.Continue Editing to the Next Stage Users can continue editing forscene-scene further according to one's own wishes. With the many menus available, users can more easily edit videos according to their own wishes (Smith & Magnani, 2019). Users can continue editing forscene-scene further according to one's own wishes. With the many menus available, users can more easily edit videos according to their own wishes (Smith & Magnani, 2019). 275 Learning Arabic with Animaker-Based Learning Videos for Elementary School Students ACKNOWLEDGEMENT The researcher would like to thank Mrs. Marnita Indra Dauly as the principal of SD IT Mutiara Hati who has given the opportunity and allowed the researcher to conduct research at SD IT Mutiara Hati. Then the researcher also expressed a thousand thanks to the teacher and supervisor in completing this research, namely Ustadz Adam Mudinillah, M. Pd. Not forgetting especially the family, Appa and Mother who have given an abundance of love so that researchers can complete this research. Lastly, the researcher would also like to thank his friends who always put a smile on his face. Hopefully this research can be useful for SD IT Mutiara Hati, UIN Mahmud Yunus Batusangkar, and all parties who can learn from this research. 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W4353007551.txt	https://ieeexplore.ieee.org/ielx7/9552933/9712344/10229197.pdf	en		Global Performance Guarantees for Localized Model Predictive Control	arXiv (Cornell University)	2,023	cc-by	10,954	This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 Received 20 March 2023; revised ; date of current version 18 August 2023. Digital Object Identifier 10.1109/OJCSYS.2023.Doi Number Global Performance Guarantees for Localized Model Predictive Control JING SHUANG (LISA) LI1 , CARMEN AMO ALONSO1 1 Department of Computing and Mathematical Sciences, California Institute of Technology, Pasadena, CA 91106 USA CORRESPONDING AUTHOR: J. S. Li (e-mail: jslisali@umich.edu) The work of J. S. Li was supported in part by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada [NSERC PGSD3-557385-2021]. ABSTRACT Recent advances in model predictive control (MPC) leverage local communication constraints to produce localized MPC algorithms whose complexities scale independently of total network size. However, no characterization is available regarding global performance, i.e. whether localized MPC (with communication constraints) performs just as well as global MPC (no communication constraints). In this paper, we provide analysis and guarantees on global performance of localized MPC — in particular, we derive sufficient conditions for optimal global performance in the presence of local communication constraints. We also present an algorithm to determine the communication structure for a given system that will preserve performance while minimizing computational complexity. The effectiveness of the algorithm is verified in simulations, and additional relationships between network properties and performance-preserving communication constraints are characterized. A striking finding is that in a network of 121 coupled pendula, each node only needs to communicate with its immediate neighbors to preserve optimal global performance. Overall, this work offers theoretical understanding on the effect of local communication on global performance, and provides practitioners with the tools necessary to deploy localized model predictive control by establishing a rigorous method of selecting local communication constraints. This work also demonstrates — surprisingly — that the inclusion of severe communication constraints need not compromise global performance. INDEX TERMS Distributed control, large-scale systems, model predictive control, optimal control. I. INTRODUCTION Distributed control is crucial for the operation of largescale networks such as power grids and intelligent transport systems. Distributed model predictive control (MPC) is of particular interest, since MPC is one of the most powerful and commonly used control methods. Many formulations for distributed MPC involve open-loop policies (i.e. optimize directly over states and inputs) [1]–[8]. Distributed closed-loop approaches (i.e. optimize over policies) are rarer and typically require strong assumptions, such as the existence of a static structured stabilizing controller [9] or decoupled subsystems [10]. However, distributed closedloop approaches tend to be more amenable for extension to robust formulations — this motivated the development of the distributed and localized MPC (DLMPC) formulation, introduced in previous work [11]–[13]. DLMPC is unique among distributed MPC methods in that it computes structured closed-loop policies, can be solved at scale via distributed optimization, and requires no strong assumptions on the system: it may be used on arbitrary linear systems. In the context of system level synthesis, upon which DLMPC is based, DLMPC is also the first work to extend to system level formulation to the distributed online (i.e. predictive) setting. Additionally, DLMPC enjoys minimally conservative feasibility and stability guarantees [13]. However, DLMPC requires the inclusion of local communication constraints, whose effects on performance is, thus far, unexplored — this is the focus of this work. The key benefits of DLMPC are facilitated by the inclusion of local communication constraints, which are typically encapsulated in a single parameter d (rigorously defined in Section II). The question of how to select this param- This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME XX, 2023 1 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 J. S. LI ET AL.: GLOBAL PERFORMANCE GUARANTEES FOR LOCALIZED MODEL PREDICTIVE CONTROL eter remains unresolved, as two opposing forces come into play: smaller values of d represent stricter communication constraints, which correspond to decreased complexity — however, overly strict communication constraints may render the problem infeasible, or compromise system performance. In this work, we address this problem by providing a rigorous characterization of the impact of local communication constraints on performance. The analysis in this work is enabled by the unique formulation of DLMPC with regards to communication constraints. While previous distributed MPC approaches typically 1) focus on iterative distributed solutions of the centralized problem [1], [3], [5], [8] or 2) reshape performance objectives for distributed optimization [2], [4], DLMPC does neither. In DLMPC, we begin with a reparametrization of the standard centralized MPC problem, then introduce an additional constraint of local communication, which manifests as a sparsity constraint on the decision variables. No changes to the performance objective are required. The resulting localized MPC problem can be exactly and optimally solved via distributed optimization — that is, we can apply the alternating direction method of multipliers (ADMM) [14] to split the problem into subproblems, which are solved in parallel. No assumptions are required on the underlying system graph, which is constructed solely using system matrices (see Definition 1). However, when the system graph is sparse (see Section V for an example), the local communication constraints confer additional scalability upon the distributed problem — in particular, the size of subproblems do not depend on the total size of the network [11], [12], [15]. In this work, we rigorously analyze the effect of this local constraint by comparing the performance of the localized MPC problem to the standard global MPC problem. We restrict analysis to the linear setting, and focus on cases in which optimal global performance (with respect to any convex objective function with its minimum at the origin) may be obtained with local communication. In other words, the performance of the system is unchanged by the introduction of communication constraints. A striking finding is that in a network of coupled pendula, optimal global performance can be achieved with relatively strict local communication constraints — in fact, if every subsystem is actuated, then each subsystem only needs to communicate with its immediate neighbors to preserve optimal global performance. Prior work: For large networked systems, several studies have been conducted on the use of offline (i.e. fixed-policy) controllers with local communication constraints. Local communication can facilitate faster computational speed [16] and convergence [17], particularly in the presence of delays [18] — however, this typically comes at the cost of supoptimal global performance [19]. In [20], a trade-off between performance and decentralization level (i.e. amount of global communication) is found for a truncated linear quadratic regulator. In system level synthesis, the offline predecessor of DLMPC [15], localization is typically associated with reduced performance of around 10% relative to the global controller. More generally, for both global and localized control, the topology of the network and actuator placement [21] plays a role in achievable controller performance [22], [23] and convergence [24]. In the realm of predictive control, communication constraints are important considerations [25]. However, the improved computational speeds offered by local predictive controllers typically come at the cost of suboptimal global performance and lack of stability and convergence guarantees [26]. The novel DLMPC method [11]–[13] overcomes some of these drawbacks, providing both stability and convergence guarantees — however, thus far, its performance has not been substantially compared to that of the global, full-communication controller.1 . In the few instances that it has, it performed nearly identically to the global controller despite the inclusion of strict communication constraints [27], prompting further investigation. Contributions: This work contains two key contributions. First, we provide a rigorous characterization of how local communication constraints restrict (or preserve) the set of trajectories available under predictive control, and use this to provide guarantees on optimal global performance for localized MPC. Secondly, we provide an exact method for selecting an appropriate locality parameter d for localized MPC. To the best of our knowledge, these are the first results of this kind on local communication constraints; our findings are useful to theoreticians and practitioners alike. Paper structure: We first provide the appropriate mathematical formulations for global MPC and localized MPC in Section II, then leverage these formulations to provide rigorous characterizations of global and localized MPC performance in Section III — this is the main theoretical contribution of the paper. We then use these results to create an algorithm that determines the optimal local communication parameter d in Section IV. We run this algorithm on a powersystem inspired grid of coupled pendula, and observe the dependence of d on various system parameters in Section V, and conclude with directions of future work in Section VI. A. NOTATION Lower-case and upper-case letters such as x and A denote vectors and matrices respectively, although lower-case letters might also be used for scalars or functions (the distinction will be apparent from the context). An arrow above a matrix #» quantity denotes vectorization, i.e. A is the vectorization of A. Boldface lower case letters such as x denote finite horizon signals. Unless otherwise stated, boldface upper case letters such as K denote causal (i.e. lower block triangular) finite 1 Prior work focuses on comparisons between centralized and distributed optimization schemes for localized MPC 2 VOLUME XX 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 horizon operators:    x0 K0,0  x1   K1,1    x =  . ,K =  .  ..   .. xT KT,T The MPC problem at each timestep τ is defined as follows:  K1,0 .. . .. ... KT,1     . xt ,ut ,γt KT,0 where each Ki,j is a matrix. Unless required, dimensions are not stated, and compatible dimension can be assumed. For matrix A, (A)i,: denotes the ith row of A, (A):,j denotes the j th column, and (A)i,j denotes the element in the ith row and j th column. (A)i:,: denotes the rows of A starting from the ith row. Calligraphic letters such as Y denote sets, and script letters such as R denote a subset of Z+ , e.g. R = {1, . . . , n} ⊂ Z+ . For matrix A, (A)R,C denotes the submatrix of A composed of the rows and columns specified by R and C, respectively; for vector x, (x)R denotes the vector composed of the elements specified by R. Bracketed indices denote timestep of the real system — for example, the system state is x(τ ) at time τ , not to be confused with xt which denotes the predicted state x in t timesteps. Square bracket notation, e.g. [x]i , denotes the components of x corresponding to subsystem i. For ease of variable manipulation, we also introduce augmented variables. For any matrix Z , the corresponding augmented matrix Z blk is defined as a block-diagonal matrix containing Nx copies of Z , i.e. Z blk := blkdiag(Z, . . . Z). For any matrix Y = ZΛ, the corresponding vectorization #» #» can be written as Y = Z blk Λ . For state x0 , the corresponding augmented state X is defined as X(x0 ) := (x0 )1 I (x0 )2 I . . . (x0 )Nx I . For notational simplicity, we write X instead of X(x0 ); #» dependence on x0 is implicit. For any matrix Λ, Λx0 = X Λ . II. LOCALIZED MPC We begin with a brief summary of global MPC and localized MPC [11]–[13]. Consider the standard linear time-invariant discrete-time system: x(t + 1) = Ax(t) + Bu(t) (2) where x ∈ RNx is the state and u ∈ RNu is the control input. System (2) can be interpreted as N interconnected subsystems, each equipped with its own sub-controller. Definition 1. G(A,B) (E, V) is the system graph, which is undirected and unweighted. Assign each state k ∈ {1 . . . Nx } and actuation l ∈ {1 . . . Nu } to some subsystem i ∈ {1 . . . N }. Each subsystem i is identified with a vertex vi ∈ V , and an edge eij ∈ E exists whenever [A]ij ̸= 0 or [B]ij ̸= 0 for subsystems i and j . A natural choice is to assign each state to its own subsystem, and to assign actuation to the states they act upon. Occasionally, two or more states may be assigned to the same subsystem — for example, when two states represent two different measurements (e.g. phase and frequency) on the same physical location. See Section V for an example. T −1 X min (1) ft (xt , ut ) + fT (xT ) (3a) t=0 s.t. x0 = x(τ ), xt+1 = Axt + But , xT ∈ XT , xt ∈ Xt , ut ∈ Ut , ut = γt (x0:t , u0:t−1 ), t = 0, ..., T − 1 (3b) (3c) (3d) (3e) where ft (·, ·) and fT (·) are assumed to be closed, proper, and convex, with the minimum at the origin; γt (·) is a measurable function of its arguments; and sets XT , Xt , and U are assumed to be closed and convex sets containing the origin for all t. Remark 1. Problem (3) is the closed-loop version of the standard MPC problem — they are mathematically equivalent. Policies γt are time-varying and capture all possible causal combinations of state xt and input ut .2 In localized MPC, we impose communication constraints such that each subsystem can only communicate with a other subsystems within its local region. We shall refer to these constraints interchangeably as local communication constraints or locality constraints. Let N (i) denote the set of nodes that subsystem i can communicate with them. Then, to enforce locality constraints on (3), we replace constraint (3e) with [ut ]i = γi,t ([x0:t ]j∈N (i) , [u0:t−1 ]j∈N (i) ) (4) Most SLS-based works use the concept of d-local neighborhoods, replacing N (i) with Nd (i): Definition 2. Nd (i) denotes the d-local neighborhood of subsystem i. Subsystem j ∈ Nd (i) if there exists a path of d or less edges between subsystems i and j in G(A,B) (E, V). The inclusion of local communication constraints renders problem (3) difficult to solve. To ameliorate this, we can apply ideas from the system level synthesis framework [15] to reparametrize the problem [12] — we now introduce relevant ideas from this framework. First, we define Â := blkdiag(A, ..., A), B̂ := blkdiag(B, ..., B), and Z , the block-downshift matrix — with identity matrices along the first block sub-diagonal and zeros elsewhere. The behavior of system (2) over time horizon t = 0 . . . T can be written in terms of closed-loop maps in signal domain: x = (I − Z(Â + B̂K))−1 w =: Ψx w (5a) u = KΨx w =: Ψu w (5b) where w represents driving noise; in our case, where no driving noise is present, it consists ⊤ only of the initial con⊤ dition, i.e. w = x0 0 . . . 0 . The key idea is to use 2 Readers are referred to [12] for details. VOLUME XX 2023 3 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 J. S. LI ET AL.: GLOBAL PERFORMANCE GUARANTEES FOR LOCALIZED MODEL PREDICTIVE CONTROL closed-loop maps Ψx , Ψu as optimization variables, subject to appropriate constraints. Theorem 1. (Theorem 2.1 of [15]) For system (2) with causal state feedback control policy u = Kx, 1) The affine subspace of causal closed-loop maps Ψx =I ZAB Ψu Φx ∈ Lx , Φu ∈ Lu (6) parametrizes all achievable closed-loop maps Ψx , Ψu . Here, ZAB := I − Z Â −Z B̂ 2) For any closed-loop maps Ψx , Ψu obeying (6), the controller K = Ψu Ψ−1 x achieves the desired closedloop response as per (5). Theorem 1 allows us to reformulate a control problem over state and input signals into an equivalent problem over closed-loop maps Ψx , Ψu . In the case with no driving noise, only the first block column of Ψx , Ψu need to be computed due to the zeros in w. We can rewrite (5) as x = Φx x0 and u = Φu x0 , where Φx and Φu correspond to the first block columns of Ψx and Ψu , respectively. Then, we can rewrite (6) as Φx I ZAB = (7) Φu 0 Notice that for any Φx satisfying constraint (7), (Φx )1:Nx ,: = I . This is due to the structure of ZAB . Also, constraint (7) is always feasible, with solution space of dimension Nu T . Tosee this, notice that I rank(ZAB ) = rank ZAB always holds, since 0 ZAB has full row rank due to the identity blocks on its diagonal; apply the Rouché-Capelli theorem [28] to get the desired result. We now apply this closed-loop parametrization to (3), as is done in [12]: min Φx ,Φu s.t. i and j for all j ̸= i. The relationship between closed-loop maps and communication constraints are further detailed in [12]. Thus, to incorporate local communication into this formulation, we introduce an additional constraint: f (Φx x0 , Φu x0 ) (8a) x0 = x(τ ), I Φx = , ZAB Φu 0 (8b) Φx x0 ∈ X , Φu x0 ∈ U (8d) (8c) Objective f is defined such that (3a) and (8a) are equivalent; similarly, constraint sets X , U are defined such that (3d) and (8d) are equivalent. Remark 2. Problem (8) is mathematically equivalent to problem (3). Both are alternative — but equivalent — formulations of the standard MPC problem. In (8), Φx and Φu not only represent the closed-loop maps of the system, but also the communication structure of the system. For instance, if [Φx ]i,j = 0 and [Φu ]i,j = 0 ∀i ̸= j , then node i requires no knowledge of (x0 )j̸=i — consequently, no communication is required between nodes (9) where Lx and Lu are sets with some prescribed sparsity pattern that is compatible with the desired local communication constraints. Φx For simplicity, we define decision variable Φ := , Φu which has NΦ := Nx (T + 1) + Nu T rows. We also rewrite locality constraints (9) as Φ ∈ L. From here on, we shall use global MPC to refer to (3), or equivalently, (8). We shall use localized MPC to refer to (8) with constraint (9). We remark that for appropriately chosen locality constraints, localized MPC confers substantial scalability benefits [12]. In particular, we can use ADMM [14] to split the localized MPC problem into subproblems, which are solved in parallel, i.e. each subsystem solves its own subproblem. When the system graph is sparse (see Section V for an example), the size of subproblems do not depend on the total size of the network [11], [12], [15]. III. GLOBAL PERFORMANCE OF LOCALIZED MPC In this section, we analyze the effect of locality constraints Φ ∈ L on MPC performance. We are especially interested in scenarios where localized MPC achieves optimal global performance, i.e. f ∗ = fL∗ , where f ∗ and fL∗ are the solutions to the global MPC problem and localized MPC problem, respectively, for some state x0 . First, we must analyze the space of available trajectories from state x0 for both globaland localized MPC. We denote x1:T an available trajectory y := . u Definition 3. Trajectory set Y(x0 ) denotes the set of available trajectories from state x0 under dynamics (2): I Y(x0 ) := {y : ∃Φ s.t. ZAB Φ = , y = (Φ)Nx +1:,: x0 } 0 Localized trajectory set YL (x0 ) denotes the set of available trajectories from state x0 under dynamics (2) and locality constraint Φ ∈ L: I YL (x0 ) := {y : ∃Φ s.t. ZAB Φ = , 0 Φ ∈ L, y = (Φ)Nx +1:,: x0 } Proposition 1. (Optimal global performance) For state x0 , if the local communication constraint set L is chosen such that Y(x0 ) = YL (x0 ), then the localized MPC problem will attain optimal global performance. Proof: 4 VOLUME XX 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 Global MPC problem (3) can be written as min x,u s.t. f (x, u) (10a) x0 = x(τ ), x ∈ X , u ∈ U, x1:T ∈ Y(x0 ) y := u (10b) (10c) The localized MPC problem can also be written in this form, by replacing Y(x0 ) in constraint (10c) with YL (x0 ). Thus, if Y(x0 ) = YL (x0 ), the two problems are equivalent and will have the same optimal values. Remark: This is a sufficient but not necessary condition for optimal global performance. Even if this condition is not satisfied, i.e. YL (x0 ) ⊂ Y(x0 ), the optimal global trajectory may be contained within YL (x0 ). However, this is dependent on objective f . Our analysis focuses on stricter conditions which guarantee optimal global performance for any objective function. We now explore cases in which Y(x0 ) = YL (x0 ), i.e. the localized MPC problem attains optimal global performance. Localized trajectory set YL (x0 ) is shaped by the dynamics and locality constraints: I ZAB Φ = (11a) 0 Φ∈L (11b) To obtain a closed-form solution for YL (x0 ), we will parameterize these constraints. Two equivalent formulations are available. The dynamics-first formulation parameterizes constraint (11a), then (11b), and the locality-formulation parameterizes the constraints in the opposite order. The dynamics-first formulation clearly shows how local communication constraints affect the trajectory space; the localityfirst formulation is less clear in this regard, but can be implemented in code with lower computational complexity than the dynamics-first formulation. We now derive each formulation. A. DYNAMICS-FIRST FORMULATION We first parameterize (11a), which gives a closed-form expression for trajectory set Y(x0 ). Lemma 1. (Image space representation of trajectory set) 1) The trajectory set from state x0 is described by: Y(x0 ) = {y : y = Zp x0 + Zh Xλ, λ ∈ RNΦ } I † where Zp := (ZAB )Nx +1:,: 0 and † Zh := (I − ZAB ZAB )Nx +1:,: and the size of the trajectory set is dim(Y(x0 )) = rank(Zh X) 2) If x0 has at least one nonzero value, then dim(Y(x0 )) = Nu T Proof: As previously shown, (11a) always has solutions. We can parameterize the space of solutions Φ as: I † † + (I − ZAB ZAB )Λ (12) Φ = ZAB 0 where Λ is a free variable with the same dimensions as Φ. Recall that Φ1:Nx ,: = I always holds — thus, we can omit the first Nx rows of (12). Define Φ2 := (Φ)Nx +1:,: and consider Φ2 = Zp + Zh Λ (13) Combining (13) and the definition of y, we have y = Φ2 x0 = Zp x0 + Zh Λx0 (14) Making use of augmented state X , rewrite this as #» y = Φ2 x0 = Zp x0 + Zh X Λ (15) This gives the desired expression for Y(x0 ) and its size. To prove 2), notice that if x0 has at least one nonzero value, then rank(Zh X) = rank(Zh ) due to the structure of X . All that is left is to show rank(Zh ) = Nu T . First, note that rank(ZAB ) = Nx (T + 1) due to the identity blocks on the diagonal of ZAB . It follows that † † ZAB ) = Nx (T + 1). Thus, ) = rank(ZAB rank(ZAB † rank(I − ZAB ZAB ) = NΦ − Nx (T + 1) = Nu T . Recall that † Zh is simply I − ZAB ZAB with the first Nx rows removed; this does not result in decreased rank, since all these rows are zero (recall that Φ1:Nx ,: is always equal to I ). Thus, † rank(Zh ) = rank(I − ZAB ZAB ) = Nu T . To write the closed form of localized trajectory set YL (x0 ), we require some definitions: Definition 4. Constrained vector indices L denote the set of indices of Φ2 := (Φ)Nx +1:,: that are constrained to be zero by the locality constraint (11b), i.e. #» (Φ2 )L = 0 ⇔ Φ ∈ L Let NL be the cardinality of L. We now parameterize (11b) and combine this with Lemma 1, which gives a closed-form expression for localized trajectory set YL (x0 ). Lemma 2. (Image space representation of localized trajectory set) Assume there exists some Φ that satisfies constraints (11a) and (11b). Then, the localized trajectory set from state x0 is described by the following: YL (x0 ) = {y : y = Zp x0 + Zh XF † g † + µ ∈ RNL } #» g := −(Zp )L Zh X(I − F F )µ, where F := (Zhblk )L,: and and the size of the localized trajectory set is dim(YL (x0 )) = rank(Zh X(I − F † F )) Proof: Using the augmented matrix of Zh , we can write the vectorization of (13) as # » #» #» Φ2 = Zp + Zhblk Λ (16) VOLUME XX 2023 5 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 J. S. LI ET AL.: GLOBAL PERFORMANCE GUARANTEES FOR LOCALIZED MODEL PREDICTIVE CONTROL #» where Λ is a free variable. Incorporate locality constraint (11b) using the constrained vector indices: #» #» (17) (Zp + Zhblk Λ)L = 0 #» #» #» blk This is equivalent to (Zp )L + (Zh )L,: Λ = 0, or F Λ = g . We can parameterize this constraint as #» Λ = F † g + (I − F † F )µ (18) where µ is a free variable. Plugging this into (15) gives the desired expression for YL (x0 ) and its size. We remark that there is no need to consider Φ1 = I in relation to the locality constraints, since the diagonal sparsity pattern of the identity matrix (which corresponds to self-communication, i.e. node i ‘communicating’ to itself) satisfies any local communication constraint. Theorem 2. (Optimal global performance) If x0 has at least one nonzero value, then localized MPC attains optimal global performance if 1) there exists some Φ that satisfies constraints (11a) and (11b), and 2) rank(Zh X(I − F † F )) = Nu T Proof: By definition, YL (x0 ) ⊆ Y(x0 ). Equality is achieved if and only if the two sets are of equal size. Applying Lemmas 1 and 2 shows that the conditions of the theorem are necessary and sufficient for YL (x0 ) and Y(x0 ) to be equal. Then, apply Proposition 1 for the desired result. This theorem provides a criterion to assess how the inclusion of locality constraints affects the trajectory set. It also allows us to gain some intuition on the effect of these constraints, which are represented by the matrix F . If no locality constraints are included, then F has rank 0; in this case, rank(Zh X(I − F † F )) = rank(Zh X) = Nu T , via Lemma 1. The rank of F increases as the number of locality constraints increases; this results in decreased rank for I − F † F , and possibly also decreased rank for Zh X(I − F † F ). However, due to the repetitive structure of Zh X , this is not always the case — it is possible to have locality constraints that do not lead to decreased rank for Zh X(I − F † F ). We provide a detailed numerical example of this in Section C. Unfortunately, checking the conditions of Theorem 2 is computationally expensive. In particular, we must assemble and compute the rank of matrix Zh X(I − F † F ). The complexity of this operation is dependent on the size of the matrix, which increases as Φ becomes more sparse (as enforced by locality constraints). This is a problem, since it is generally preferable to use very sparse Φ, as previously mentioned — this would correspond to an extremely large matrix Zh X(I −F † F ), which is time-consuming to compute with. Ideally, sparser Φ should instead correspond to lower complexity; this is the motivation for the next formulation. B. LOCALITY-FIRST FORMULATION We first parameterize (11b), then (11a). This directly gives a closed-form expression for localized trajectory set YL (x0 ). First, some definitions: Definition 5. Support vector indices M denote the set of #» #» indices of Φ such that (Φ)M ̸= 0 is compatible with locality constraint (11b). Let NM be the cardinality of M. Remark: this is complementary to Definition 4. Instead of looking at which indices are constrained to be zero, we now look at which indices are allowed to be nonzero. A subtlety is that this definition considers the entirety of Φ, while Definition 4 omits the first Nx rows of Φ. Lemma 3. (Image space representation of localized trajectory set) Assume there exists some Φ that satisfies constraints (11a) and (11b). Then, the localized trajectory set from state x0 is described by the following: YL (x0 ) = {y : y = (X2 ):,M H † k + † (X2 ):,M (I − H H)γ, γ ∈ RNM } where X2 := (X)Nx +1:,: # » I and H := and k := 0 and the size of the localized trajectory set is blk (ZAB ):,M dim(YL (x0 )) = rank((X2 ):,M (I − H † H)) Proof: Future trajectory y can be written as #» #» y = X2 Φ = (X2 ):,M (Φ)M (19) where the first equality arises from the definitions of y and X , and the second equality arises from the fact that zeros #» in Φ do not contribute to y; thus, we only need to consider #» nonzero values (Φ)M . Using the augmented matrix of ZAB , constraint (11a) can #» blk #» be rewritten as ZAB Φ = k . Nonzero values (Φ)M must obey #» H(Φ)M = k (20) Constraint (20) is feasible exactly when constraints (11a) and (11b) are feasible. By assumption, solutions exist, so we can parameterize the solution space as #» (Φ)M = H † k + (I − H † H)γ (21) where γ is a free variable. Substituting (21) into (19) gives the desired expression for YL (x0 ) and its size. Theorem 3. (Optimal global performance) If x0 has at least one nonzero value, then localized MPC attains optimal global performance if 1) there exists some Φ that satisfies constraints (11a) and (11b), and 2) rank((X2 ):,M (I − H † H)) = Nu T Proof: 6 VOLUME XX 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 𝑢1 𝑥1 FIGURE 1. 𝑢2 𝑥2 𝑥3 actuation plant Example system with three nodes, two of which are actuated. Similar to Theorem 2; instead of applying Lemma 2, apply Lemma 3. To check the conditions of Theorem 3, we must assemble and compute the rank of matrix (X2 ):,M (I − H † H). The complexity of this operation is dependent on the size of this matrix, which decreases as Φ becomes more sparse (as enforced by locality constraints). This is beneficial, since it is preferable to use very sparse Φ, which corresponds to a small matrix (X2 ):,M (I −H † H) that is easy to compute with. This is in contrast with the previous formulation, in which sparser Φ corresponded to increased complexity. However, from a theoretical standpoint, this formulation does not provide any intuition on the relationship between the trajectory set Y(x0 ) and the localized trajectory set YL (x0 ). For completeness, we now use the locality-first formulation to provide a closed-form expression of Y(x0 ). The resulting expression is equivalent to — though decidedly more convoluted than — the expression in Lemma 1: Lemma 4. (Image space representation of trajectory set) The trajectory set from state x0 is described by: Y(x0 ) = {y : blk y = X2 ZAB k X2 (I − + blk† blk ZAB )γ, ZAB γ ∈ RNΦ } Proof: In the absence of locality constraints, M includes all indices #» of Φ since all entries are allowed to be nonzero. Here, NM = #» #» blk NΦ , (X2 ):,M = X2 , (Φ):,M = Φ, and H = ZAB . Substitute these into the expression in Lemma 3 to obtain the desired result. blk Remark: By definition, X2 ZAB k = Zp x0 and X2 (I − blk† blk ZAB ZAB ) = Zh X . Substituting these quantities into Lemma 4 recovers Lemma 1. C. NUMERICAL EXAMPLE To provide some intuition on the results from the previous subsections, we present a simple numerical example. We work with a system of three nodes in a chain interconnection, as shown in Fig. 1. The system matrices are:     1 2 0 1 0 A = 3 4 5 , B = 0 0 (22) 0 6 7 0 1 ⊤ We set initial state x0 = 1 1 1 , and choose a predictive horizon size of T = 1. Here, NΦ = Nx (T + 1) + Nu T = 8. We choose locality constraints L such that each node may only communicate with its immediate neighbors; node 1 with node 2, node 2 with both nodes 1 and 3, and node 3 with node 2. Then, locality constraint (11b) is equivalent to  ∗ ∗  0  ∗ Φ= ∗  0  ∗ 0  ∗ 0 ∗ ∗  ∗ ∗  ∗ 0  ∗ ∗  ∗ ∗  ∗ 0 ∗ ∗ (23) where ∗ indicate values that are allowed to be nonzero. The support vector indices are M = {1, 2, 4, 5, 7, 9 − 16, 18, 19, 21, 22, 24}, and the constrained vector indices are L = {3, 5, 11, 14} (recall that indices in L do not include the first Nx rows of Φ). We confirm that there exists some Φ that satisfies both dynamics constraints (11a) and locality constraints (11b) by checking that constraint (20) is feasible. We start with dynamics-first formulation. In our case, Zh = 05×3 c1 05×1 c2 c1 c2 (24) where c1 and c2 are defined as     1 0 0 0 1  1  0 1 , c2 :=  (25) c1 :=     2  2  1 0 0 1 Zh has a rank of 2. Then, Zh X = Zh Zh Zh , also has a rank of 2. Per Lemma 1, this is the size of the trajectory set Y(x0 ), which is exactly equal to Nu T , as expected. We write out Zh X and Zh X(I − F † F ) in full in equations (26) and (27). Boxed columns represent columns zeroed out as a result of locality constraints, i.e. if we replace the boxed columns in Zh X with zeros, we obtain Zh X(I − F † F ). In our example, Zh X(I − F † F ) also has a rank of 2; by Theorem 2, the local trajectory set is equal to the trajectory set, and by Theorem 2, the localized MPC problem attains optimal global performance. Two observations are in order. First, we notice that the rank of Zh X (= 2) is low compared to the number of nonzero columns (= 12), especially when x0 is dense. Additionally, the structure of Zh X is highly repetitive; the only two linearly independent columns are c1 and c2 , and each appears 6 times in Zh X . Furthermore, the specific values of x0 do not affect the rank of these matrices — only the placement of nonzeros and zeros in x0 matters. Second, notice that post-multiplying Zh X by (I − F † F ) effectively zeros out columns of Zh X . However, due to the repetitive structure of Zh X , this does not result in decreased rank for Zh X(I − F † F ). In fact, it is difficult to find a feasible locality constraint that results in decreased rank. This observation is corroborated by simulations in Section V, in which we find that locality constraints that are feasible also typically preserve global performance. For more complex systems and larger predictive horizons, postmultiplication of Zh X by (I − F † F ) no longer cleanly VOLUME XX 2023 7 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 J. S. LI ET AL.: GLOBAL PERFORMANCE GUARANTEES FOR LOCALIZED MODEL PREDICTIVE CONTROL Zh X = 05×3 c1 05×1 c2 c1 c2 05×3 c1 05×1 c2 c1 c2 05×3 c1 05×1 c2 c1 c2 (26) Zh X(I − F † F ) = 05×3 c1 05×2 c1 05×4 c1 05×1 c2 c1 c2 05×5 c2 05×1 c2 (X2 ):,M (I − H † H) = 05×2 c1 05×1 c1 05×3 c1 05×1 c2 c1 c2 05×3 c2 c2 corresponds to zeroing out columns, but similar intuition applies. We now apply the locality-first formulation. To check the conditions of Theorem 3, we must construct the matrix (X2 ):,M (I − H † H) and check its rank. This matrix is written out in equation (28). As expected, the rank of this matrix is also equal to two. Additionally, notice that (X2 ):,M (I − H † H) contains the same nonzero columns as Zh X(I − F † F ): c1 and c2 are each repeated four times, in slightly different orders. This is unsurprising, as the two formulations are equivalent. IV. ALGORITHMIC IMPLEMENTATION OF OPTIMAL LOCALITY SELECTION Leveraging the results of the previous section, we introduce an algorithm that selects the appropriate locality constraints for localized MPC. For simplicity, we restrict ourselves to locality constraints corresponding to d-local neighborhoods, though we remark that Subroutine 1 is applicable to arbitrary communication structures. The localized MPC problem can be solved via distributed optimization techniques; the resulting distributed and localized MPC problem enjoys complexity that scales with locality parameter d, as opposed to network size N [12]. Thus, when possible, it is preferable to use small values of d to minimize computational complexity. For a given system and predictive horizon length, Algorithm 1 will return the optimal locality size d — the smallest value of d that attains optimal global performance. As previously described, the specific values of x0 do not matter — only the placement of nonzeros and zeros in x0 matters. We will restrict ourselves to considering dense values of x0 . For simplicity, our algorithm will work with the vector of ones as x0 — the resulting performance guarantees hold for any dense x0 . To check if a given locality constraint preserves global performance, we must check the two conditions of Theorem 3. First, we must check whether there exists some Φ that satisfies both dynamics and locality constraints; this is equivalent to checking whether (20) is feasible. We propose to check whether ∥H(H † k) − k∥∞ ≤ ϵ (29) for some tolerance ϵ. Condition (29) can be distributedly computed due to the block-diagonal structure of H . Define partitions [H]i such that H = (27) (28) blkdiag([H]1 , [H]2 . . . [H]N )3 . Then, (29) is equivalent to ∥[H]i ([H]†i [k]i ) − [k]i ∥∞ ≤ ϵ ∀i (30) If this condition is satisfied, then it remains to check the second condition of Theorem 3. To so, we must construct matrix J := (X2 ):,M (I − H † H) and check its rank. Notice that J can be partitioned into submatrices Ji , i.e. J := J1 J2 . . . JN , where each block Ji can be constructed using only information from subsystem i, i.e. [H]i , [k]i , etc. Thus, J can be constructed in parallel — each subsystem i performs Subroutine 1 to construct Ji . Subroutine 1 Local sub-matrix for subsystem i inputs: [H]i , [k]i , ϵ output: Ji † 1: Compute w = [H]i [k]i 2: if ∥[H]i w − [k]i ∥∞ > ϵ : Ji ← False else : Ji ← I − [H]†i [H]i return Subroutine 1 checks whether the dynamics and locality constraints are feasible by checking (30), and if so, returns the appropriate submatrix Ji . Notice that the quantity [H]i is used in both the feasibility check and in Ji . Also, x0 does not appear, as we are using the vector of ones in its place. Having obtained J corresponding to a given locality constraint, we need to check its rank to verify whether global performance is preserved, i.e. rank(J) = Nu T , as per Theorem 3. As previously described, we restrict ourselves to locality constraints of the d-local neighborhood type, preferring smaller values of d as these correspond to lower complexity for the localized MPC algorithm [12]. Thus, in Algorithm 1, we start with the smallest possible value of d = 1, i.e. subsystems communicate only with their immediate neighbors. If d = 1 does not preserve global performance, we iteratively increment d, construct J , and check its rank, until optimal global performance is attained. In Step 1 of Algorithm 1, we call Subroutine 1 to check for feasibility and construct submatrices Ji . If infeasibility is encountered, or if optimal global performance is not attained, we increment d; otherwise, we return optimal locality size d. 3 H should have Nx blocks, where Nx is the number of states. Since N ≤ Nx and one subsystem may contain more than one state, we are able to partition H into N blocks as well. 8 VOLUME XX 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 Algorithm 1 Optimal local region size inputs: A, B , T , ϵ output: d for d = 1 . . . N : 1: for i = 1 . . . N : Construct [H]i , [k]i Run Subroutine 1 to obtain Ji if Ji is False : continue 2: Construct J = J1 . . . JN 3: if rank(J) = Nu T : return d A. COMPLEXITY To analyze complexity, we first make some simplifying scaling assumptions. Assume that the number of states Nx and inputs Nu are proportional to the number of subsystems N , i.e. O(Nx + Nu ) = O(N ). Also, assume that the number of nonzeros NM for locality constraint corresponding to parameter d are related to one another as O(NM ) = O(N dT ). Steps 1 and 3 determine the complexity of the algorithm. In Step 1, each subsystem performs operations on matrix [H]i , which has size of approximately Nx (T + 1) by dT — the complexity will vary depending on the underlying implementations of the pseudoinverse and matrix manipulations, but will generally scale according to O((dT )2 Nx T ), or O(T 3 d2 N ). In practice, d and T are typically much smaller than N , and this step is extremely fast; we show this in the next section. In Step 3, we perform a rank computation on a matrix of size (Nx +Nu )T by NM . The complexity of this operation, if Gaussian elimination is used, is O((Nx +Nu )1.38 T 1.38 NM ), or O(T 2.38 N 2.38 d). Some speedups can be attained by using techniques from [29], which leverage sparsity — typically, J is quite sparse, with less than 5% of its entries being nonzero. In practice, Step 3 is the dominating step in terms of complexity. We remark that this algorithm needs only to be run once offline for any given localized MPC problem. Given a system and predictive horizon, practitioners should first determine the optimal locality size d using Algorithm 1, then run the appropriate online algorithm from [12]. V. SIMULATIONS We first present simulations to supplement runtime characterizations of Algorithm 1 from the previous section. Then, we use the algorithm to investigate how optimal locality size varies depending on system size, actuation density, and prediction horizon length. We find that optimal locality size is primarily a function of actuation density. We also verify in simulation that localized MPC performs identically to global MPC when we use the optimal locality size provided by Algorithm 1, as expected. Code needed to replicate all simulations can be found at https://github.com/flyingpeach/ FIGURE 2. An example of an 11 by 11 grid topology generated using the described random procedure. Each node on the grid is associated with two states (angle and frequency). LocalizedMPCPerformance. This code makes use of the SLS-MATLAB toolbox [30], which includes an implementation of Algorithm 1. Note on MATLAB implementation: When implementing subroutine 1 in MATLAB, use of the backslash operator (i.e. H\k) is faster than the standard pseudoinverse function (i.e. pinv(H)*k). The backslash operator also produces Ji matrices that are as sparse as possible, which facilitates faster subsequent computations. A. SYSTEM AND PARAMETERS We start with a two-dimensional n by n square mesh. Every pair of neighboring nodes have a 40% probability to be connected by an edge. The expected number of edges is 0.8 × n × (n − 1). We consider only fully connected graphs4 . An example is given in Fig. 2. The underlying assumption of this random procedure is that each node has some physical location, and edges are only formed between nodes that are physically nearby — many other real-world systems (e.g. transport systems, water canals) exhibit similar connectivity and sparsity patterns. Each node i represents a two-state subsystem of linearized and discretized swing equations X θ(t) θ(t + 1) + [B]i [u]i (31a) [A]ij = ω(t) j ω(t + 1) i j∈N1 (i) ∆t , di 1 − mi ∆t 0 0 0 [A]ij = kij , [B]i = 1 ∆t 0 mi [A]ii = 1 ki −m ∆t i (31b) where [θ]i , [ω]i , and [u]i are the phase angle deviation, frequency deviation, and control action of the controllable load of bus i. Parameters are m−1 (inertia), di (damping), i and kij (coupling); they are randomly drawn from uniform distributions over [0, 2], [1, 1.5] P and [0.5, 1] respectively. Self term ki is defined as ki := j∈N1 (i) kij . Discretization step size ∆t is 0.2. 4 Unconnected system graphs imply some amount of dynamical decoupling, which makes distributed control easier than the fully connected case. We consider only the harder case in simulations — naturally, our method will do just as well on the easier case. VOLUME XX 2023 9 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 J. S. LI ET AL.: GLOBAL PERFORMANCE GUARANTEES FOR LOCALIZED MODEL PREDICTIVE CONTROL 100 100 10-2 10-2 20 40 60 80 100 120 10 20 30 6 6 6 5 5 5 4 4 4 3 3 3 2 2 2 1 FIGURE 3. Runtime of matrix construction (Step 1, green) and rank determination (Step 3, pink) of Algorithm 1 vs. network size and horizon length. Parallelized (i.e. per-subsystem) runtimes are shown for matrix construction. The algorithm was run for grids containing 16, 25, 36, 64, and 121 subsystems. For each point, we run the algorithm on five different systems, and plot the average and standard deviation — here, the standard deviation is so small that it is barely visible. As expected, the rank determination step dominates total runtime, while the matrix construction step is extremely fast. Under the given parameter ranges, the system is typically neutrally stable, with a spectral radius of 1. The baseline system parameters are n = 5 (corresponding to a 5 × 5 grid containing 25 nodes, or 50 states) and 100% actuation. Note: this does not correspond to ‘full actuation’ in the standard sense; it means that each subsystem (which contains 2 states) has one actuator — 50% of states are actuated. We use a prediction horizon length of T = 15 unless otherwise stated. B. ALGORITHM RUNTIME We plot the runtime of Algorithm 1 in Fig. 3 for different system sizes and horizon lengths. We separately consider runtimes for matrix construction (Step 1) and rank determination (Step 3). The former is parallelized, while the latter is not. Runtime for matrix construction is extremely small. Even for the grid with 121 subsystems (242 states), this step takes less than a millisecond. Interestingly, matrix construction runtime also stays relatively constant with increasing network size, despite the worst-case runtime scaling linearly with N , as described in the previous section. This is likely due to the sparse structure of H . Conversely, matrix construction runtime increases with increasing horizon length. Runtime for rank determination dominates total algorithm runtime, and increases with both system size and horizon length. Rank determination runtime appears to increase more sharply with increasing horizon length than with increasing system size. Further runtime reductions may be achieved by taking advantage of techniques described in the previous section; however, even without additional speedups, the runtime is no more than 10 seconds for the grid with 242 states. C. OPTIMAL LOCALITY SIZE AS A FUNCTION OF SYSTEM PARAMETERS We characterize how optimal locality size changes as a function of the system size and horizon length — the results 1 0.2 0.4 0.6 0.8 1 1 20 40 60 80 100 120 10 20 30 FIGURE 4. Optimal locality size as a function of various parameters. Each point represents the average over five different systems; standard deviations are shown by the fill area. (Left) Optimal locality size vs. actuation density. The two are inversely correlated. (Center) Optimal locality size vs. network size for 60% actuation (pink), 80% actuation (blue), and 100% actuation (green). For 60% and 80% actuation, optimal locality size roughly increases with network size. For 100% actuation, the optimal locality size is always 1, independent of network size. (Right) Optimal locality size vs. predictive horizon length for 60% actuation (pink), 80% actuation (blue), and 100% actuation (green). For 60% and 80% actuation, optimal locality size increases with horizon size up until T = 10, then stays constant afterward. For 100% actuation, the optimal locality size is always 1. are summarized in Fig. 4. Actuation density is the main factor that affects optimal locality size. Remarkably, at 100% actuation, the optimal locality size always appears to be d = 1, the smallest possible size (i.e. communication only occurs between nodes that share an edge). As we decrease actuation density, the required optimal locality size increases. This makes sense, as unactuated nodes must communicate to at least the nearest actuated node, and the distance to the nearest actuated node grows as actuation density decreases. The optimal locality size also increases as a function of system size — but only when we do not have 100% actuation. At 60% actuation, for 121 nodes, the optimal locality size is around d = 5; this still corresponds to much less communication than global MPC. Predictive horizon length does not substantially impact optimal locality size. At short horizon lengths (T ≤ 10), we see some small correlation, but otherwise, optimal locality size stays constant with horizon size. Similarly, the stability of the system (i.e. spectral radius) appears to not affect optimal locality size; this was confirmed with simulations over systems with spectral radius of 0.5, 1.0, 1.5, 2.0, and 2.5 for 60%, 80%, and 100% actuation (not included in the plots). D. LOCALIZED PERFORMANCE From the previous section, we found that with 100% actuation, the optimal locality size is always 1. This means that even in systems with 121 subsystems, each node need only communicate with its immediate neighbors (i.e. 4 or less other nodes) to attain optimal global performance. This 10 VOLUME XX 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 is somewhat surprising, as this is a drastic (roughly 30fold) reduction in communication compared to global MPC. To confirm this result, we ran simulations on 20 different systems of size N = 121 and 100% actuation. We use LQR objectives with random positive diagonal matrices Q and R, and state bounds θi ∈ [−4, 4] for phase states and ωi ∈ [−20, 20] for frequency states. We use random initial conditions where each value (x0 )i is drawn from a uniform distribution over [−2, 2]. For each system, we run localized MPC with d = 1, then global MPC, and compare their costs over a simulation of 20 timesteps. Over 20 simulations, we find the maximum cost difference between localized and global MPC to be 5.6e-6. Thus, we confirm that the reported optimal locality size is accurate, since the cost of localized and global MPC are nearly identical. As previously described, localized MPC enjoys additional scalability benefits. In this particular case, the size of each subproblem is very small, since it involves at most 10 states5 . This is the size of the subproblem for any system size — if there are 10 nodes, each node will solve a 10-state subproblem in parallel; if there are 100, or even 1000 nodes, each node will still solve a 10-state subproblem in parallel, and the resulting runtime and complexity will be the same [12]. This is in contrast with standard (i.e. global) MPC, in which the complexity will increase with system size. E. FEASIBILITY VS. OPTIMALITY OF LOCALITY CONSTRAINTS In Algorithm 1, a given locality size is determined to be suboptimal if (1) the locality constraints are infeasible, or (2) the locality constraints are feasible, but matrix J has insufficient rank. The example from Section C suggests that the second case is rare — to further investigate, we performed 200 random simulations, in which all parameters were randomly selected from uniform distributions — grid size N from [4, 11] (corresponding to system sizes of up to 121 subsystems), actuation density from [0.2, 1.0], spectral radius from [0.5, 2.5] and horizon length from [3, 20]. In these 200 simulations, we encountered 4 instances where a locality constraint was feasible but resulted in insufficient rank; in the vast majority of cases, if a locality constraint was feasible, the rank condition was satisfied as well. VI. CONCLUSIONS In this work, we provided analysis and guarantees on locality constraints and global performance. We presented lemmas, theorems, and an algorithm to certify optimal global performance — these are the first results of their kind, to the best of our knowledge. We then leveraged these theoretical results to provide an algorithm that determines the optimal locality constraints that will expedite computation while preserving the performance — this is the first exact method to compute the optimal locality parameter d for DLMPC. 5 Each subproblem consists of up to 5 subsystems: the self subsystems plus a maximum of 4 neighbors. Each subsystem has 2 states Several directions of future work may be explored: 1) The results in this work can be leveraged to investigate the relationship between network topology and optimal locality constraints, i.e. the strictest communication constraints that still preserve optimal global performance. Certain topologies may require long-distance communication between handful of nodes; others may require no long-distance communications. A more thorough characterization will help us understand the properties of systems that are suited for localized MPC. 2) Algorithm 1 considers d-local communication constraints; a natural extension is to consider non-uniform local communication constraints, which are supported by the theory presented in this work. A key challenge of this research direction is the combinatorial nature of available local communication configurations; insights from the research direction suggested above will likely help narrow down said set of configurations. 3) Simulations suggest that feasibility of a given locality constraint overwhelmingly coincides with optimal global performance. This poses the question of whether feasibility of a given locality constraint is sufficent for J to be full rank under certain conditions, and what these conditions may be. Additional investigation could reveal more efficient implementations of Algorithm 1, as bypassing the rank checking step would save a substantial amount of computation time. 4) This work focuses on nominal trajectories. Additional investigation is required to characterize the impact of locality constraints on trajectories robust to disturbances. For polytopic disturbances, the space (or minima) of available values of Ξg from (10) in [12] is of interest. Due to the additional variables in the robust MPC problem, we cannot directly reuse techniques from this paper, though similar ideas may be applicable. REFERENCES [1] A. Venkat, I. Hiskens, J. Rawlings, and S. Wright, “Distributed MPC strategies with application to power system automatic generation control,” IEEE Trans. Control Syst. Tech., vol. 16, no. 6, pp. 1192– 1206, 2008. [2] Y. Zheng, S. Li, and H. Qiu, “Networked coordination-based distributed model predictive control for large-scale system,” IEEE Trans. Control Syst. Tech., vol. 21, no. 3, pp. 991–998, 2013. [3] P. Giselsson, M. D. Doan, T. Keviczky, B. D. Schutter, and A. Rantzer, “Accelerated gradient methods and dual decomposition in distributed model predictive control,” Automatica, vol. 49, no. 3, pp. 829–833, 2013. [4] C. Conte, C. N. Jones, M. Morari, and M. N. Zeilinger, “Distributed synthesis and stability of cooperative distributed model predictive control for linear systems,” Automatica, vol. 69, pp. 117–125, 2016. [5] R. E. Jalal and B. P. Rasmussen, “Limited-Communication Distributed Model Predictive Control for Coupled and Constrained Subsystems,” IEEE Trans. Control Syst. Tech., vol. 25, no. 5, pp. 1807–1815, 2017. [6] Z. Wang and C.-J. Ong, “Distributed MPC of constrained linear systems with online decoupling of the terminal constraint,” in Proc. IEEE ACC, 2015, pp. 2942–2947. VOLUME XX 2023 11 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Open Journal of Control Systems. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/OJCSYS.2023.3308009 J. S. LI ET AL.: GLOBAL PERFORMANCE GUARANTEES FOR LOCALIZED MODEL PREDICTIVE CONTROL [7] A. N. Venkat, J. B. Rawlings, and S. J. Wright, “Stability and optimality of distributed model predictive control,” in Proc. IEEE CDC, 2005, pp. 6680–6685. [8] Y. R. Sturz, E. L. Zhu, U. Rosolia, K. H. Johansson, and F. Borrelli, “Distributed learning model predictive control for linear systems,” in Proc. IEEE CDC, 2020, pp. 4366–4373. [9] C. Conte, M. N. Zeilinger, M. Morari, and C. N. Jones, “Robust distributed model predictive control of linear systems,” in Proc. IEEE ECC, 2013, pp. 2764–2769. [10] A. Richards and J. P. How, “Robust distributed model predictive control,” Int. J. Control, vol. 80, no. 9, pp. 1517–1531, 2007. [11] C. Amo Alonso and N. Matni, “Distributed and localized closed-loop model predictive control via system level synthesis,” in Proc. IEEE CDC, 2020, pp. 5598–5605. [12] C. Amo Alonso, J. S. Li, N. Matni, and J. Anderson, “Distributed and localized model predictive control. Part I: Synthesis and implementation,” To appear in IEEE Transactions on Control of Network Systems, 2021. [13] C. Amo Alonso, J. S. Li, J. Anderson, and N. Matni, “Distributed and localized model predictive control. Part II: Theoretical guarantees,” To appear in IEEE Transactions on Control of Network Systems, 2022. [14] S. Boyd, N. Parikh, E. Chu, B. Peleato, J. Eckstein et al., “Distributed optimization and statistical learning via the alternating direction method of multipliers,” Foundations and Trends® in Machine learning, vol. 3, no. 1, pp. 1–122, 2011. [15] J. Anderson, J. C. Doyle, S. H. Low, and N. Matni, “System level synthesis,” Annual Reviews in Control, vol. 47, pp. 364–393, 2019. [16] A. Gasparri and A. Marino, “A distributed framework for kk-hop control strategies in large-scale networks based on local interactions,” IEEE Transactions on Automatic Control, vol. 65, no. 5, pp. 1825– 1840, 2020. [17] L. Ballotta and V. Gupta, “Faster consensus via sparser controller,” arXiv preprint arXiv:2302.01021, 2023. [18] L. Ballotta, M. R. Jovanović, and L. Schenato, “Can decentralized control outperform centralized? the role of communication latency,” IEEE Transactions on Control of Network Systems, pp. 1–11, 2023. [19] J. Jiao, H. L. Trentelman, and M. K. Camlibel, “Distributed linear quadratic optimal control: Compute locally and act globally,” IEEE Control Systems Letters, vol. 4, no. 1, pp. 67–72, 2020. [20] S. Shin, Y. Lin, G. Qu, A. Wierman, and M. Anitescu, “Near-optimal distributed linear-quadratic regulator for networked systems,” arXiv preprint arXiv:2204.05551, 2022. [21] T. Summers and J. Ruths, “Performance bounds for optimal feedback control in networks,” in Proc. IEEE ACC, 2018, pp. 203–209. [22] H. K. Mousavi and N. Motee, “Explicit characterization of performance of a class of networked linear control systems,” IEEE Transactions on Control of Network Systems, vol. 7, no. 4, pp. 1688– 1699, 2020. [23] W. Tang and P. Daoutidis, “The role of community structures in sparse feedback control,” in Proc. IEEE ACC, 2018, pp. 1790–1795. [24] J. S. Baras and P. Hovareshti, “Effects of topology in networked systems: Stochastic methods and small worlds,” in Proc. IEEE CDC, 2008, pp. 2973–2978. [25] S. Lucia, M. Kögel, P. Zometa, D. Quevedo, and R. Findeisen, “Predictive control in the era of networked control and communication - a perspective,” IFAC-PapersOnLine, vol. 48, no. 23, pp. 322–331, 2015. [26] A. Bemporad and D. Barcelli, Decentralized Model Predictive Control. London: Springer London, 2010, pp. 149–178. [27] J. S. Li, C. Amo Alonso, and J. C. Doyle, “Frontiers in Scalable Distributed Control: SLS, MPC, and beyond,” in IEEE American Control Conference, 2021, pp. 2720–2725. [28] I. R. Shafarevich and A. O. Remizov, Linear algebra and geometry. Springer Science & Business Media, 2012. [29] H. Y. Cheung, T. C. Kwok, and L. C. Lau, “Fast matrix rank algorithms and applications,” Journal of the ACM (JACM), vol. 60, no. 5, pp. 1– 25, 2013. [30] J. S. Li, “SLS-MATLAB: Matlab toolbox for system level synthesis,” 2019. [Online]. Available: https://github.com/sls-caltech/sls-code JING SHUANG (LISA) LI (Student Member, IEEE) received a B.A.Sc. in engineering science from the University of Toronto in 2018, and a Ph.D. in Control and Dynamical Systems from California Institute of Technology in 2023, where she was supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada. Her research interests include distributed control and its applications to biological modeling and cyberphysical systems. CARMEN AMO ALONSO (Student Member, IEEE) received a B.Sc. in aerospace engineering from the Polytechnic University of Madrid (Spain) in 2016, an M.Sc. in space engineering from California Institute of Technology in 2017, and a Ph.D. in Control and Dynamical Systems from California Institute of Technology in 2023, where she was an AI4Science Amazon/Caltech fellow. Her research interests include distributed optimal control, convex optimization, parallel programming, and the applications of control theory to language models. 12 VOLUME XX 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/
https://openalex.org/W2034308921	https://europepmc.org/articles/pmc3231033?pdf=render	English	null	Novel Designs for Application Specific MEMS Pressure Sensors	Sensors	2,010	cc-by	9,943	⋆Authors to whom correspondence should be addressed; ⋆Authors to whom correspondence should be addressed; E-Mail: giulio.fragiacomo@nanotech.dtu.dk (G.F.); Tel.: +45 4525 6316; Fax +45 4588 7762; E-Mail: kasper.reck@nanotech.dtu.dk (K.R.); Tel.: +45 4525 6311; Fax: +45 4588 7762. E-Mail: giulio.fragiacomo@nanotech.dtu.dk (G.F.); Tel.: +45 4525 6316; Fax +45 4588 7762; E-Mail: kasper.reck@nanotech.dtu.dk (K.R.); Tel.: +45 4525 6311; Fax: +45 4588 7762. Received: 2 August 2010; in revised form: 25 September 2010 / Accepted: 20 October 2010 / Published: 28 October 2010 Received: 2 August 2010; in revised form: 25 September 2010 / Accepted: 20 October 2010 / Published: 28 October 2010 Abstract: In the framework of developing innovative microfabricated pressure sensors, we present here three designs based on different readout principles, each one tailored for a speciﬁc application. A touch mode capacitive pressure sensor with high sensitivity (14 pF/bar), low temperature dependence and high capacitive output signal (more than 100 pF) is depicted. An optical pressure sensor intrinsically immune to electromagnetic interference, with large pressure range (0–350 bar) and a sensitivity of 1 pm/bar is presented. Finally, a resonating wireless pressure sensor power source free with a sensitivity of 650 KHz/mmHg is described. These sensors will be related with their applications in harsh environment, distributed systems and medical environment, respectively. For many aspects, commercially available sensors, which in vast majority are piezoresistive, are not suited for the applications proposed. Keywords: micromachined; pressure; capacitive; optical; wireless; silicon; MEMS; MOEMS ywords: micromachined; pressure; capacitive; optical; wireless; silicon; MEMS; Sensors 2010, 10, 9541-9563; doi:10.3390/s101109541 OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Article Novel Designs for Application Speciﬁc MEMS Pressure Sensors Giulio Fragiacomo ⋆, Kasper Reck ⋆, Lasse Lorenzen and Erik V. Thomsen Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345E, DK-2800 Kgs. Lyngby, Denmark; E-Mails: lasse.lorenzen@nanotech.dtu.dk (L.L.); erik.v.thomsen@nanotech.dtu.dk (E.V.T.) ⋆Authors to whom correspondence should be addressed; E-Mail: giulio.fragiacomo@nanotech.dtu.dk (G.F.); Tel.: +45 4525 6316; Fax +45 4588 7762; E-Mail: kasper.reck@nanotech.dtu.dk (K.R.); Tel.: +45 4525 6311; Fax: +45 4588 7762. Received: 2 August 2010; in revised form: 25 September 2010 / Accepted: 20 October 2010 / Published: 28 October 2010 Sensors 2010, 10, 9541-9563; doi:10.3390/s101109541 Giulio Fragiacomo ⋆, Kasper Reck ⋆, Lasse Lorenzen and Erik V. Thomsen Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345E, DK-2800 Kgs. Lyngby, Denmark; E-Mails: lasse.lorenzen@nanotech.dtu.dk (L.L.); erik.v.thomsen@nanotech.dtu.dk (E.V.T.) Sensors 2010, 10 Sensors 2010, 10 the sensing element which can easily be translated into a pressure signal. This principle has been tested widely in the past 40 years and is often preferred over other approaches, thanks to the fact that only standard cleanroom process such as ion implantation, oxidation and etching are involved. Compatibility to IC processes and therefore cheap process development are not the only reasons to prefer a piezoresistive sensor, also excellent linearity and simple conditioning circuits are important and quality have been the key of the success of these kind of devices. Nonetheless, strong temperature dependence of the piezoresistive coefﬁcient π44, limited pressure range and scalability (due to necessity of accommodating the piezoresistors on the membrane), necessity of a DC conditioning circuit, dependence to packaging induced stress and low sensitivity have ruled out these sensors in many cases. In this work, we will ﬁrst present a touch mode capacitive pressure sensor (TMCPS) with a sensitivity of 14 pF/bar. A mathematical model that describes the behavior of TMCPS and can be used to ﬁt experimental data will be described. Given its ﬂat sensing surface, this sensor can easily be coated with a protective layer which makes it well suited for harsh environment applications. Furthermore, since the readout is capacitive, only an AC signal is needed and therefore the power dissipation is limited to the small amount given by the parasitic resistive contribution (i.e., the connectors). We will describe a microfabrication step that can be introduced in these type of sensors in order to reduce drastically the hysteresis without compromising their sensitivity. Moreover temperature characterization of this device has been carried out showing a very low temperature dependence in both normal and touch mode. Also the all-optical Bragg grating sensor which is presented in Section III is very well suited for harsh environments and lends itself to distributed sensing. All-optical sensors are unaffected by electromagnetic ﬁelds and noise and have low power consumption combined with good signal transmission, making them ideal for remote sensing. These properties makes them particularly well-suited for a number of demanding applications, e.g., sensing in the proximity of electrical generators, power lines and inside MRI scanners as well as sensing in fuel tanks and for oil explorations. 1. Introduction Commercially available micromachined pressure sensors are by a vast majority based on the piezoresistive effect, where a mechanical deformation causes a change in the electrical resistance of 9542 Sensors 2010, 10 The sensor we propose has been designed speciﬁcally for oil explorations, with focus on extremely large pressure ranges, up to 350 bar, and its frequency modulated output signal makes it the perfect candidate for distributed systems. A mathematical model which can be applied to Bragg grating optical sensors has also been developed. Finite elements analysis has been used to characterize the last device we propose: a wireless pressure sensor for medical applications. The footprint of the sensor is small, so it can be placed inside the bladder using a catheter through the urethra. This transducer has been encapsulated in silicone in order to be implantable in the bladder tissue. A mathematical model which relates the change in pressure to a shift of the resonant frequency of the sensor has been formulated. Here, a new design based on the optimization of the fabrication parameters and the introduction of a bossed plate as the sensing element will be illustrated. All three sensors proposed open up new possibilities for pressure measurements and have the scope to offer a solution for very sensitive pressure measurements, pressure monitoring in harsh environment or pressure monitoring inside the human body. Sensors 2010, 10 9543 2. Capacitive Pressure Sensors in Silicon Technology Capacitive sensing has been historically a mechanical engineering ﬁeld, mainly concerning positioning, pressure and sounds measurements [1]. In the beginning of the 70’s Heerens [2] reviewed various techniques to mechanically design capacitive sensors; in the same years Jones and Richards [3] described an ultra sensitive capacitive micrometer and in 1982 Hugill [4] presented a capacitive displacement transducer. These sensors usually employed a transformer ratio bridge circuit in order to achieve the maximum possible sensitivity and a high degree of immunity to parasitic capacitance. Transformer ratio bridges, together with charge transfer circuits and more complicated methods such as switched capacitor circuits are still used to interface capacitive transducers [5–7]. While signal conditioning methods has always relayed on analog or mixed-signals electronics, the capacitive senor design, due to the needs of miniaturization, power consumption and costs issues, has shifted from being realized by bulk mechanical parts to microfabricated silicon dies. An early example of this device was fabricated for medical purposes by Frobenius et al. [8] in 1973. In 1986 Ko [9] compares the capacitive pressure sensor with the well established piezoresistive device. In many respects capacitive sensing results more promising than its counterpart. In 1993, Puers [10], is of the same opinion as Ko and identiﬁes pressure as the main ﬁeld of application for capacitive measurements. In 1997, Baxter [11] wrote a book which threat thoroughly the wast subject of capacitive sensing but, with respect to silicon technology, he refers to the article written by Puers four years earlier. At the same time Eaton and Smith [12] reviewed the state of the art in micromachined pressure sensor technology, in their opinion capacitive micromachined pressure sensors were still under development. Only this century saw the ﬁrst commercialized devices as pointed out by Gao and Zhang [13] in 2004. Some example are the tire pressure monitoring (TPM) sensor developed by Motorola, VTI absolute pressure sensor for implantable medical devices and Vaisala barometric sensor. Finally, in 2007, Wise [14] predicts a future were MEMS of different type will be part of wireless integrated microsystems that will serve as the front-ends of information networks used in a huge variety of contexts; the capacitive pressure sensor is seen as one of the most probable candidate to be integrated in these systems. 2.1. Micromachined Touch Mode Capacitive Pressure Sensors: Models and Developme A micromachined capacitive pressure sensor which allows the sensing element to ﬂatten onto an insulation layer deposited on the bottom electrode is known as touch mode capacitive pressure sensor (TMCPS). The TMCPS was ﬁrstly introduced in 1990 by Ding et al. [15], while an array of capacitive sensing elements in parallel was presented in the work of Dudaicevs et al. [16] in 1994. We have developed a TMCPS which is made of an array of elements supported by a SiO2 honeycomb structure. The fabrication process, where the top plate is given by the device layer of a silicon on insulator wafer, was previously described by Pedersen et al. [17]. In Figure 1 an artistic view of the sensor is shown; the old design, Figure 1(a), which has a ﬂat substrate surface is compared to the new solution, Figure 1(b), where part of the membrane has been removed in order to make the newly introduced nanopillars structure visible. This type of sensor has the advantage of eliminating interconnection between elements, thus minimizing the active area and the parasitic capacitance. Furthermore, its ﬂat sensing surface is well 9544 Sensors 2010, 10 suited for coating with a corrosion resistant layer, thus making this sensor a good candidate for harsh environment applications where also a low power consumption is needed. Figure 1. Artistic view of a touch mode capacitive pressure sensors based on an array of capacitive elements. (a) Artistic view of the old design, the membrane (active area) has been made transparent in order to show the support structure. (b) Artistic view of the new design, part of the membrane has been removed in order to show the nanopillars structure. (a) (b) (a) (b) Moreover, an analytical solution for the deﬂection of the membrane in all the operation regimes has been found and used to ﬁt the experimental data [18]. In normal mode, i.e., when the maximum deﬂection of the membrane is less than its distance from the substrate, the governing equation for the deﬂection, (a) (b) (b) (a) Moreover, an analytical solution for the deﬂection of the membrane in all the operation regimes has been found and used to ﬁt the experimental data [18]. Sensors 2010, 10 Sensors 2010, 10 Figure 2. Schematic of the touch mode regime. The plate comes into contact with the insulation layer deposited on the substrate. The touching area and the suspended area of the plate are deﬁned by the pressure dependent variables ab(p) and av(p) which are linked by the radius of the plate a0. A polar coordinate system with the origin placed in the center of the unloaded plate has been used. The center deﬂection of the plate is measured along the negative z direction. Figure 2. Schematic of the touch mode regime. The plate comes into contact with the insulation layer deposited on the substrate. The touching area and the suspended area of the plate are deﬁned by the pressure dependent variables ab(p) and av(p) which are linked by the radius of the plate a0. A polar coordinate system with the origin placed in the center of the unloaded plate has been used. The center deﬂection of the plate is measured along the negative z direction. In Figure 3 it is possible to see the results given by the approximate model proposed; the blue dots represent the data points measured on the TMCPS described in [17] while the solid line is the ﬁtting curve. From this curve parameters such as the parasitic capacitance, the ﬂexural rigidity and the thickness of the insulation layer used to prevent short circuit when working in touch mode has been extracted. An excellent match between them and the measured ones was reported in [18] where also a solution for the integral C = Z 2π 0 Z a0 0 ϵ0ϵoxrdrdθ tox + ϵox(g −w(r, p)) (6) (6) has been found for both operating modes. has been found for both operating modes. has been found for both operating modes. Figure 3. Capacitance as a function of the pressure. The analytical model (solid line) ﬁts the experimental data (circles) and allows the extraction of important sensor parameters such as the parasitic capacitance, the insulating layer thickness and the ﬂexural rigidity. As expected, this curve is highly nonlinear, especially in the transition region (see inlet) between normal mode and touch mode operations. The capacitance of the sensor is measured with a HP 4294A Precision Impedance Analyzer which accuracy is better than 0.1% in the range of interest. The pressure is varied with a Druck DPI 520 pressure controller which accuracy is 0.025% of the reading. 2.1. Micromachined Touch Mode Capacitive Pressure Sensors: Models and Developme In normal mode, i.e., when the maximum deﬂection of the membrane is less than its distance from the substrate, the governing equation for the deﬂection, w, as a function of the radial distance, r, and the pressure, p, of a clamped circular isotropic plate is well known [19] 2 w(r, p) = pa4 0 64D 1 − r a0 2!2 (1) (1) where a0 is the radius of the plate and D is the ﬂexural rigidity of the plate given by D = Et3 12(1 −ν2) (2) (2) It will be shown that, in normal mode operations, the sensor proposed can be modeled under the assumption of linear elasticity and therefore described by Equation (1). A good approximation for the shape of the plate when working in touch mode (see Figure 2) is instead given by w(r, p) =      g 0 < r < ab(p) g 1 − r−ab(p) av(p) 22 ab(p) < r < a0 (3) (3) where g is the distance between the top electrode and the substrate at zero pressure, ab is the radius of the part of the membrane touching the substrate and av is calculated with av = a0 −ab (4) (4) The radius of the plate touching the bottom of the cavity, ab, is a function of pressure as is also the part of the diaphragm not touching the bottom plate, av. In the simpliﬁed model proposed, the radius av is simply given by the center deﬂection being ﬁxed at The radius of the plate touching the bottom of the cavity, ab, is a function of pressure as is also the part of the diaphragm not touching the bottom plate, av. In the simpliﬁed model proposed, the radius av is simply given by the center deﬂection being ﬁxed at av(p) ≡ 64Dg p 1/4 (5) (5) 9545 Sensors 2010, 10 Sensors 2010, 10 9546 At this point, based on the amount of research done in the ﬁeld and the fact that the working principle of the TMCPS has been fully investigated and tested, we can state that this is deﬁnitely the most developed of the devices proposed. None the less, process reliability, conditioning circuit complexity and hysteresis are some of the obstacles for the commercialization of this sensor at a level comparable to its piezoresistive counterpart. We present, with the new design, a solution to the hysteresis problem of TMCPS. The process sequence for the fabrication of the new device is shown in Figure 4. Firstly a silicon and a SOI (Silicon On Insulator) wafer with 2 µm are subjected to a one-hour phosphorous pre deposition in the ﬁrst processing step, Figure 4(a). A 600 nm thick oxide is grown on the silicon wafer and the cavity is etched to form the hexagonal structure mentioned earlier, Figure 4(b). The nanopillars are then developed on the bottom of the cavities with the process described later in this section, Figure 4(c). A second oxidation is performed to grow a 30 nm SiO2 layer around the pillars. This SiO2 layer serves as a short circuit protection when the sensor works in touch mode operation. In the next step a 10 µm wide groove is etched all the way through the device layer of the SOI wafer to the buried oxide such that it will serve as an electrical insulation between the active part of the sensor, i.e., the membrane area and the rest of the highly doped device layer. If these regions are not separated from each other, the area outside the membrane will add a considerable parasitic capacitance to the system. Then, the SOI wafer is turned upside down and fusion bonded with DSP wafer. Figure 4(d) shows the result of etching the insulation groove, bonding and removing the handle wafer. Finally, the separated part of the device layer,the upper and lower electrode are contacted by means of Al evaporation, Figure 4(e). Figure 4. Process sequence for touch mode capacitive pressure sensors with low hysteresis. (a) Phosphorous doping of both the double side polished (DSP) silicon wafer and the top (SOI) wafer. (b) Growing of a 600 nm SiO2 layer and etching of the cavity. (c) Etching of the nanopillar structure on the bottom of the cavity and performing a second oxidation. Sensors 2010, 10 Therefore the error bars cannot be seen given the high accuracy of the instrument used. Figure 3. Capacitance as a function of the pressure. The analytical model (solid line) ﬁts the experimental data (circles) and allows the extraction of important sensor parameters such as the parasitic capacitance, the insulating layer thickness and the ﬂexural rigidity. As expected, this curve is highly nonlinear, especially in the transition region (see inlet) between normal mode and touch mode operations. The capacitance of the sensor is measured with a HP 4294A Precision Impedance Analyzer which accuracy is better than 0.1% in the range of interest. The pressure is varied with a Druck DPI 520 pressure controller which accuracy is 0.025% of the reading. Therefore the error bars cannot be seen given the high accuracy of the instrument used. Sensors 2010, 10 (d) Etching of the insulation groove, fusion bonding and etching of the handle wafer. (e) Metalization of the contacts. Sensors 2010, 10 Sensors 2010, 10 9547 It must be noticed that, in order to reduce the amount of hysteresis, whether due to stiction forces or to poor quality of the insulating layer, a reduction in the contact area between the top electrode and the insulation layer is needed. Two different types of corrugation can serve this purpose, a deﬁnite one obtained with a lithographic step or a random corrugated surface obtained without the use of a mask. Even though the ﬁrst option requires an extra mask step (illustrated in Figure 4(c)), it must be preferred if the process involve fusion bonding of two wafers in order to keep a sufﬁciently smooth bonding surface. Furthermore, in the ﬁrst case, the lithography limits when deﬁning structures in a cavity (i.e., with a proximity distance between the mask and the photoresist) must be considered. Experimental results has proven to be extremely difﬁcult to approach the theoretical critical dimension, CD, given by [20] CD ≡2 3 s λ d −tr 2 , (7) (7) where λ is the wavelength of the source used, d is the proximity distance between the mask and the photoresist and tr is the photoresist thickness. In our case, with a photoresist thickness of 1.5 µm, a wavelength of 365 nm and a proximity distance equal to the gap distance between the two electrodes (600 nm) the theoretical limit was calculated to be 1.3 µm. However, to achieve good results pillars of 6 µm radius and around 50 nm height were fabricated with an isotropic SF6/O2 plasma etch (STS ICP Advanced Silicon Etcher). Figure 5 shows a contact proﬁlometer (Dektak8) measurement of the nanopillars structure fabricated on the bottom of a 600 nm cavity etched in SiO2. Figure 5. Proﬁlometer measurement of the pillar structure etched on the bottom of the cavities. The 50 nanometer pillars are measured inside the walls of the honeycomb structure which height is roughly 600 nm. A SEM image of a TMCPS fabricated with the pillar structure and diced afterwards is shown in Figure 6. A SEM image of a TMCPS fabricated with the pillar structure and diced afterwards is shown in Figure 6. A SEM image of a TMCPS fabricated with the pillar structure and diced afterwards is shown in Figure 6. 9548 Sensors 2010, 10 The choice of technology is a trade-off between the high ﬂexibility and sensing capabilities of electronics and the elimination of parasitic capacitance, extremely low transmission loss and immunity to electromagnetic interference of all-optical sensors. Several designs for optical pressure sensors have been suggested in Sensors 2010, 10 Sensors 2010, 10 Figure 6. A SEM image of a fabricated chip. Part of the top plate (A) has been removed in order to see the underlying pillar structure (B) which has been fabricated on the bottom of the cavities etched in the silicon wafer (C). In Figure 7 capacitance as a function of the pressure at different temperature is reported, from these curves 14 pF/bar sensitivity in touch mode (where there is a nearly linear relation) was calculated. In Figure 7 capacitance as a function of the pressure at different temperature is reported, from these curves 14 pF/bar sensitivity in touch mode (where there is a nearly linear relation) was calculated. Furthermore, a relative temperature coefﬁcient, α, of 0.008 %/◦C, 0.023 %/◦C and 0.044 %/◦C have been calculated near 40 ◦C, 60 ◦C and 80 ◦C using α ≡ CT −CT0 CT0(T −T0) · 100 (8) (8) where CT is the output signal at the temperature T and CT0 is the output signal at room temperature, T0. Pressure cycles (where the pressure has been risen up to 10 Bar and lowered back to 500 mbar) have been carried on in order to evaluate the hysteresis, κ, of the sensor as κ ≡Cdown(p) −Cup(p) Cup(p) · 100 (9) (9) where Cdown(p) is the value of the capacitance when the pressure is lowered and Cup(p) is the value of the capacitance when the pressure is raised. In Figure 8 a hysteresis comparison between a sensor fabricated without the nanopillars structure and one were the nanopillars were added in the process ﬂow is presented. In the letter case, the hysteresis was reduced to less than 1% in the entire pressure range. Moreover the hysteresis peak at touch point has been eliminated. These new results bring the TMCPS closer to the actual needs required for its commercialization. Sensors 2010, 10 9549 Sensors 2010, 10 Figure 7. Measured capacitance as a function of the pressure at varying temperatures. From these curves the relative pressure coefﬁcient in the different operating modes has been extracted. The capacitance of the sensor is measured with a HP 4294A Precision Impedance Analyzer which accuracy is better than 0.1% in the range of interest. The pressure is varied with a Druck DPI 520 pressure controller which accuracy is 0.025% of the reading. Therefore the error bars cannot be seen given the high accuracy of the instrument used. Figure 7. Measured capacitance as a function of the pressure at varying temperatures. From these curves the relative pressure coefﬁcient in the different operating modes has been extracted. The capacitance of the sensor is measured with a HP 4294A Precision Impedance Analyzer which accuracy is better than 0.1% in the range of interest. The pressure is varied with a Druck DPI 520 pressure controller which accuracy is 0.025% of the reading. Therefore the error bars cannot be seen given the high accuracy of the instrument used. Figure 8. Hysteresis, κ, comparison between two chips of the same batch, one of them fabricated with the pillar structure (black crosses), the other without (green triangles). Capacitances curves have been added to show qualitatively the hysteresis of the sensors. Figure 8. Hysteresis, κ, comparison between two chips of the same batch, one of them fabricated with the pillar structure (black crosses), the other without (green triangles). Capacitances curves have been added to show qualitatively the hysteresis of the sensors. 3. Optical Pressure Sensors 3. Optical Pressure Sensors Optical pressure sensors can in general be divided by detection method (amplitude or frequency modulated) and technology (electro-optical or all-optical design). Since amplitude modulation can be an on/off-signal, the sensitivity can be very high as in [21], where cantilever deﬂections of 10 pm is detected. However, an on/off-signal is not very well suited for multiplexing as each sensor requires its own transmission line. The sensitivity of frequency modulation is limited by the resolution of the readout system and while several frequency modulated optical sensor systems have been studied, the sensitivity is in general not as good as what can be obtained in amplitude modulated sensors. Sensors 2010, 10 Sensors 2010, 10 Sensors 2010, 10 literature, though with no or only very limited commercialization. While some of these sensors are made using III-IV technology [29], the ﬂexibility of silicon technology makes this a more obvious choice. An example of an electro-optical amplitude modulated pressure sensor has been given by Hall et al. [28] which combines a vertical-cavity surface-emitting laser (VCSEL), a membrane with integrated diffraction grating and photodetectors into a microphone capable of detecting down to 24 dB(A). Even though electro-optical designs show very good performance, they have none of the advantages that optical sensors have compared to electrical sensors (e.g., immunity to electromagnetic ﬁelds, low signal transmission loss and no risk of short-circuits), and are thus in direct competition with electrical sensors. In the domain of all-optical pressure sensors, the most prominent designs are the Fabry-Perot cavity [26] and the Mach-Zender interferometer [25,27] pressure sensors. In Fabry-Perot pressure sensors the deformation of a membrane causes a change in the width of a Fabry-Perot cavity which again changes the period and amplitude of the interference fringes. Depending on membrane size this can be a relatively sensitive design and using sensors with different cavity lengths, also suited for multiplexing. The Mach-Zender design utilizes the phase shift between a waveguide crossing a deﬂected membrane and a reference waveguide to measure pressure. The sensitivity of Mach-Zender sensors can be very high [22], but they are not easily applicable to distributed sensing systems. A common advantage to these two designs is that both are passive components i.e., there are no power requirements for the sensor itself. A closely related alternative to the Fabry-Perot sensor is the Bragg grating sensor, which creates a change in wavelength due to deformation of a waveguide with integrated Bragg grating. The Bragg grating sensor is also all-optical and frequency modulated and therefore has all the same inherent characteristics as the Fabry-Perot sensor. Contrary to the Fabry-Perot sensor, the Bragg grating sensor does not have multiple material interfaces in the light path, which reduces loss due to Fresnel reﬂection and it can be structured more ﬂexible, allowing for mechanical force ampliﬁcation. An example of a Bragg grating sensor is the ﬁber Bragg grating sensors (FBGs) [23], where ﬁbers with UV-written gratings act as sensing elements. Sensors 2010, 10 Fiber Bragg grating pressure sensors with detection limits of 0.36 kPa have been achieved [24], however, using MEMS technology, much better control of material and structural properties than what can be achieved in ﬁber technology would allow for much higher sensitivities [30]. 3. Optical Pressure Sensors Optical pressure sensors can in general be divided by detection method (amplitude or frequency modulated) and technology (electro-optical or all-optical design). Since amplitude modulation can be an on/off-signal, the sensitivity can be very high as in [21], where cantilever deﬂections of 10 pm is detected. However, an on/off-signal is not very well suited for multiplexing as each sensor requires its own transmission line. The sensitivity of frequency modulation is limited by the resolution of the readout system and while several frequency modulated optical sensor systems have been studied, the sensitivity is in general not as good as what can be obtained in amplitude modulated sensors. The choice of technology is a trade-off between the high ﬂexibility and sensing capabilities of electronics and the elimination of parasitic capacitance, extremely low transmission loss and immunity to electromagnetic interference of all-optical sensors. Several designs for optical pressure sensors have been suggested in 9550 3.1. All-optical MOEMS Bragg Grating Pressure Sensor It is in many situations in oil exploration critical to obtain real-time measurements of the pressure at different locations several kilometers under ground, far from the actual readout point. Also pressure and temperature at these depths can be very high. Electrical sensors are not well suited for this scenario as as they in general do not lend themselves towards remote distributed sensing due to transmission loss and parasitic capacitances and furthermore they carry the risk of a potential short circuit causing ignition, which could be fatal in this environment. Here we present an all-optical Bragg grating sensor for high pressure and remote distributed sensing. The basic principle behind Bragg grating sensors is mechanically induced modulations of the period of a Bragg grating, causing a change in the wavelength 9551 Sensors 2010, 10 of the reﬂected wave from the Bragg grating. The Bragg wavelength, λB, is expressed through the equation of the reﬂected wave from the Bragg grating. The Bragg wavelength, λB, is expressed through the equation λB = 2neﬀΛ (10) (10) where neﬀis the effective refractive index and Λ is the period of the Bragg grating. The relative change in wavelength of such a sensor is given by where neﬀis the effective refractive index and Λ is the period of the Bragg grating. The relative change in wavelength of such a sensor is given by ∆λB λB = (α + ζ)∆T + (1 −pe)εl (11) (11) where α is the thermal expansion coefﬁcient of the part of the waveguide containing the grating, εl is the longitudinal strain, ζ is the thermooptic coefﬁcient, pe is the photoelastic coefﬁcient for longitudinal strain and ∆T is the temperature change. Hence, the wavelength change is proportional to both temperature and the refractive index change due to strain. We will assume constant temperature, that the minimum detectable wavelength shift is 1 pm and that the Bragg wavelength is 1550 nm, hence the strain should be larger than approximately 10−6. 3.1. All-optical MOEMS Bragg Grating Pressure Sensor Considering a circular plate with a waveguide with integrated Bragg grating on top and under uniform load, p, see Figure 9, the strain components in polar coordinates, r, θ, are [31] εr = −σr −νσθ E = −3 8 (3r2 −a2 + ν2a2 −3r2ν2)p h2E (12) εθ = −σθ −νσr E = −3 8 (r2 −a2 + ν2a2 −r2ν2)p h2E (13) where ν is the Poisson ratio, σ is the stress, E is Young’s module, a is the plate radius and h is the plate thickness. The two strains have been plotted in Figure 10. (12) (13) where ν is the Poisson ratio, σ is the stress, E is Young’s module, a is the plate radius and h is the plate thickness. The two strains have been plotted in Figure 10. Figure 9. The optical pressure sensor, which 3D sketch (left ﬁgure) and cross section (right ﬁgure) are shown, consists of a circular plate with a waveguide on top. A Bragg grating is integrated into the half-circle of the waveguide. When the plate deforms the tangential strain in the grating causes a uniform change in grating period. 9552 Sensors 2010, 10 Figure 10. The radial and tangential strain in a circular plate in arbitrary units. Since the radial strain is not constant the Bragg grating would experience a non-uniform strain if it was place radially on the plate, causing lower reﬂection and broader reﬂection peak. However, while the tangentially strain is generally lower than the radial strain it would give a uniform strain in the Bragg grating was it to be placed along this direction. Since the tangential strain is monotonically decreasing with r, the grating should be placed as close to the center of the plate as possible, while the waveguide bending loss is kept at a minimum. The grating will still experience the radial strain, but only at a perpendicular angle. Due to the ﬁnite width of the waveguide there will be a difference in tangential strain across the waveguide. For typical waveguide widths, this will however be negligible. The minimum radius of curvature for which the waveguide can still carry a mode is [32] Since the radial strain is not constant the Bragg grating would experience a non-uniform strain if it was place radially on the plate, causing lower reﬂection and broader reﬂection peak. 3.1. All-optical MOEMS Bragg Grating Pressure Sensor However, while the tangentially strain is generally lower than the radial strain it would give a uniform strain in the Bragg grating was it to be placed along this direction. Since the tangential strain is monotonically decreasing with r, the grating should be placed as close to the center of the plate as possible, while the waveguide bending loss is kept at a minimum. The grating will still experience the radial strain, but only at a perpendicular angle. Due to the ﬁnite width of the waveguide there will be a difference in tangential strain across the waveguide. For typical waveguide widths, this will however be negligible. The minimum radius of curvature for which the waveguide can still carry a mode is [32] Rmin = λ 8 1 q n2core n2 cladding −1 −cos−1 ncladding ncore (14) (14) This puts a theoretical maximum limit on the sensitivity of this device. Assuming that the core is made of SiON (ncore = 1.47) and that the cladding is made of SiO2 (ncladding = 1.45) the minimum radius is Rmin = 128 µm. The power transmitted through this waveguide as function of its radius of curvature [32] is shown in Figure 11. This puts a theoretical maximum limit on the sensitivity of this device. Assuming that the core is made of SiON (ncore = 1.47) and that the cladding is made of SiO2 (ncladding = 1.45) the minimum radius is Rmin = 128 µm. The power transmitted through this waveguide as function of its radius of curvature [32] is shown in Figure 11. For a loss of less than 0.5 dB, the radius should be larger than 405 µm. From equations 12 and 13 the two unknowns, a and h, fulﬁlling the requirements of pressure range, bending loss and max pressure, i.e., \|ε(r = a, p = pmax, r = Rmin)\| ≤σsafe/E and εθ(r, pmin, r = Rmin) ≥ϵmin, can be found as a = r psafepmin psafepmin + 2εminEpmax Rmin (15) h = s 3pminpmax(1 −ν2) 8εminEpmax + 4σsafepmin Rmin (16) (15) (16) 9553 Sensors 2010, 10 Sensors 2010, 10 where σsafe is the maximum allowable stress, pmax and pmin are the maximum and minimum pressure the sensor should be able to detect, respectively and εmin is the minimum detectable strain change. In oil exploration situations pressures up to 350 bar can occur. If we assume a pressure range from 0 to 350 bar (pmax = 350 bar) with a resolution of pmin = 1 bar, a maximum allowable stress of σsafe = 500 MPa, minimum strain of εmin = 10−6, a silicon membrane with E = 170 GPa and ν = 0.28, one obtains h = 102 µm and a = 464 µm. A SEM image of a sensor realized using the previous design rules is seen in Figure 12. The Bragg grating is located in the half circular part of the waveguide while the ﬁber connectors are located at the two ends of the waveguide. The waveguide and Bragg grating are made using a combination of deep reactive ion etching (DRIE) and electron beam lithography. Figure 11. The power through an half circumference shaped 2 µm wide waveguide as a function of its radius. Figure 11. The power through an half circumference shaped 2 µm wide waveguide as a function of its radius. Figure 12. SEM image of circular plate pressure sensor and waveguide with integrated Bragg grating. Figure 12. SEM image of circular plate pressure sensor and waveguide with integrated Bragg grating. 9554 Sensors 2010, 10 By using different period in each optical sensor in combination with low loss optical ﬁbers, several sensors can be multiplexed on one single ﬁber transmission line. Also the small form factor (approx 2 × 2 mm2) and compatibility with high pressure and harsh environments make this sensor ideal for a number of applications, speciﬁcally underground oil exploration. While the temperature dependence has been neglected so far, any temperature related measurement errors can easily be compensated by integrating a pressure insensitive Bragg grating (i.e., placed outside the membrane) and measuring the differential wavelength shift. For zero strain, the two reﬂection peaks will shift equally in wavelength when subjected to temperature changes. For non-zero strain, the two peaks will separate proportional to the strain, hence temperature and strain can be measured uncoupled. 4. Wireless Pressure Sensors in Silicon Technology Wireless sensing of physical and chemical parameters is highly desirable when physical access is limited, e.g., devices for the biomedical industry and for hazardous environments [33,34]. Wireless sensing can be achieved both in an active or in a passive way. An active device has to be implemented with a microchip powered by a battery or inductively coupled power supply to make measurements [35,36], while a passive wireless sensor can be made both with Surface Acoustic Waves (SAW) or with LC resonators [33,37]. These can be measured remotely without any implemented power supply, giving passive devices the possibility of unlimited lifetime. The drawback is a limited interaction distance. The wireless development is closely linked to the medical industry since it opens new possibilities for monitoring of human parameters inside the body. In 1967 Collins et al. [33] made an intraocular pressure sensor based on the LC resonance method with a sensitivity of 1 MHz/mmHg. As the IC technology was developed it gave engineers a toolbox for developing more compact systems. In 1998 Park et al. [38] made a wafer level hermetically sealed wireless pressure sensor with an integrated LC resonator in bulk micro machining, where electroplating, anodic bonding and an implanted boron etch stop were used to achieve a footprint of 3 × 3 × 1.6 mm. The sensitivity was estimated to 2 MHz/mmHg with a resonance frequency of approximately 500 MHz. In 2001 Akar. et al. [39] presented a batch fabricated sealed pressure sensor with a measured sensitivity of 120 kHz/mmHg in a pressure range of 0–50 mmHg. The development has continued to produce passive wireless sensors for measuring the cerebral ventricle pressure [40] and a commercially CardioMEMS product to measure aneurysm sac pulse pressure [41]. Sensors 2010, 10 Sensors 2010, 10 9555 In the medical industry new products are under heavy restrictions when it comes to lifetime and choice of material. This limits the sensor in many different ways, e.g., the device has to be biological compatible, the form factor has to be small and the design has to take diffusion and mechanical fatigue into account. From these limitations a design has been made to accommodate the different restrictions. The sensing principle is capacitive, where the varying capacitance is included in a LC circuit. The capacitor plates are a circular membrane coated with metal on one side, and an electroplated copper spiral on a glass substrate on the other side. Furthermore, the spiral also functions as the inductor for the LC circuit. Combining the coil as one of the capacitor plates enables the device to become smaller and more compact compared to similar designs. The ﬁrst conceptual design was a simple structure, made by a KOH etched membrane, copper electroplated coil, e-beam evaporated capacitor plate and anodic bonding, see Figure 13(a). To improve the design, simulation of a bossed membrane is used to optimize sensitivity, Q-factor and resonance frequency. The new design can be seen in Figure 13(b). According to classical electrodynamics this circuit has angular resonance frequency, ωres, and a Q-factor, Q, that can be calculated knowing the inductance, L, the capacitance, C, and the resistance, R, of the circuit ωres = 1 √ LC (17) Q = 1 R r L C (18) (17) (18) Figure 13. The ﬁgures shows the old and the new design of the passive bladder pressure sensor. The bottom plate and membrane are fabricated in silicon, 2, and the top plate is a glass substrate, 1. The inductor is electroplated copper, 3, and the opposite capacitor plate is made on the pressure sensitive membrane with a deposited gold ﬁlm 4. (a) A 3D sketch of the old design. Notice that the copper coil, 3, continues to the center of the sensor. The top plate, 1, is a glass substrate. The bottom plate, 2, is a silicon substrate with a etched membrane covered with a gold layer, 4. (b) A 3D sketch of the new design. Notice the bossed membrane in the the center of the sensor, 5, and the electroplated copper coil, 3, that is solid in the center. 4.1. Wireless Implantable Pressure Sensor An application for passive wireless pressure sensor is to measure the pressure inside the urinary bladder, to monitor it for patients with urinary incontinence [42]. Urinary incontinence is a common problem but it is often concealed and associated with great discomfort [42]. Sensing the pressure in the urinary bladder can, via a feedback stimulation system, control the bladder, hence give control over the involuntarily excretion of urine as is characteristic for urinary incontinence [43]. Conventional sensing systems have been based on wired pressure sensors with the wires penetrating the bladder wall, causing undesirable side effects [44]. In these systems a wireless pressure sensor is designed to make reliable measurements without damaging the tissue inside the bladder. Sensors 2010, 10 Sensors 2010, 10 9556 To measure this angular resonance frequency through an external measuring circuit, the circuit is coupled to the sensor through a mutual inductance using a tuned transformer coupling, see Figure 14. Here the resonance of the sensor is reﬂected in the external measuring circuit through the coupling constant, k, and the external coil, L1. The impedance of the system seen from the external terminals is given by Z (jω) = R1 + j ω ω01 2 + 1 ωC1 + jωL1k2 ω ω02 2 j ω Q2ω02 − ω ω02 2 + 1 , (19) (19) where Q2 is the Q-factor of the sensor, j is the imaginary constant, ω is the angular frequency, ω01 and ω02 are the resonance angular frequencies of the external system and the sensor, respectively. Figure 14. Equivalent circuit diagram of a transformer circuit where the oscillation circuit on the primary side, made up by C1 and L1, can be tuned to that of the secondary side C2 and L2. Scaling the sensor implies scaling the radius of the sensor, as the inductance of a spiral is proportional with the radius cubed, whereas the capacitor is proportional to the radius squared. This implies that the Q-factor of the sensor increases with its dimensions. This also gives rise to an increase in the impedance of the system, see Equation 19. Unfortunately, due to the biological application of the device the size is restricted to avoid excessive discomfort and side effects. A trade off between these has to be made. The old design is 6 × 6 mm wide and has a thickness below 1 mm, the same outer dimensions as the new design. This is small enough to avoid disturbing the bladder, and large enough to produce a measurable signal. The inductor and capacitor are also important; since the capacitance change governs the sensitivity and the inductor governs the signal strength there is a trade-off between these as well. Finite element method (FEM) simulation is made using the commercial software COMSOL in order to optimize the sensor. Figure 15 shows a structural mechanical simulation made with the parameters for the new design shown in Table 1 at 1 bar and used to extract the deﬂection of the sensor. Sensors 2010, 10 The membrane deﬂects in the region, 4, that is situated between the bossed structure, 5, and the silicon substrate, 2. (c) A 2D sketch of the old design. (d) A 2D sketch of the new design. (a) (b) (c) (d) (b) (a) (b) (a) (d) (c) (d) (c) Sensors 2010, 10 Then the parallel plate capacitor approximation is used to calculate the capacitance.The quasi-static magnetic module in COMSOL, see Figure 16, is then used to simulate the inductance by solving the magnetic vector potential using Maxwell equations. The inductor is enclosed in a sphere with radius twice as large as the inductor radius, in order to encapsulate all of the electromagnetic ﬁelds. These two FEM simulations are programmed to investigate the resonance frequency, sensitivity and Q-factor as a function of the design parameters shown in 9557 Sensors 2010, 10 Sensors 2010, 10 Sensors 2010, 10 Table 1. The simulation are made for an absolute pressure range of 0 to 330 mmHg, which for safety reasons is 10 times higher than the pressure range of interest. Figure 15. A structural mechanical simulation made in COMSOL for 1 bar pressure is shown. The black outline is the unstrained structure. Notice that the largest stress is located in the thin membrane, and the bossed structure is only moving vertically movement according to the center defection. Axial symmetry is employed in this simulation, the y-axis is considered the axis of rotation. Table 1. In this table the parameters used for the simulations are shown, both for the old and the new design. Table 1. In this table the parameters used for the simulations are shown, both for the old and the new design. Parameter Old Design New Design Membrane thickness 70 µm 50 µm Gap distance at vacuum 40 µm 30 µm Radius of the membrane 3 mm 3 mm Spiral width 50 µm 50 µm Spiral spacing 50 µm 50 µm Spiral thickness 50 µm 50 µm Radius of bossed structure - 1.5 mm Thickness of bossed structure - 150 µm From the simulations it can be seen that the sensitivity is monotonically increasing as the bossed structure gets thicker and membrane gets thinner and plate distance decreases, whereas the Q-factor is monotonically decreasing. More interesting are the variations of the radius of the bossed structure on the new design, see Figure 17(a). Here the optimal radius, in terms of sensitivity, can be read from the numerical simulations shown on the graph and is approximately 1.2 mm, whereas the Q-factor, see 9558 Sensors 2010, 10 Figure 17(b), is increasing up to a radius of approximately 2.4 mm. It can be seen that a higher sensitivity, if compared to the old design, can be reached implementing a radius of approximately 1 to 1.5 mm, still keeping a satisfactory Q-factor. According to the simulation made with the new design parameters shown in Table 1 a sensitivity of 650 kHz/mmHg, Q-factor of 240 and a resonance frequency of 86 MHz can be reached compared to the old design which had a sensitivity of 30 kHz/mmHg, Q-factor of 150 and a resonance frequency of 63 MHz. Figure 16. A static electrical simulation made in COMSOL is shown. The inductor is enclosed in an air domain to encapsulate all of the electromagnetic ﬁeld. The cutout section in the red box shows a zoom of the inductor, that is approximated with concentric rings. Axial symmetry is employed in this simulation, the y-axis is considered the axis of rotation. From the work done it has been shown that a more sensitive sensor can be produced by adding a bossed structure and still keeping the original features of an hermetically sealed small sensor. The design modiﬁcations introduced lead to a more complicated fabrication process but, with the improvements on the performances of the sensor, this is of secondary importance. Finally, the simulation tool presented allows a fast evaluation of the impact on the performances of these sensors given by a modiﬁcation of their fabrication parameters. From the work done it has been shown that a more sensitive sensor can be produced by adding a bossed structure and still keeping the original features of an hermetically sealed small sensor. The design modiﬁcations introduced lead to a more complicated fabrication process but, with the improvements on the performances of the sensor, this is of secondary importance. Finally, the simulation tool presented allows a fast evaluation of the impact on the performances of these sensors given by a modiﬁcation of their fabrication parameters. From the work done it has been shown that a more sensitive sensor can be produced by adding a bossed structure and still keeping the original features of an hermetically sealed small sensor. The design modiﬁcations introduced lead to a more complicated fabrication process but, with the improvements on the performances of the sensor, this is of secondary importance. Sensors 2010, 10 Finally, the simulation tool presented allows a fast evaluation of the impact on the performances of these sensors given by a modiﬁcation of their fabrication parameters. 9559 Sensors 2010, 10 Sensors 2010, 10 Figure 17. The ﬁgures shows simulations of a bossed membrane structure with parameters: gap distance at vacuum = 80 µm, radius of membrane = 3 mm, spiral width = spiral spacing = spiral thickness = 50 µm and thickness of bossed structure = 100 µm. The design parameters are not used because it would limit the range, and in these simulations the general behavior is investigated. The internal radius of the bossed structure is varied from 0 to 3 mm. (a) Sensitivity vs. boss radius. An optimal internal radius of the bossed membrane is around 1.2 mm. (b) Q-factor vs. boss radius. A clear peak is seen at approximately 2.4 mm. (a) (b) (b) 9560 Sensors 2010, 10 Sensors 2010, 10 5. Discussion and Conclusions A TMCPS, an all-optical pressure sensor and a wireless pressure sensor have been presented. Their design has been validated either with an analytical or with a ﬁnite element model. Furthermore, one or more ﬁelds of application for each sensor have been identiﬁed. The possibility of using them in harsh environments, as well as low power consumption (especially compared to the piezoresistive pressure sensor), are the intrinsic common features of the devices proposed. None the less, while the TMCPS is in a more advanced stage of development and has been characterized with respect to temperature, the MOEMS pressure sensor has the unique feature of being immune to electromagnetic interference and is also expected to have a good temperature insensitivity. The wireless device, being a passive transducer, can be used for applications where, obviously, the other two solutions cannot be used. Table 2 summarizes all these aspects and speciﬁes the pressure ranges and sensitivities of the three designs. An experimental validation of the designs proposed was possible only for the TMCPS. However the guidelines given for the fabrication of all three type of sensors lead to a step forward in the development of pressure sensors for application speciﬁc purposes. Table 2. Mathematical model, characteristics and applications for the three designs proposed. Sensor type Model Pressure range Sensitivity Technology status Applications TMCPS analytical 0–10 bar 14 pF/bar prototyping harsh environment, low power consumption All-optical pressure sensor analytical 0–350 bar 1 pm/bar proof of concept harsh environment, low power consumption, distributed systems Wireless passive pressure sensor FEM 0–330 mmHg 650 KHz/mmHg proof of concept harsh environment, low power consumption, medical thematical model, characteristics and applications for the three designs proposed. 4. Hugill, A.L. Displacement transducers based on reactive sensors in transformer ratio bridge circuits. J. Phys. E Sci. Instrum. 1982, 15, 597-606. 3. Jones, R.V.; Richards, J.C. Design and some applications of sensitive capacitance micrometers. J. Phys. E Sci. Instrum. 1973, 6, 589-600. 5. Huang, S.M.; Stott, A.L.; Green, R.G.; Beck, M.S. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/.) c⃝2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/.)
https://openalex.org/W2163416021	https://figshare.com/ndownloader/files/40001701	English	null	Fine-tuning Tumor Immunity with Integrin Trans-regulation	Cancer immunology research	2,015	cc-by	2,446	E25 NEO E26 E27 E28 E24 XbaI XbaI XbaI Knockin 4.23k Wild type 12.5k XbaI 5’probe 3’probe Knockin 7.75k BglII BglII Knockin 5.3k Wild type 3.2k NEO probe S988A Knock-in construct wt wt wt wt XbaI digest 5’probe XbaI digest 3’probe BglII digest 3’probe BglII digest NEOprobe 12.5k 7.75k 12.5k 4.23k 5.3k 3.2k 5.3k (S988A) (S988A) (S988A) (S988A) Southern blot 0.25 7.3 7.3 0.29 0.11 0.04 25 0.17 0.49 0.51 0.66 0.38 0.8 α4 (wt) 10.1 20 75 4.3 0.1 0.02 982 9.5 17 44 46 18 39 Std. Dev. E25 NEO E26 E27 E28 E24 XbaI XbaI XbaI Knockin 4.23k Wild type 12.5k XbaI 5’probe 3’probe Knockin 7.75k BglII BglII Knockin 5.3k Wild type 3.2k NEO probe S988A Knock-in construct wt wt wt wt XbaI digest 5’probe XbaI digest 3’probe BglII digest 3’probe BglII digest NEOprobe 12.5k 7.75k 12.5k 4.23k 5.3k 3.2k 5.3k (S988A) (S988A) (S988A) (S988A) Southern blot E25 NEO E26 E27 E28 E24 XbaI XbaI XbaI Knockin 4.23k Wild type 12.5k XbaI 5’probe 3’probe Knockin 7.75k BglII BglII Knockin 5.3k Wild type 3.2k NEO probe S988A Knock-in construct wt wt wt wt XbaI digest 5’probe XbaI digest 3’probe BglII digest 3’probe BglII digest NEOprobe 12.5k 7.75k 12.5k 4.23k 5.3k 3.2k 5.3k (S988A) (S988A) (S988A) (S988A) Southern blot E25 NEO E26 E27 E28 E24 XbaI XbaI XbaI Knockin 4.23k Wild type 12.5k XbaI 5’probe 3’probe Knockin 7.75k BglII BglII Knockin 5.3k Wild type 3.2k NEO probe S988A Knock-in construct Knock-in construct Knock-in construct XbaI wt wt wt wt XbaI digest 5’probe XbaI digest 3’probe BglII digest 3’probe BglII digest NEOprobe 12.5k 7.75k 12.5k 4.23k 5.3k 3.2k 5.3k (S988A) (S988A) (S988A) (S988A) Southern blot S1 A A B Bone marrow B cells 0 1.0 2.0 3.0 4.0 Total B Pro B Pre B Immature B Cells (millions) α4 wildtype α4 S988A Thymus T cells 0 10 20 30 40 50 60 70 %CD3+ % CD4+ %DP %CD8+ % of total cells Splenic B cells 0 10 20 30 40 50 60 70 80 Total B Immature B MZ B Follicular B Cells (millions) Splenic T cells 0 20 40 60 80 100 120 140 160 Total cells Total T CD4 T CD8 T Cells (millions) α4 wildtype α4 S988A C Immunogen: TNP-KLH 0 100 200 300 400 500 600 Pre-Immune 1 week 2 weeks 3 Absorbance x Dilution Factor α4 wildtype α4 S988A WBC (103/μL) Neut (%) Lymphs (%) Monos (%) Eos (%) Baso (%) Platelet (103/μL) RBC (106/μL) HgB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) α4(S988A) 10.3 29 68 3.6 0.1 0.04 1021 10 17 44 43 17 39 Std. Dev. 0.25 7.3 7.3 0.29 0.11 0.04 25 0.17 0.49 0.51 0.66 0.38 0.8 α4 (wt) 10.1 20 75 4.3 0.1 0.02 982 9.5 17 44 46 18 39 Std. Dev. 0.17 4.2 4.3 0.73 0.06 0.03 78 0.16 0.82 0.86 0.53 0.59 1.4 p-value 0.28 0.095 0.12 0.11 0.92 0.56 0.37 0.001 0.49 0.48 0.001 0.04 0.60 WBC (103/μL) Neut (%) Lymphs (%) Monos (%) Eos (%) Baso (%) Platelet (103/μL) RBC (106/μL) HgB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) α4(S988A) 10.3 29 68 3.6 0.1 0.04 1021 10 17 44 43 17 39 Std. Dev. E25 NEO E26 E27 E28 E24 XbaI XbaI XbaI Knockin 4.23k Wild type 12.5k XbaI 5’probe 3’probe Knockin 7.75k BglII BglII Knockin 5.3k Wild type 3.2k NEO probe S988A Knock-in construct wt wt wt wt XbaI digest 5’probe XbaI digest 3’probe BglII digest 3’probe BglII digest NEOprobe 12.5k 7.75k 12.5k 4.23k 5.3k 3.2k 5.3k (S988A) (S988A) (S988A) (S988A) Southern blot 0.17 4.2 4.3 0.73 0.06 0.03 78 0.16 0.82 0.86 0.53 0.59 1.4 p-value 0.28 0.095 0.12 0.11 0.92 0.56 0.37 0.001 0.49 0.48 0.001 0.04 0.60 Bone marrow B cells 0 0 0 0 0 Total B Pro B Pre B Immature B α4 wildtype α4 S988A Thymus T cells 0 0 0 0 0 0 0 0 %CD3+ % CD4+ %DP %CD8+ Splenic B cells 0 10 20 30 40 50 60 70 80 Total B Immature B MZ B Follicular B Cells (millions) Splenic T cells 0 20 40 60 80 100 120 140 160 Total cells Total T CD4 T CD8 T Cells (millions) α4 wildtype α4 S988A C Immunogen: TNP-KLH 0 100 200 300 400 500 600 Pre-Immune 1 week 2 weeks 3 weeks Absorbance x Dilution Factor α4 wildtype α4 S988A Bone marrow B cells otal B Pro B Pre B Immature B α4 wildtype α4 S988A Thymus T cells CD3+ % CD4+ %DP %CD8+ Splenic B cells 0 10 20 30 40 50 60 70 80 Total B Immature B MZ B Follicular B Cells (millions) Splenic T cells 0 20 40 60 80 100 120 140 160 Total cells Total T CD4 T CD8 T Cells (millions) α4 wildtype α4 S988A C Immunogen: TNP-KLH 0 100 200 300 400 500 600 Pre-Immune 1 week 2 weeks 3 weeks Absorbance x Dilution Factor α4 wildtype α4 S988A B Bone marrow B cells 0 1.0 2.0 3.0 4.0 Total B Pro B Pre B Immature B Cells (millions) α4 wildtype α4 S988A Thymus T cells 0 10 20 30 40 50 60 70 %CD3+ % CD4+ %DP %CD8+ % of total cells Splenic B cells 0 10 20 30 40 50 60 70 80 Total B Immature B MZ B Follicular B Cells (millions) Splenic T cells 0 20 40 60 80 100 120 140 160 Total cells Total T CD4 T CD8 T Cells (millions) α4 wildtype α4 S988A C 0 100 200 300 400 500 600 Pre-Immune Absorbance x Dilution Factor α4 wil α4 S9 Splenic B cells 0 10 20 30 40 50 60 70 80 Total B Immature B MZ B Follicular B Cells (millions) Splenic T cells 0 20 40 60 80 100 120 140 160 Total cells Total T CD4 T CD8 T Cells (millions) α4 wildtype α4 S988A Bone marrow B cells Pre B Immature B α4 wildtype α4 S988A Thymus T cells D4+ %DP %CD8+ Splenic B cells 0 10 20 30 40 50 60 70 80 Total B Immature B MZ B Follicular B Cells (millions) Splenic T cells 0 20 40 60 80 100 120 140 160 Total cells Total T CD4 T CD8 T Cells (millions) α4 wildtype α4 S988A C Immunogen: TNP-KLH 0 100 200 300 400 500 600 Pre-Immune 1 week 2 weeks 3 weeks Absorbance x Dilution Factor α4 wildtype α4 S988A C B S2 α4 (S988A) (Ly5.2) Splenocyte mixture 24 h Spleen Periton. E25 NEO E26 E27 E28 E24 XbaI XbaI XbaI Knockin 4.23k Wild type 12.5k XbaI 5’probe 3’probe Knockin 7.75k BglII BglII Knockin 5.3k Wild type 3.2k NEO probe S988A Knock-in construct wt wt wt wt XbaI digest 5’probe XbaI digest 3’probe BglII digest 3’probe BglII digest NEOprobe 12.5k 7.75k 12.5k 4.23k 5.3k 3.2k 5.3k (S988A) (S988A) (S988A) (S988A) Southern blot lavage Flow cytometry Rag1-/- Rag1-/- ioglycollate, i.p. Rag1-/- 4-18h α4 (wildtype) (Ly5.1) 0 10 20 30 40 50 60 70 80 4.5h 18h 4.5h 18h Fraction a4(S988A Cells (%) Spleen Peritoneum All T cells CD8+ T cells *** * α4 (S988A) (Ly5.2) Splenocyte mixture 24 h Spleen Periton. lavage Flow cytometry Rag1-/- Rag1-/- collate, i.p. Rag1-/- 4-18h α4 (wildtype) (Ly5.1) 0 10 20 30 40 50 60 70 80 4.5h 18h 4.5h 18h Fraction a4(S988A Cells (%) Spleen Peritoneum All T cells CD8+ T cells *** * Spleen Periton. E25 NEO E26 E27 E28 E24 XbaI XbaI XbaI Knockin 4.23k Wild type 12.5k XbaI 5’probe 3’probe Knockin 7.75k BglII BglII Knockin 5.3k Wild type 3.2k NEO probe S988A Knock-in construct wt wt wt wt XbaI digest 5’probe XbaI digest 3’probe BglII digest 3’probe BglII digest NEOprobe 12.5k 7.75k 12.5k 4.23k 5.3k 3.2k 5.3k (S988A) (S988A) (S988A) (S988A) Southern blot lavage S3 S4 ICAM ICAM+VCAM ICAM+VCAM (+anti-α4) Migration (%) T Cells 0 5 10 15 20 25 30 35 40 45 4 (wt) 4 (S988A) p=0.01 p=0.0005 B Cells 0 10 20 30 40 50 60 70 4 (wt) 4 (S988A) p=0.05 p=0.3 Migration (%) T Cells 0 5 10 15 20 25 30 35 40 45 4 (wt) 4 (S988A) p=0.01 p=0.0005 B Cells 0 10 20 30 40 50 60 70 4 (wt) 4 (S988A) p=0.05 p=0.3 S4 0 0.05 0.1 0.15 0.2 0.25 α4(S988A) α4(wt) Tumor Mass (g) * 0 0.05 0.1 0.15 0.2 0.25 α4(S988A) α4(wt) Tumor Mass (g) * S5 0 10 20 30 40 50 α4(wt) α4(S988A) Cells (% of T cells) Anti-CD8 Isotype * * Anti-CD4/CD8/NK Isotype -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Effect of α4(S988A) mutation on tumor mass (%) Anti-CD8 Isotype * *** * 4 CD8 CD4 CD8 Anti-CD4/CD8/NK Isotype B 4(S988A) 4(wt) -90 -80 -70 -60 -50 -40 -30 -20 -10 0 10 Effect of α4(S988A) mutation on tumor mass (%) Anti-CD4/CD8/NK Isotype A B 0 10 20 30 40 50 α4(wt) α4(S988A) Cells (% of T cells) Anti-CD8 Isotype * * -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Effect of α4(S988A) mutation on tumor mass (%) Anti-CD8 Isotype Depletion efficiency -90 -80 -70 -60 -50 -40 -30 -20 -10 0 10 Effect of α4(S988A) mutation on tumor mass (%) Anti-CD4/CD8/NK Isotype * * *** * 0 20 40 60 80 100 120 CD4 CD8 CD4 CD8 Cells (% of Isotype) Anti-CD4/CD8/NK Isotype A 4(S988A) 4(wt) -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Effect of α4(S988A) mutation on tumor mass (%) Anti-CD8 Isotype 0 10 20 30 40 50 α4(wt) α4(S988A) Cells (% of T cells) Anti-CD8 Isotype * * Depletion efficiency * * *** * 0 20 40 60 80 100 120 CD4 CD8 CD4 CD8 Cells (% of Isotype) Anti-CD4/CD8/NK Isotype 4(S988A) 4(wt) S6 0 1 2 3 4 5 6 7 8 α4(S988A) α4(wt) % Migration ICAM ICAM +VCAM 0 1 2 3 4 5 6 7 8 α4(S988A) α4(wt) % Migration ICAM ICAM +VCAM 0 1 2 3 4 5 6 7 8 α4(S988A) α4(wt) % Migration ICAM ICAM +VCAM S7 Supplemental Figure Legends Supplemental Figure 1 Generation of the α4(S988A) mouse. A DNA fragment encoding α4(S988A) integrin with a Neomycin resistance gene was transfected into ES cells. ES clones heterozygous for knock-in were confirmed by treatment of restriction-digested DNA with P32- labeled DNA probes specific for 3’ or 5’ flanking sequences or neomycin resistance cassette. One α4(wt) sample is compared with DNA from two α4(S988A) ES clones. (B) Hematological analysis. Error bars are S.E.M. of n=5 for each group. (C) Selectively increased lymphocyte migration in α4(S988A) mice. Supplemental Figure 2. (A) Hematological analysis. Blood was collected from adult (>9 weeks) α4(S988A) or control α4(wt) mice and analyzed by an automated cell counter and manual differential cell counting of stained smears. Error bars are S.E.M. of n=4 mice for each group. Two parameters (RBC and MCV) were statistically significant different, but fell within normal ranges for BL6 mice. (B) Naïve lymphoid organ lymphocyte subsets. Bone marrow, thymus, and spleen were isolated from adult (>9 weeks) α4(S988A) or control α4(wt) mice, made into single-cell suspensions, treated with RBC lysis buffer, and counted. Bone marrow cells were stained with combinations of antibodies for the following B cell subsets and analyzed by flow cytometry: Total B (B220+) Pro-B (CD43+ B220+), Pre-B (B220+ IgM- IgD-), and Immature B (B220+IgM+IgD-). Likewise, thymocytes were stained for T-cell subsets: Total T- cells (CD3+), CD4 SP (CD3+CD4+CD8-), CD8 SP (CD3+CD8+CD4-), and DP (CD4+CD8+). Splenic T and B cells subsets were identified as: Total B (B220+), Immature B (CD21/35-CD23- ), Marginal Zone (MZ) B (CD21/35hi CD23-), Follicular B (CD23+ CD21/35lo), Total T (CD3+), CD4 T (CD3+CD4+CD8-), and CD8 T (CD3+CD8+CD4-). (C) Humoral immune response. Adult α4(S988A) or control α4(wt) mice were immunized i.p. with 100 g Trinitrophenol-Keyhole Lympet Hemocyanin (TNP-KLH) emulsified in Complete Freund’s Adjuvant (CFA). Plasma from pre-immune and weeks 1, 2, and 3 bleeds were tested by direct specific ELISA for the presence of anti-TNP IgG. Similar results were seen with anti-IgM responses. Error bars are S.E.M. of n=4 for each group Supplemental Figure 3. Intrinsic homing advantage of α4(S988A) splenocytes. Splenocytes from α4(S988A) or control α4(wt) Ly5.1 congenic mice were mixed and injected i.v. into Rag1-/- mice that had been challenged 24h earlier with 1ml thioglycollate medium. Mice were sacrificed at 4.5h or 18h after splenocyte transfer; peritoneal lavage and spleen were counted, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry. Supplemental Figure Legends The ratio of α4(S988A) to control α4(wt) splenocytes was determined by measuring the ratio of Ly5.1- negative to Ly5.1-positive T-cells (CD3+). Errors bars indicate SEM from n=3 (4.5h) and n=2 (18h) mice per group. p< 0.02, p<0.01, *p<0.04 (one-tailed t-test Supplemental Figure 4. Migration of α4(S988A lymphocytes in vitro. B and T-cells were purified from spleens of α4(S988A) or control α4(wt) mice. Chemotactic migration towards SDF- 1 (15ng/ml) was assessed using a modified Boyden Chamber assay in wells coated with ICAM-1 (5 μg/ml) +/- VCAM-1 (0.02 μg/ml). For anti-integrin antibody blocking, cells were treated with 10μg/ml of anti-4 or integrin prior to the assay. Error bars are S.E.M. of n=4 for each group Supplemental Figure 5. Lewis Lung Carcinoma Tumor Growth. Lewis Lung Carcinoma (LLC) tumor cells (1x106) were injected subcutaneously in the hind flank of α4(S988A) or control α4(wt) mice. On day 15, mice were sacrificed and tumors weighed. Error bars are S.E.M. from 10-11 mice per group. p<0.02 (one-tailed Mann-Whitney test). Similar results were obtained with inoculation of 5x106 tumor cells. Supplemental Figure 6. Tumor growth after depletion of lymphoid cells in α4(S988A) mice. B16 tumors were grown in α4(S988A) or control α4(wt) mice as in Figure 3. Two days before and 5 days after tumor cell inoculation, anti-CD8 (A), or a combination of anti-CD4, anti-CD8, and anti-NK1.1 (B) depleting antibodies were injected i.p. On day 15, excised tumors were weighed and compared between α4(S988A) and control α4(wt) genotypes in depleted or in antibody isotype-treated mice. Upper bar graphs show percentage change in tumor mass in α4(S988A) vs. α4(wt) mice. Lower graphs are depletion efficiency of T cells in the spleen on day 15. n ≥ 5 mice per group. p<0.03, p<0.015, **p<0.001 (one-tailed) Supplemental Figure 7. Migration of α4(S988A) macrophages. Macrophages were differentiated from bone marrow of α4(S988A) or control α4(wt) mice by culture for one week in 30% L929 supernatant medium. Chemotactic migration towards SDF-1 and MCP-1 (15ng/ml each) was assessed using a modified Boyden Chamber assay in wells coated with VCAM-1 alone (2 ug/ml), or ICAM-1 (5 ug/ml) +/- VCAM-1 (0.02 ug/ml). Bar graphs summarize migration from n= 3 mice per group; no integrin transregulation or migrational differences were observed.
W4392381348.txt	https://stemeducationjournal.springeropen.com/counter/pdf/10.1186/s40594-024-00475-6	en		Beyond STEM attrition: changing career plans within STEM fields in college is associated with lower motivation, certainty, and satisfaction about one’s career	International journal of STEM education	2,024	cc-by	14,807	Rosenzweig et al. International Journal of STEM Education (2024) 11:15 https://doi.org/10.1186/s40594-024-00475-6 International Journal of STEM Education Open Access RESEARCH Beyond STEM attrition: changing career plans within STEM fields in college is associated with lower motivation, certainty, and satisfaction about one’s career Emily Q. Rosenzweig1* , Xiao‑Yin Chen1, Yuchen Song1, Amy Baldwin2, Michael M. Barger1, Michael E. Cotterell3, Jonathan Dees4, Allison S. Injaian5, Nandana Weliweriya6, Jennifer R. Walker7, Craig C. Wiegert6 and Paula P. Lemons2 Abstract Background Research and policy often focus on reducing attrition from educational trajectories leading to careers in science, technology, engineering, and mathematics (STEM), but many students change career plans within STEM. This study examined how changing career plans within STEM fields was associated with psychological indicators of career readiness. We conducted a large online survey of undergraduate students (N = 1,727) across 42 courses covering every major STEM discipline at a large U.S. research-intensive public university. Students reported about their career plans, whether plans had changed, motivation for those career plans, and satisfaction with and certainty of per‑ sisting with those plans. A trained team of coders classified whether students reported having STEM career plans at the time of the survey and at the beginning of college. Results Students who said they had changed career plans within STEM fields during college also reported lower motivation for their new career plans, satisfaction with those plans, and certainty of persisting in them, compared to students who retained consistent STEM career plans. With few exceptions, these associations held across stu‑ dents’ gender, race, year in school, and STEM field of study. Within-STEM career plan changes were very common, reported by 55% of fourth-year STEM students. Women reported changing career plans within STEM fields more often than men. Implications Results suggest that changing career plans within STEM is an important phenomenon to consider in preparing a qualified and diverse STEM workforce. Students who change career plans within STEM fields may need additional supports for their career motivation and satisfaction compared to students who do not change plans. Keywords Career planning, Expectancy-value theory, Gender, Motivation, STEM education Correspondence: Emily Q. Rosenzweig Emily.rosenzweig@uga.edu Full list of author information is available at the end of the article © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Rosenzweig et al. International Journal of STEM Education (2024) 11:15 Introduction Significant national resources have been devoted to understanding and reducing STEM attrition, or the phenomenon of students leaving STEM or STEM-related career paths for non-STEM ones. Researchers argue that studying STEM attrition is essential both to increase students’ participation in careers where there are labor market shortages, and to help address ongoing gender and racial/ethnic disparities in STEM career participation (Chen, 2012; National Science Board, 2022; President’s Council of Advisors on Science & Technology, 2012). Yet, a sole focus on reducing attrition is not likely to be sufficient to prepare diverse students for STEM workforce success. The STEM economic sector includes hundreds of potential career options, and during college many students change career plans within STEM (Rosenzweig et al., 2021a). For example, students may shift from wanting to be an engineer to wanting to be a computer scientist. Students who shift plans within STEM often go undiscussed in workforce policy and research, which can seem to imply that students are “on track” for career success so long as they stay in STEM. The present study challenges this assumption, examining whether college students who change career plans within STEM fields report lower scores on various psychological indicators of motivation and readiness for post-graduation career plans compared to their peers who retain consistent STEM career plans throughout college. Situated expectancy value theory and STEM career readiness All students who plan to pursue STEM career paths are not equally likely to achieve STEM career success. In considering what factors predict students’ satisfaction with, persistence in, and engagement with STEM career paths, Eccles and colleagues’ situated expectancy-value theory of motivation is a useful theoretical framework. According to situated expectancy-value theory, individuals’ achievement-related decisions and engagement with achievement-related tasks are most proximally influenced by their motivational beliefs, and these beliefs are the major lenses through which individuals internalize their past experiences, socialization histories, and broader self-beliefs with respect to thinking about upcoming achievement tasks (Eccles & Wigfield, 2020). Situated expectancy-value theory suggests that there are two major types of motivational beliefs that determine engagement with and choices of academic activities, including preparing for and persisting in an STEM career: competence-related beliefs and task values (Eccles [Parsons] et al., 1983; Eccles & Wigfield, 2020). Competence-related beliefs are individuals’ beliefs about whether they are capable of success in a particular Page 2 of 18 career. Competence-related beliefs are often described as relating to the question of “Can I do this task?”; if students think they cannot achieve success on a task, they will not be motivated to engage with it (Eccles & Wigfield, 2020). Traditionally, situated expectancy-value theory defined competence-related beliefs only in terms of expectations of success on an upcoming task (Eccles [Parsons] et al., 1983). However, expectancies of success overlap empirically with a number of broader competence-related beliefs including individuals’ academic selfconcepts in particular fields, broader ability beliefs, and self-efficacy (Bong & Skaalvik., 2003; Marsh et al., 2019). For this reason, many researchers including us examine the broader concept of competence-related beliefs instead of studying one particular belief (e.g., Muenks et al., 2018; Simpkins et al., 2006). Extensive research shows that individuals’ competence-related beliefs for STEM fields of study, majors, or careers predict varied outcomes including their emotions during STEM courses (Jiang et al., 2018), their performance in STEM courses during high school and college (Guo et al., 2015a, 2015b; Jiang et al., 2018; Perez et al., 2014, 2019), their choices of whether or not to pursue STEM coursework or majors (Guo et al., 2015b; Lent et al., 2003; Musu-Gillette et al., 2015; Perez et al., 2019), and their plans to take or choices to pursue STEM career paths (Guo et al., 2015a; Lauermann et al., 2017; Rosenzweig & Chen, 2023). Task values refer to individuals’ beliefs about whether a career is worthwhile and generally these beliefs relate to the question of “Do I want to do this task?” Even if students believe they are capable of success at a particular task, they may not be motivated to engage with it if it lacks perceived value. Researchers working within situated expectancy-value theory often discuss value in terms of four components: how useful a career is assumed to be (i.e., utility value), how relevant it is to one’s identity (i.e., attainment value), how interesting or enjoyable it is (i.e., intrinsic value), and, conversely, how much it requires individuals to sacrifice time, effort, or emotional resources (i.e., cost) (Eccles-Parsons et al., 1983; Eccles & Wigfield, 2020). Each of the four components of task values has also been found to predict varied learning and engagement outcomes, including students’ engagement with and emotions during STEM courses (Jiang et al., 2018), academic performance (Guo et al., 2015a, 2015b; Jiang et al., 2018), STEM course-taking choices or major persistence (Guo et al., 2015a; Guo et al., 2015b; MusuGilette et al., 2015; Safavian, 2019; Safavian & Conley, 2016), and STEM career intentions or career pursuit (Guo et al., 2015a; Lauermann et al., 2017). Though each of the major constructs of situated expectancy-value theory significantly predicts a variety of achievement-related outcomes, the beliefs do not Rosenzweig et al. International Journal of STEM Education (2024) 11:15 all predict all types of outcomes in the same ways. There are some general trends in the literature suggesting that different specific beliefs most strongly predict unique types of STEM achievement outcomes. First, competence-related beliefs, compared to task values, tend to predict grades and academic performance more strongly, whereas task values tend to predict more strongly academic choices and decisions (e.g., courses to take, majors to pursue, whether or not to persist in STEM fields) (e.g., Bong et al., 2012; Meece et al., 1990; Safavian & Conley, 2016; Safavian, 2019; see Wigfield & Eccles, 2020 for review). Second, within task values, intrinsic value tends to predict momentary decisions like engagement and emotions, whereas utility and attainment value tend to predict longer-term decisions like what courses or majors to pursue (Rosenzweig et al., 2022). Third, perceptions of cost have been studied less than the other components of task values, and thus the outcomes likely to be affected by cost are less well-understood. However, cost perceptions generally seem to predict individuals’ decisions to avoid or disengage from challenging tasks or drop out of particular fields of study (e.g., Jiang et al., 2018; Perez et al., 2014). The trends just noted should not be taken to suggest that certain constructs exclusively predict certain STEM outcomes. Competence-related beliefs and attainment, utility, and intrinsic value all relate positively to one another over time (and often also relate negatively to perceived cost), and students’ academic outcomes influence one another over time as well (e.g., academic performance influences participation) (Chen, 2012; Durik et al., 2006; Jacobs et al., 2002; Ost, 2010). For this reason, each of the SEVT constructs has potentially multifaceted impacts on individuals’ academic preparation and persistence. In this study, we operationalize “career motivation” in accordance with situated expectancy-value theory and examine students’ competence-related beliefs and the four dimensions of task values for their chosen career plans at the time of the study as indicators of STEM career motivation. Our key assumption, grounded by the extensive research just reviewed, is that if students report lower scores on each of these indicators of motivation for an STEM career (or higher cost perceptions), they will be less likely to persist at that career over time, less likely to enjoy and feel satisfied with it, and less likely to engage with career preparation or day-to-day work activities associated with it. Most specifically, we might expect that if students report lower competence-related beliefs for a career they might perform less well in courses or career preparation activities en route to it, if they report lower utility or attainment value for a career they might be less likely to choose to persist in it, if they report lower intrinsic value for a career they might be less satisfied Page 3 of 18 with it, and if they report higher perceived cost they may be more likely to disengage with in-the-moment career preparation tasks or opportunities. Understanding within‑STEM career plan changes to promote STEM workforce participation To help ensure that more students are on track to succeed in STEM careers, educators and researchers can identify students who have low competence-related beliefs and/or task values for STEM career plans and provide them with supports to enhance their motivation. Prior research has examined this topic, but that research predominantly focuses on one group of students with low motivation: students who intend to leave or actually do leave STEM career paths for non-STEM ones (i.e., those who are likely to engage in STEM attrition). For example, much prior research has examined what motivational beliefs predict whether students want to do STEM fields of study/career paths versus non-STEM ones (i.e., Guo et al., 2016; Lauermann et al., 2017; Musu-Gillette et al., 2015; Perez et al., 2014, 2019). This work demonstrates that students’ competence-related beliefs and task values are key predictors of students leaving STEM fields for non-STEM alternatives. Though important, this body of work overlooks the experiences of a group of students who do not leave STEM careers, but still may have lower career motivation in STEM: students who change career plans within STEM fields of study. These students are often assumed to be “on track” for STEM career success because they continue to pursue STEM career paths. However, students who change career plans within STEM fields may have lower career motivation for their new chosen career plans compared to their peers who do not change plans, for reasons discussed in the next paragraph. Having lower motivation, according to situated expectancyvalue theory, would make these students more likely not to prepare effectively for their chosen career path, not to succeed at obtaining the career they want, and/or not to persist at their chosen STEM career after college. If we as a society want a qualified STEM workforce where employees are satisfied, productive, and remain engaged with in-demand STEM careers, it is essential to identify and support all students who might have relatively low STEM career motivation, not just those who wish to leave STEM careers for non-STEM ones. There are two reasons why changing career plans within STEM fields may be associated with students having lower career motivation. First, students with lower overall motivation for an STEM field may in turn be less motivated for any careers within that field, and as a result they may be more likely to change plans within STEM more often than their peers. If individuals start college Rosenzweig et al. International Journal of STEM Education (2024) 11:15 with relatively less motivation for their chosen field of study (i.e., lower perceptions of competence-related beliefs intrinsic value, attainment value, or utility value, or higher cost perceptions) this may translate into correspondingly lower levels of those same motivational beliefs for any careers within those fields. Prior research supports this idea by showing that students’ general competence-related beliefs translate into competence-related beliefs for specific sub-tasks within those fields, and that students are more strongly motivated to engage with and put forth effort on specific tasks that are congruent with their broader interests and identities (Eccles, 2005; Master et al., 2016; Muenks et al., 2020). Thus, students who are more confident about their ability to succeed in a particular STEM domain, like computer science, are likely to feel more confident about their ability to succeed in career plans within that domain compared to students who feel less confident. Similarly, if students perceive a particular STEM domain as less central to their identity, interesting, or useful, they may be likely to think of career paths within that domain as being less valuable compared to students who perceive that domain to be more valuable. Even if students who change career plans report somewhat higher motivation for their new career path than they used to report for their old path, they still might have lower career motivation for their new path compared to those who never changed plans, because the students with consistent plans are just that highly motivated for studying their chosen career. At the same time, students with lower levels of career-related motivational beliefs may choose to change career plans more often than their peers who have relatively higher motivational beliefs, given the extensive research outlined in the prior section showing links between the different expectancyvalue motivational beliefs and choices to leave STEM career paths. Taken together, this evidence suggests that changing plans within STEM would serve as an indicator of a group of students who are particularly low on motivation for their chosen STEM careers and thus need support, but the process of changing plans itself would not have caused students to have lower motivation. A second reason why students who change career plans within STEM may report lower career motivation is that the process of changing career plans within STEM fields may cause students to be less motivated for their new career plans. Students who change career plans during college have less time to take coursework or pursue professional development that provides skillbuilding related to their new career paths. This might prevent them from having success experiences that support their skill development in their new careers. Having an opportunity to develop skills hands-on is one of the strongest factors impacting the development of students’ Page 4 of 18 competence-related beliefs for particular academic tasks, so in this case failing to have sufficient experiences to develop skills would lead students to report lower competence-related beliefs for their chosen career path (Bandura, 1997; Butz & Usher, 2015). Additionally, similar to competence-related beliefs, interest in a field is thought to develop as a result of repeated experiences over time that help students increasingly see the relevance of a particular area of study to their lives and other interests (Hidi & Renninger, 2006). Students in turn are thought to develop deeper sources of value, such as utility and attainment value, via initial experiences of interest where they connect activities to their personal goals and identities (Priniski et al., 2018). If students change career plans, they may not have as many opportunities to engage in self-relevant activities that deepen their interest in their new career paths or allow them to see how those paths fit with their identities. As a result, students may not have an opportunity to develop as much attainment and/or utility value for their new career path as they might have had they had more opportunities to engage in career preparation. There is not much existing research about the developmental antecedents of perceived cost, so it is unclear whether this phenomenon would affect cost perceptions as well. There are hundreds of career possibilities within STEM, and many college students report that career decisionmaking is a source of psychological stress (Fouad et al., 2006). These facts make the two mechanisms just discussed very salient to students during college. Indeed, even minor career shifts may indicate (or cause) differences in students’ motivation; career changes do not need to be substantive. With respect to the first potential explanation for effects, students who are feeling like they are not capable or who perceive low value in their career choice may be more likely to shift careers in both major or minor ways. With respect to the second mechanism, if students need additional experiences to prepare for any new career path (even if that new path is as minor as pursuing one type of engineering career versus another), they may still doubt their competence for the new path or have concerns about whether the new career is valuable, more so than would individuals who have always maintained consistent plans and had opportunities to explore them throughout college. Regardless of the mechanism for effects, if students who change career plans within STEM fields do report lower motivation for their new career paths compared to those retaining consistent STEM career plans, this fact has critical implications for research and practice. From a research perspective, STEM motivation research almost exclusively examines students who stay in versus leave STEM, essentially assuming that students who Rosenzweig et al. International Journal of STEM Education (2024) 11:15 remain in STEM fields are motivated and on track for career success. However, these students might need to be studied in depth to shed light on heterogeneity in career preparation among individuals in STEM fields. From a practice perspective, students who have changed plans within STEM or who are considering changing may be an essential group for whom to provide extra supports and resources to ensure that they are motivated for success in the STEM careers that they do choose. For this reason, the goal of the present study was to examine whether changing career plans within STEM was associated with career motivation, as well as with two other indicators of career readiness thought to be affected by motivation (career satisfaction and certainty). A secondary goal was to examine the prevalence of changing career plans within STEM. A recent study reported that 46% of college students pursuing biomedical careers at a 4-year U.S. state university changed career plans within biomedical fields, which was more than 50% larger than the estimate of the rate of attrition out of biomedical careers in that sample (Rosenzweig et al., 2021a). Identifying how many students change career plans within STEM can shed light on the scope of the issue of heterogeneity in career motivation among students who remain in STEM fields of study. These findings are important to provide new insights around labor market shortages in particular STEM- or STEM-related career paths (e.g., physicians or computer programmers, Association of American Medical Colleges, 2021; Xue & Larson, 2015). Any student who leaves an in-demand STEM career path contributes to shortages in that career, even if the student ultimately chooses some other STEM career path. If students change careers within STEM more often than they leave STEM, then researchers or educators might best address some career shortages by studying and supporting students who switch careers within STEM, instead of focusing solely on preventing students from leaving for non-STEM career paths. Student characteristic differences in changing career plans within STEM fields A final major study goal was to explore whether findings were robust to students’ gender, race/ethnicity, year in school, or field of study. We examined this topic because there have been differences in STEM attrition rates observed in prior studies as a function of each of these student background variables (e.g., Chang et al., 2014; Cheryan et al., 2017; Maltese & Tai, 2011; Shaw & Barbuti, 2010); these findings suggest that students’ background may shape how they respond to different types of STEM learning experiences and contexts. It is theoretically possible that each of these background characteristics are important in shaping how students think about Page 5 of 18 career planning and motivation and thus might influence students’ likelihood of retaining consistent STEM career plans versus changing plans within STEM and/or the strength of the association between changing plans and career motivation/satisfaction. We discuss each specific characteristic in turn in the paragraphs that follow. However, we also note that our goal in conducting these analyses was largely exploratory. We believed it was equally possible that the general trends just discussed would hold regardless of students’ background characteristics. Thinking about gender, we expected that there might be differences in the proportion of students changing plans as a function of gender. Inequities in STEM workforce participation persist between women and men (e.g., National Science Board, 2022). These are driven by differences in relative career participation patterns by gender within STEM fields. For example, women are underrepresented in more careers within computer science and engineering than in the life sciences (e.g., Cheryan et al., 2017). When considering why these relative differences in STEM participation exist, one contributing factor may be that women change from one STEM career plan to another more often than men do. Research shows that women receive social messages leading them on average to prefer careers that offer work–life balance and afford communal values (Diekman et al., 2017; Eccles, 2009; Starmer et al., 2019). Communally oriented careers are not traditionally associated with STEM (Diekman et al., 2010). As women learn more about required career training and preparation in college, they may be more attuned to finding which career paths within STEM most readily afford such opportunities and they may be more likely to change career paths within STEM as a result (e.g., switching from being a doctor to a physician assistant, Rosenzweig et al., 2021a). We did not expect based on the literature that gender was likely to moderate the associations between changing plans and career motivation, satisfaction, and certainty, but we examined those analyses to test for robustness of findings as a function of gender. With respect to race, we thought that the proportion of students changing plans within STEM might differ by whether of not students’ racial or ethnic identity was historically marginalized in STEM. In terms of how many students change plans within STEM, there are inequities in STEM workforce participation between White students and students from historically marginalized racial/ethnic groups (e.g., AAUW, 2010; National Science Board, 2022). In addition, within STEM, Black/African American and Hispanic/Latino/a students have larger participation gaps compared to White or Asian/Asian American students in engineering careers compared to life sciences careers (e.g., Cheryan et al., 2017; National Science Board, 2022; Pew Research Center 2021). Similar Rosenzweig et al. International Journal of STEM Education (2024) 11:15 to what was reported for gender, these trends may be due to race-based differences in changing career plans within STEM fields of study. Black and Hispanic/Latino/a students, similarly to women, have been found to receive more social messages about the importance of engaging with STEM careers that afford communal opportunities (Brown et al., 2015) and thus may also be more likely to shift career plans within STEM fields more readily to find STEM careers that meet these needs. Racially marginalized students in STEM also might experience discrimination or encounter non-welcoming professional climates that lead them to change career plans more readily within STEM fields (Seals, 2016). Like with gender, we did not have an a priori theoretical reason to expect race to moderate the association between changing plans and career motivation or career readiness, so those analyses were treated as exploratory robustness checks. With respect to field of study, we thought it would be possible to observe differences both in terms of the proportion of students changing plans within STEM, and in terms of the association between changing plans and career motivation, satisfaction, and certainty. Some fields of study in STEM (e.g., biology) are associated with a large number of career paths whereas others (e.g., engineering) lend themselves more to a specialized set of careers with particular training. Change within STEM might be more common in domains that are associated with a broader set of potential career options. In terms of associations between changing plans and outcomes, some STEM sub-fields require more commitment and preparation for careers than others. For example, in the life sciences, many students want to become doctors which entails a heavy pre-requisite course load, so the time required to prepare for a new career path may be higher than in another field. These differences may lead changing career plans to be more negatively associated with career readiness indicators in life sciences versus other subfields. Finally, with respect to year in school, we also had some reasons to expect differences in both the proportion of students changing plans within STEM fields and the associations between changing plans and career motivation, satisfaction, and certainty. For proportion of students changing plans, it would be expected that in a crosssectional data set, the percentage of students who report having made within-STEM changes would be higher at each year in school. This is because students have more time to change career plans on average the longer they are in college. For associations with outcomes, it may be more detrimental to career readiness if students change plans later in college, because there is less time to prepare for a new career. Because this data was cross-sectional, we were not able to test directly how the same students’ Page 6 of 18 decisions unfolded across college, but this data provides a starting point for comparing students’ responses at different points in the college experience. The present study Given the potential importance of changing plans within STEM for career motivation and readiness, and the potential high number of students engaging in this type of career shift, the present study aimed to examine the phenomenon in more depth. We had the following questions: 1. What proportion of college students report having changed career plans within STEM fields during college? 2. How is changing career plans within STEM fields during college associated with students’ motivation for their current career plans (i.e., perceived competence-related beliefs and perceptions of task values for that career plan), satisfaction with their current career plans, and certainty of pursuing those career plans after college? 3. Do findings differ as a function of gender, race/ethnicity, year in school, or STEM field of study? Methods Participants Participants were 1,727 students, who were both enrolled in STEM courses and pursuing STEM career plans, at a large, public Southeastern U.S. research-intensive state flagship university during the Spring 2022 semester. Participants were 68.9% women, 29.9% men, 0.9% non-binary or other gender identities, and 0.4% stated that they preferred not to disclose gender. They were 69.7% White, 23.9% Asian or Asian American, 7.7% Black or African American, 5.6% Hispanic or Latino/a, 0.4% Native American, and 0.6% other racial/ethnic identities (participants could select multiple racial/ethnic identities). They were primarily first-year students (50.4%), with 22.2% secondyear students, 17.7% third-year students, 7.7% fourth-year students, and 2.1% students not in a typical year of college. Of the sample, 18.4% indicated that they were first-generation college students (i.e., neither parent had obtained a college degree). The most common groups of majors pursued by students in the sample were majors related to biology (i.e., biology, biological science, biochemistry and molecular biology, genetics, ocean science, microbiology, plant biology, cellular biology, ecology, entomology), computer science (i.e., computer science, data science), and engineering (i.e., agricultural, biochemical, biological, civil, computer systems, electrical/electronics, environmental, mechanical). Rosenzweig et al. International Journal of STEM Education (2024) 11:15 Participants were recruited from 42 STEM courses (26 unique course titles, with some having multiple sections participate that were taught by multiple professors) across the university as well as five large listservs (pre-health, computer science, engineering, ecology, and microbiology). These courses and listservs were chosen to represent as wide a range of STEM disciplines as possible and included biology, ecology, genetics, microbiology, chemistry, physics, engineering, computer science, and mathematics. In 30 of the 42 courses, participants received a small amount of extra credit in exchange for participation. Students recruited through the other courses and listservs were offered entry into a gift card drawing in exchange for study participation. All participants were treated in accordance with APA ethical guidelines, and this study was approved via this university’s Office of Research. The final sample of 1,727 represents the number of participants after exclusions. This study focused specifically on career plan changes within STEM fields, so we only included participants in the present manuscript who had pursued STEM career plans across the course of college. Our data collection in the STEM courses initially provided 2,312 valid participants, but 422 did not have career plans that were clearly classifiable as being in STEM or non-STEM fields, and another 163 reported having non-STEM or non-classifiable career plans either at the point of the survey or when starting college (see Measures section and Additional file 1 for full description of the career classification process we used). Such students were excluded from the analyses, resulting in the final sample of 1,727 used in the present study. Procedure Students received information via electronic announcements from listservs or course professors inviting them to participate in an online study taking 15–30 min. Among other questions, students provided information about their major/field of study, their long-term career plans, their satisfaction with and certainty of pursuing those plans, whether or not those plans had changed, their motivational beliefs for pursuing those career plans, and background information. Measures Identifying students who pursued STEM career paths throughout college Only students who reported having STEM career paths both at the time of the survey and at the beginning of college were included in the analyses. To determine eligibility for this study, we used a coding process adapted from prior research (Rosenzweig et al., 2021a). First, a team of trained research assistants classified students’ Page 7 of 18 self-generated career plans as being related to STEM or non-STEM fields, by matching their write-in long-term career plans to career titles from the ONet Database, which is a career database run by the U.S. Department of Labor (see SOM for detailed description of this process). If students reported changing career plans during college, we also asked them to write in what their initial career plans were at the beginning of college and we classified both their initial and long-term career plans as being in STEM, non-STEM, or unable to classify. Any students whose career plans were not clearly in STEM fields, either initially during college or at the time of the survey, were excluded from the sample (i.e., students who did not have clearly classifiable career plans in STEM or nonSTEM at either time point, who entered STEM careers during college, who left STEM careers during college, or who never were interested in STEM careers; see SOM for complete description). Identifying students who changed career plans To identify students who changed their career plans during college, we examined students’ responses to a yes/ no question, “Have your long-term career plans changed since the beginning of college?” Students who indicated yes were categorized to have changed their career plans. In taking this approach, our goal was to examine the psychological correlates of having changed career plans within STEM in any way that seemed meaningful in students’ minds. That is, small career changes within one major (e.g., moving from being a biology Ph.D. to a biology lab technician) or within one career category (e.g., moving from one type of physician career to another) constituted a career plan change in this study, so long as students considered that to be a change in their minds. Our goal in using this approach was to capture the varied and sometimes subtle nature of career plan changes that might be meaningful to students. Such changes could not be captured well using “objective” indicators of career affiliation (e.g., looking at whether students reported pursuing the same or different majors or career categories at different points in college) because those indicators may be too coarse to capture meaningful changes that students undertake. We hoped to demonstrate that even students making subtle changes in career plans report different levels of career motivation, satisfaction, and certainty compared to their peers who report not changing plans, and that these students are worth supporting more in STEM environments. Self‑reported motivation for career plans Students completed a questionnaire assessing their motivational beliefs regarding their current long-term career plans, which was adapted from prior questionnaires Rosenzweig et al. International Journal of STEM Education (2024) 11:15 grounded in situated expectancy value theory. Items were adapted from prior studies (which typically focus on motivation for a particular subject areas or courses) to reference students’ perceptions of their career plans (e.g., becoming a biological engineer, becoming a physician assistant). All items were answered on 7-point scales ranging from Not at All True to Very True. Confirmatory factor analyses showed excellent data-model fit of the measure, and the measure showed appropriate discriminant and predictive validity (see SOM for details and complete list of items). Students’ competencerelated beliefs were assessed with four items measuring how well students thought they would do in their career (adapted from Eccles & Wigfield, 1995; α = 0.81). Students’ perceptions of intrinsic value were measured with five items assessing how exciting and enjoyable a career would be (adapted from Conley, 2012; α = 0.91), perceptions of attainment value were measured with five items assessing the personal importance of pursuing a career (adapted from Conley, 2012; α = 0.85), and perceptions of utility value were assessed with four items assessing how practical and/or useful a career would be (adapted from Conley, 2012; α = 0.76). Students’ perceptions of cost were assessed with three items evaluating the effort required by a career, the valued alternatives one must give up to pursue a career, and anticipated negative emotional consequences of engaging with a career (adapted from Beymer et al., 2021; α = 0.72). Satisfaction with and certainty of pursuing career plans Students completed one item measuring career satisfaction, “How satisfied are you with your current long-term career plans?” They also completed one item measuring career certainty, “How certain are you that you will pursue this career?” These single-item measures were created by the researchers to represent the outcomes of most direct interest with respect to career readiness. Both were answered on 7-point scales ranging from Not at All ___ to Very ___. Student background information Students self-reported their gender, race/ethnicity, firstgeneration student status, and year in school. For field of study, students wrote in their majors or intended majors at the time of the survey, after which a trained research assistant classified each response as relating to one or more of the most common fields of study represented in the sample (biology-related fields of study, engineeringrelated fields of study, computer-science-related fields of study, or other fields of study; see SOM for details). Many students who changed career plans within STEM retained the same field of study/major despite changing career plans (i.e., students could change from a career Page 8 of 18 plan of doctor to that of biomedical lab technician, but retain a biology major), but some students who changed career plans also changed majors within STEM. For those students, the field of study variable used in the present study represents students’ new fields of study after having changed plans within STEM fields. College GPA Students self-reported their college GPA on a 0–4 scale to be used as a covariate in analyses. Self-reported GPAs are argued to be reliable and valid when measured from relatively higher performers and relatively older students (e.g., college students at a somewhat selective institution, Kuncel et al., 2005), which describes the context of this study well. Analytic strategy Descriptive statistics were used to indicate the proportion of students changing career plans within STEM fields (Research Question 1). To assess how changing plans was associated with students’ motivational beliefs (Research Question 2), linear regression was used to predict each indicator (competence-related beliefs, intrinsic value, utility value, attainment value, perceived cost, career satisfaction, career certainty) from students’ reports of whether or not they changed plans (change = + 1, no change = 0), while controlling for two covariates in addition to all of the student characteristics examined in Research Question 3. To assess differences by gender, race/ethnicity, field of study, and year in school (Research Question 3), logistic regressions were used to indicate whether students’ likelihood of changing plans differed as a function of student characteristics, and interaction terms were added to the regression models for Research Question 2 to assess whether changing plans interacted with any student characteristics in predicting any career readiness indicators. Student-level characteristics were operationalized in the models as follows: gender (men = 0; women = 1), racially minoritized status (historically underrepresented in STEM = 1; not historically underrepresented = 0), year in school (a set of four dummy-codes, representing each year—2nd, 3rd, 4th, or other—compared to the first year), field of study (a set of three dummy-codes, comparing students enrolled in biology-related fields of study, coded as 0, to (a) computer-science-related fields of study, (b) engineering-related-fields of study, or (c) other fields of study, each coded as 1, respectively). All analyses controlled for college GPA (standardized) to rule out the possibility that higher academic performance in general (or group differences in academic performance) drove observed effects on career motivation/satisfaction and on plans changing. All analyses also controlled for first-generation student Rosenzweig et al. International Journal of STEM Education (2024) 11:15 status (first-generation student = 1; non-first-generation student = 0).1 For the purposes of ensuring adequate statistical power to assess each gender group in the models, we excluded non-binary individuals and those who did not disclose gender from the analyses that included gender. We also grouped together students who were racially minoritized in STEM (i.e., students who indicated a racial identity of Black, Latino/a, and/or Native American) and compared them to students whose race was not minoritized in STEM fields, to maximize power to detect effects in the race/ethnicity moderator analyses. For missing data, a very small number of students were excluded from analyses who did not answer all of the items for a particular measure (n = 8 to 39 depending on the measure). Page 9 of 18 How is changing career plans within STEM associated with indicators of career readiness? A large proportion of students, 32.5%, reported having changed their career plans within STEM fields at some point during college, despite the sample being primarily first- and second-year students. Breaking this number down by students’ year in college revealed increasingly higher proportions of students having changed plans in each successive year of college, with a majority of fourthyear students (55.4%) reporting having changed career plans within STEM fields at some point (see Table 1). Tables 4, 5, 6 report the results of regression analyses predicting students’ expectancy-value motivational beliefs (Table 4 reports competence-related beliefs, Table 5 reports task values) and career satisfaction/certainty (Table 6) from their likelihood of changing career plans. Students who changed their career plans within STEM fields during college reported significantly lower scores for competence-related beliefs, utility value, attainment value, and intrinsic value for their long-term career plans, compared to students who retained consistent STEM career plans, βs = −0.09 to −0.16, ps < 0.001; see Fig. 1 for visualization. Similarly, students who changed their career plans within STEM fields reported lower career satisfaction and lower certainty of pursuing their chosen STEM careers, βs = −0.20 to −0.22, ps < 0.001; see Fig. 1. Perceived cost was the only variable not aligned with this pattern. Changing career plans within STEM fields was associated with students reporting lower perceived cost for their new careers (to be consistent with the other findings given the theoretical nature of this construct, one would expect students to report higher perceived cost), β = −0.10, p < 0.001. All effects just noted are small, but they represent the unique effects of plans changing, after controlling for students’ gender, racially minoritized status, year in school, field of study, self-reported GPA, and first-generation student status. Descriptive statistics and correlations among continuous variables Do findings differ by gender, race, year in school, or field of study? Descriptive statistics and correlations among key study variables are reported in Table 2; descriptive statistics for each variable by whether or not students changed plans are reported in Table 3. All correlations were of the expected magnitudes and directions, with the exception of the correlation between perceived cost and attainment value, which was not significant. On average, students were highly motivated for their chosen career paths, with low perceptions of cost. On average, students who had not changed career plans within STEM fields reported higher scores for each outcome measure compared to students who had changed plans. Logistic regression analyses were used to predict the likelihood of students changing career plans within STEM during college from their gender, race/ethnicity, year in school, and field of study, controlling for self-reported college GPA and first-generation college student status. Table 7 reports the results of these analyses. Gender was a significant predictor of plans changing within STEM fields, with women being significantly more likely than men to change career plans within STEM fields compared to retaining consistent STEM career plans, B = 0.31, S.E. = 0.14, p = 0.021, odds ratio = 1.37. Year in school was also a significant predictor, with each year corresponding to a significantly higher likelihood of changing career plans within STEM fields compared to the first year of college, Bs = 0.87 to 1.61 for the second through fourth years versus the first year, S.E.’s = 0.14 to 0.20, ps < 0.001, odds ratios = 2.38 to 5.01. Finally, students in engineering-related fields of study at the time of the survey were more likely to report having changed career plans than were students in biology-related fields of study, as were students in “other” fields of study (i.e., not biology, computer science, or engineering) compared Results How many students changed career plans within STEM fields throughout college? 1 Originally, it was our intention to test first-generation student status as another moderating variable in Research Question 3, but when included in models the predictors’ variance inflation factor statistics (an indicator of collinearity issues) became too high (> 6). We therefore chose to test only race/ethnicity as a moderator and not first-generation status in the present study, which eliminated the collinearity issues. If analyses omit the covariates of GPA and first generation status, this does not change the significance of any effects, except for one interaction between year in school and plans changing which became non-significant (p = .054 in the models without covariates included) and which is not interpreted as a key finding in the present study. Rosenzweig et al. International Journal of STEM Education (2024) 11:15 Page 10 of 18 Table 1 Who changes career plans within STEM fields in college Changed career plans Overall Did not change career plans Total 561 32.5% 1166 67.5% 1727 First 176 20.7% 674 79.3% 850 Second 143 38.1% 232 61.9% 375 Third 128 42.8% 171 57.2% 299 Fourth 72 55.4% 58 44.6% 130 Other 26 76.5% 8 23.5% 34 Woman 386 33.1% 781 66.9% 1167 Man 152 30.0% 354 70.0% 506 8 38.1% 13 61.9% 21 72 31.4% 157 68.6% 229 472 32.2% 992 67.8% 1464 195 28.3% 495 71.7% 690 Computer science-related field of study 56 32.7% 115 67.3% 171 Engineering-related field of study 82 36.9% 140 63.1% 222 223 35.1% 413 64.9% 636 Year in school Gender Non-binary or prefer not to say Race/ethnicity Historically marginalized in STEM Not historically marginalized in STEM Field of study Biology-related field of study Other field of study Percentages reflect the percentage of students in each sub-category who changed versus did not change plans, among those who provided data for each item Table 2 Correlations and descriptive statistics for key study variables Variable 1 2 3 4 5 6 7 1. Competence-related beliefs 2. Intrinsic value .578* 3. Attainment value .496 .613 4. Utility value .514 .591 .599 5. Perceived cost −.204 −.180** .019 −.052* 6. Career satisfaction .480 .572 .431 .419 −.160 7. Career certainty .464 .472 .402 .385 −.103 .710** M 5.379 6.221 5.908 5.889 3.725 5.770 5.500 S.D. 0.922 0.792 0.918 0.847 1.282 1.184 1.261 n 1705 1705 1705 1705 1705 1727 1727 p < .05; *p < .01 Rosenzweig et al. International Journal of STEM Education (2024) 11:15 Table 3 Mean scores for career motivation, satisfaction, and certainty as a function of plans changing within STEM Variable Changed career plans Did not change career plans M M S.D. S.D. Competence-related beliefs 5.27 0.97 5.43 0.89 Intrinsic value 6.06 0.86 6.30 0.75 Attainment value 5.75 0.98 5.98 0.88 Utility value 5.76 0.91 5.95 0.81 Perceived cost 3.54 1.27 3.81 1.28 Career satisfaction 5.47 1.31 5.91 1.09 Career certainty 5.19 1.35 5.65 1.19 Table 4 Results of regressions predicting competence-related beliefs for expected STEM career plans Variable Intercept B S.E. 5.54 β p 0.09 Plans changing −0.19 0.05 −0.09 Gender −0.12 0.05 −0.06 .025 0.01 0.07 0.00 .889 −0.04 0.06 −0.02 .505 Racially minoritized status First-generation status < .001 Year in school Second versus first 0.14 0.06 0.06 .019 Third versus first 0.19 0.06 0.08 .002 Fourth vs. first 0.25 0.09 0.07 .005 Other vs. first 0.37 0.17 0.06 .030 < .001 Field of study C.S.-related vs. Bio.-related −0.34 0.08 −0.11 Eng.-related vs. Bio.-related −0.03 0.07 −0.01 .705 Other fields vs. Bio.-related −0.05 0.05 −0.03 .315 0.07 0.03 0.07 .006 College GPA Plans Changing: Yes = 1, No = 0. Gender: Woman = 1, Man = 0. Racially Minoritized Status: Historically marginalized with respect to racial/ethnic identity in STEM = 1, Non-historically marginalized = 0. First-generation status: first-generation student = 1; non-first-generation student = 0. Year in School: Dummy codes representing each year versus the first year (focal year coded as 1, all other groups coded as 0, with first year as the reference group). Field of Study: Dummy codes representing each field of study versus biology-related fields of study (focal field of study coded as 1, all other fields of study coded as 0, with biology-related fields of study as the reference group). College GPA is standardized for analysis to students in biology-related fields of study, Bs = 0.33 to 0.38, S.E.’s = 0.13 to 0.18, ps = 0.037 to 0.014, odds ratios = 1.39 to 1.47. Although there were some differences in the likelihood of changing plans within STEM by student-level characteristics, there were few interactions suggesting that the relation between changing plans and the psychological outcome variables differed as a function of student characteristics. The SOM provides complete description of Page 11 of 18 the statistical output of these analyses and visualization of the few observed interactions. To summarize the findings, no associations between changing plans and career motivation, satisfaction, and certainty differed significantly as a function of race/ethnicity, and there was only one observed interaction across seven possible outcome measures suggesting that results differed by gender. There were only four interactions across the set of all four of the dummy codes for year in school predicting any of the seven outcome measures (i.e., only four significant effects out of 28 possible interactions), and even when accounting for those interactions the same overall trends still were observed. That is, some effects were slightly stronger among second-year compared to first-year students, but the same overall patterns held for almost all outcomes that changing career plans was associated with lower scores on the various measures. Similarly, for field of study, there were only three observed interactions across the set of three dummy codes predicting any of the seven outcome measures (i.e., 21 possible interactions), and the same overall trends were still observed even accounting for those interactions. Discussion Students who reported changing career plans within STEM fields during college also reported lower scores on multiple psychological indicators of career motivation— namely competence-related beliefs, intrinsic value, utility value, and attainment value—as well as on satisfaction with and certainty of persisting at their current careers, compared to students who reported retaining consistent STEM career plans throughout college. A large proportion of students also had changed career plans within STEM, and women reported changing more often than did men. These findings demonstrate that researchers, educators, and policy-makers should examine students who switch career plans within STEM fields, not just those who leave STEM careers, to contribute to STEM workforce participation and equity. The importance of studying students who change career plans within STEM On every indicator of career motivation examined except perceived cost, individuals who changed career plans within STEM reported lower scores than individuals who retained consistent career plans. Changing one’s career plans is not in and of itself a bad thing – research focused on both STEM attrition (e.g., Seymour & Hunter, 2019; Rosenzweig et al., 2021b; Thoman et al., 2017) and within-STEM career plan shifts (Rosenzweig et al., 2021a) suggests that many students are attracted to new career paths positively, because the new plans align better with students’ interests and preferences. The findings Rosenzweig et al. International Journal of STEM Education (2024) 11:15 Page 12 of 18 Table 5 Results of regressions predicting task values for expected STEM career plans Intrinsic value Variable Intercept Plans changing B Attainment value S.E. β p B 6.16 0.08 −0.27 0.04 −0.16 Gender 0.15 0.04 0.09 Racially minoritized status 0.00 0.06 0.00 First-generation status 0.01 0.05 S.E. β Utility value p B Perceived cost S.E. β p B S.E. β p 5.69 0.09 5.79 0.08 3.67 0.12 < .001 −0.27 0.05 −0.14 < .001 −0.22 0.05 −0.12 < .001 −0.27 0.07 −0.10 < .001 < .001 0.21 0.05 0.11 < .001 0.13 0.05 0.07 .008 0.06 0.07 0.02 .423 .958 −0.04 0.07 −0.01 .559 −0.01 0.06 0.00 .912 0.16 0.09 0.04 .086 0.01 .795 0.14 0.06 0.06 .013 0.04 0.05 0.02 .437 0.22 0.08 0.07 .006 Year in school Second versus first 0.06 0.05 0.03 .228 0.10 0.06 0.05 .070 0.05 0.05 0.03 .316 0.11 0.08 0.04 .172 Third versus first 0.16 0.05 0.08 .002 0.18 0.06 0.08 .004 0.15 0.06 0.07 .008 0.16 0.09 0.05 .069 Fourth vs. first 0.27 0.07 0.09 < .001 0.31 0.09 0.09 < .001 0.13 0.08 0.04 .105 −0.10 0.12 −0.02 .395 Other vs. first 0.04 0.14 0.01 .772 0.13 0.16 0.02 .443 0.01 0.16 0.00 .960 −0.28 0.23 −0.03 .226 < .001 −0.30 0.08 −0.10 < .001 −0.61 0.12 −0.14 < .001 .768 −0.47 0.10 −0.12 < .001 .348 −0.31 0.07 −0.12 < .001 Field of study −0.26 0.07 −0.10 < .001 −0.33 0.08 −0.11 Eng.-related vs. Bio.-related −0.05 0.06 −0.02 C.S.-related vs. Bio.-related .409 −0.21 0.07 −0.08 Other fields vs. Bio.-related College GPA .004 0.02 0.07 0.01 0.03 0.01 0.04 0.01 .814 −0.03 0.05 −0.02 .585 0.04 0.05 0.01 0.02 0.01 .791 .315 0.00 0.02 −0.01 0.02 0.02 0.03 .845 0.07 0.04 0.05 .060 Plans Changing: Yes = 1, No = 0. Gender: Woman = 1, Man = 0. Racially Minoritized Status: Historically marginalized with respect to racial/ethnic identity in STEM = 1, Non-historically marginalized = 0. First-generation status: first-generation student = 1; non-first-generation student = 0. Year in School: Dummy codes representing each year versus the first year (focal year coded as 1, all other groups coded as 0, with first year as the reference group). Field of Study: Dummy codes representing each field of study versus biology-related fields of study (focal field of study coded as 1, all other fields of study coded as 0, with biology-related fields of study as the reference group). College GPA is standardized for analysis Table 6 Results of regressions predicting career satisfaction and certainty for expected STEM career plans Variable Career satisfaction B Intercept S.E. Career certainty β p B S.E. β p 5.64 0.11 5.48 0.12 −0.51 0.06 −0.20 < .001 −0.59 0.07 −0.22 Gender 0.14 0.07 0.06 .034 0.04 0.07 0.02 .532 Racially minoritized status 0.01 0.08 0.00 .882 −0.02 0.09 −0.01 .816 First-generation status 0.03 0.07 0.01 .681 −0.06 0.08 −0.02 .444 Plans changing < .001 Year in school Second versus first 0.03 0.07 0.01 .668 0.28 0.08 0.10 < .001 Third versus first 0.21 0.08 0.07 .008 0.52 0.08 0.16 < .001 Fourth vs. first 0.53 0.11 0.12 < .001 0.79 0.12 0.17 < .001 Other vs. first 0.43 0.21 0.05 .044 0.43 0.22 0.05 .053 Field of study C.S.-related vs. Bio.-related −0.11 0.11 −0.03 .307 -0.03 0.11 −0.01 .780 Eng.-related vs. Bio.-related 0.10 0.09 0.03 .274 -0.09 0.10 −0.02 .382 Other fields vs. Bio.-related College GPA 0.19 0.06 0.08 .003 0.13 0.07 0.05 .059 0.06 0.03 0.05 .057 0.07 0.03 0.05 .048 Plans Changing: Yes = 1, No = 0. Gender: Woman = 1, Man = 0. Racially Minoritized Status: Historically marginalized with respect to racial/ethnic identity in STEM = 1, Non-historically marginalized = 0. First-generation status: first-generation student = 1; non-first-generation student = 0. Year in School: Dummy codes representing each year versus the first year (focal year coded as 1, all other groups coded as 0, with first year as the reference group). Field of Study: Dummy codes representing each field of study versus biology-related fields of study (focal field of study coded as 1, all other fields of study coded as 0, with biology-related fields of study as the reference group). College GPA is standardized for analysis Rosenzweig et al. International Journal of STEM Education (2024) 11:15 Page 13 of 18 Fig. 1 Comparing students who changed career plans within STEM fields to students who retained consistent STEM career plans on indicators of their current career motivation, satisfaction, and certainty of persisting. Unadjusted mean scores shown here; error bars represent + −1 SE of the mean. In regressions adjusting for student-level characteristics, all mean differences between groups are significant at p < .001 Table 7 Logistic regression analyses predicting likelihood of changing career plans within STEM Variable B Intercept −1.61 0.23 Gender S.E. Wald p Odds Ratio 0.31 0.14 5.35 .021 1.37 Racially minoritized status −0.24 0.17 1.98 .159 0.79 First-generation status −0.12 0.15 0.68 .409 0.89 Year in school Second vs. first 0.87 0.14 37.68 < .001 2.38 Third vs. first 1.12 0.15 56.66 < .001 3.07 Fourth vs. first 1.61 0.20 64.42 < .001 5.01 Other vs. first 2.62 0.43 36.85 < .001 13.69 Field of study C.S.-related vs. Bio.-related 0.06 0.22 0.07 .793 1.06 Eng.-related vs. Bio.related 0.38 0.18 4.35 .037 1.47 Other fields vs. Bio.-related 0.33 0.13 6.64 .010 1.39 −0.07 0.06 1.38 .240 0.93 College GPA Plans Changing: Yes = 1, No = 0. Gender: Woman = 1, Man = 0. Racially Minoritized Status: Historically marginalized with respect to racial/ethnic identity in STEM = 1, Non-historically marginalized = 0. First-generation status: first-generation student = 1; non-first-generation student = 0. Year in School: Dummy codes representing each year versus the first year (focal year coded as 1, all other groups coded as 0, with first year as the reference group). Field of Study: Dummy codes representing each field of study versus biology-related fields of study (focal field of study coded as 1, all other fields of study coded as 0, with biology-related fields of study as the reference group). College GPA is standardized for analysis of this study should not be taken to imply that we should prevent students from changing career plans within STEM fields. However, these results do show that not all students who retain an STEM career plan across college are equally motivated to succeed their chosen careers. As was discussed in the Introduction, much prior research shows that competence-related beliefs and intrinsic, attainment, and utility value each are predictive of individuals’ satisfaction with their achievement activities, performance in them, engagement with them over time, and likelihood of persisting in them (e.g., Guo et al., 2015a, 2015b; Lauermann et al., 2017; Perez et al., 2014). There are particularly strong links reported in the literature between each of these motivational beliefs and certain career-related outcomes (e.g., competencerelated beliefs most strongly predict performance, utility and attainment value most strongly predict persistence, and intrinsic value most strongly predicts satisfaction), but there are also likely to be multifaceted links of each construct to a variety of career-related outcomes. Given these prior findings, it would be expected that individuals with lower self-reported scores on each indicator of career motivation as defined by situated expectancyvalue theory (or higher cost) would also report lower career satisfaction and certainty of persisting at their careers, and indeed in this study we also found that students who changed career plans within STEM reported lower scores on these two indicators of career readiness. Findings have important implications for existing research and policy efforts, which focus predominantly on understanding why students leave STEM careers for non-STEM fields (particularly in the motivational research field). If we want a qualified and successful STEM workforce where employees are satisfied and put forth effort, especially in specific STEM career paths where there is a high need for additional employees, students who change career plans within STEM fields comprise a group that may block such goals. It may not be Rosenzweig et al. International Journal of STEM Education (2024) 11:15 sufficient to focus research and policy on trying to prevent students from leaving STEM altogether. Instead, it is important to identify students who are likely to or who actually do change plans within STEM fields, study them further to understand their experiences, and consider how we can provide students like these with additional supports throughout college to help them feel more motivated for, satisfied with, and certain of persisting in their chosen STEM career paths. There are two explanations for why individuals who changed plans within STEM fields may have reported lower career motivation than individuals who retained consistent career plans. First, as was discussed in the Introduction, there may have been pre-existing differences in motivation to engage with a particular STEM field between students who changed career plans within STEM compared to those who did not. According to this explanation, changing plans would not cause lower motivation among students who change plans within STEM, but instead changing career plans within STEM would be an indicator of which students started off with relatively lower motivation for their STEM fields of interest. Having lower perceived competence or intrinsic, utility, and attainment value for a particular STEM field of study could lead these students to change plans more often, and it could lead these students to report lower corresponding levels of motivational beliefs for any careers within that field, compared to individuals who had not changed plans. Even if students who change career plans within STEM fields are more motivated for their new STEM careers than they were for their original STEM careers, that does not mean they are as highly motivated for their new careers as students are who have always been motivated to pursue a particular STEM career are for their long-term desired career paths. For example, a student who has always loved zoology and wants to be a veterinarian might be more strongly motivated for that career path compared to a student who feels moderately about zoology and decides to become a veterinarian instead of a doctor halfway through college. The second possible explanation is that changing career plans may have caused students to report lower career motivation. That is, the process of changing career plans may harm students’ motivation for their new career pursuits by interfering with students’ opportunities to develop competence-related beliefs and intrinsic, attainment, and utility value. As was noted in the Introduction, interest develops over time via repeated engaging experiences in relevant contexts, followed by opportunities to connect in a meaningful personal way with a particular activity/career path (Hidi & Renninger, 2006). In turn, experiences where students make relevance connections are what can develop over time into more long-term Page 14 of 18 perceptions of value like attainment and utility value (Priniski et al., 2018). Students who change to a new career path may have emerging interest but they may not have as many opportunities to experience relevant activities within it (e.g., job shadowing, internship activities) or as many chances to engage in a personal way with course material (e.g., by choosing paper topics that are relevant to their future careers) compared to students who have always had that career interest. This could interfere with the development of intrinsic, attainment, and utility value for one’s new career path. Similarly, students who change career plans within college may have fewer experiences with hands-on successes relevant to a career path and fewer opportunities to develop their skills. Hands-on success activities are the strongest predictors of students’ competence-related beliefs (Butz & Usher, 2015). Thus students who change career plans within STEM are likely not to have as many opportunities to develop such beliefs as do students who retain consistent career plans. Our cross-sectional findings cannot shed light on which of these explanations is more likely. We hypothesize that both explanations are likely to be true, given that there is a relevant base of prior literature to support both arguments. This should be confirmed in subsequent research by using a longitudinal study. Longitudinal research also can examine whether the differences in career readiness and certainty that were observed here are likely to persist over time, once students have a chance to engage in more hands-on activities within their new career. It is possible that students who changed plans actually will be more motivated than those who did not change plans once in the workforce, because they are confident that they prefer this career over alternatives and have engaged in meaningful identity exploration (Perez et al., 2014). Regardless of whether effects persist, findings of the present study suggest that students in college who change career plans within STEM fields can benefit from motivational supports for their chosen career paths at the time when they are enrolled in college, when their motivational beliefs for their careers are lower than their peers’ beliefs. Instead of primarily trying to prevent students from leaving STEM careers, or trying to motivate students towards STEM versus non-STEM pursuits, educators and administrators should think more specifically about how to support students who change career plans within STEM. They might identify students who have changed plans within STEM (e.g., through regular advising meetings or surveys) and ensure these students (a) know what coursework and career preparation activities are required for success in their new career path, to help promote their competence-related beliefs (e.g., Butz & Usher, 2015) and (b) have opportunities to engage in career preparation experiences (e.g., job shadowing, Rosenzweig et al. International Journal of STEM Education (2024) 11:15 internships), to engage in sustained hands-on activities to build interest and value (e.g., Hidi & Renninger, 2006). More broadly, proactive structural supports for all students’ career motivation can be helpful in ensuring that students feel optimally motivated for their chosen paths in STEM and are less likely to switch in the first place. To that end, educators or administrators might consider adapting existing interventions that have helped college students reflect on the utility value or attainment value of what they are learning in a course (e.g., Harackiewicz & Prinsiki, 2018; Perez et al., 2022) to focus on career plans, or by helping students perceive more belonging in their career paths, for example by providing mentoring opportunities (e.g., Dollinger et al., 2019) or more connections to STEM role models (see Gladstone & Cimpian, 2021, for review). They might also consider providing better infrastructure to support career development within the requirements for STEM majors, for example by providing courses that introduce students to various career pathways in a major, or by requiring an internship. The recommendations for future research and career development supports just provided are all the more important given the large proportion of students who changed career plans within STEM fields in this study. Despite a sample of mostly first- and second-year students, more than 30% of students in our study reported having changed career plans when surveyed, and among fourth-year students, a majority reported having changed career plans within STEM fields. This study provides an initial estimate of the magnitude of within-STEM career shifts happening during college at a 4-year state research university, with findings suggesting that such shifts are likely to be equally if not more common than attrition out of STEM majors (i.e., up to about half of students are estimated to leave STEM fields during college; Chen, 2012). By revealing a large number of students who do not leave STEM yet risk lower career readiness, results underscore the consequences of ignoring students who change career plans within STEM fields in workforce education efforts and interventions. The only result that did not follow the trends noted above was the finding that students who changed career plans within STEM fields reported lower perceptions of cost compared to students retaining consistent career plans. Our initial hypotheses predicted the opposite pattern, which is that perceptions of cost would be higher among students who changed plans (either because they had higher initial perceptions of cost or because the process of changing plans made students perceive more cost). Relative to the other motivational beliefs in situated expectancy-value theory, perceived cost has been studied much less and its developmental antecedents are less well-understood (Eccles & Wigfield, 2020; Flake Page 15 of 18 et al., 2015; Wigfield et al., 2017). It is possible, conceptually, that changing career plans could help students reappraise the challenges of a new career in a more positive light, which made those challenges seem less costly than original career challenges (Rosenzweig et al., 2020). This would imply that one motivational benefit of changing career plans within STEM is perceiving that the new plan does not require one to “give up” as much to persist at it. However, more research is needed on cost and career planning to understand this particular effect most fully. Student characteristic differences in career plan changes within STEM fields We had only exploratory hypotheses about student-level characteristics that might shape students’ experiences in a way that would affect the relations we observed in this study, and in general we found few effects suggesting that the trends just discussed differed as a function of any of the characteristics we examined. Results broadly speaking point to the robustness of key findings across different types of students. The only major finding that does not fit that description was that women were more likely to change career plans within STEM fields than men were. This finding extends prior research showing the same gender difference in biomedical fields with a different sample (Rosenzweig et al., 2021a). Although women are not underrepresented in all STEM fields, they are underrepresented in many specific STEM sub-fields, particularly computer science and engineering. In thinking about how to address these issues, researchers often examine why women leave STEM altogether; however, there also seem to be systematic differences in how women negotiate career options within STEM fields that lead to them changing career plans more readily during college compared to men. These findings can be due to a variety of explanations, including (but not limited to) the idea that women may be more attuned to careers that afford communal opportunities due to their past socialization about this topic and change plans more readily to seek out careers that afford such opportunities and align with their values. To understand the precise mechanism of the effect observed requires more research, but this finding does point to the possibility that women might be more likely to have lower motivation for their chosen STEM career paths, either because they began college less motivated for their STEM field of choice due to past experiences and socialization that shaped their perception of career value affordances, or because their shift in plans may make them more likely to lose career motivation. Gendered within-STEM career shifts may explain some of the disproportionate patterns of participation as a function of gender in particular career areas within STEM, such as the life sciences versus Rosenzweig et al. International Journal of STEM Education (2024) 11:15 engineering fields. At the same time, within-STEM career shifting also can occur even in STEM sub-fields where women are not underrepresented overall (e.g., biological sciences), leading to women leaving certain types of specific career paths within those sub-fields at a higher rate than men (e.g., women may leave the career path of doctor more often than men, despite still pursuing some kind of biomedical career path; Rosenzweig et al., 2021a). Such patterns may shed new light on particular types of career shortages and whether there is equity in who pursues versus leaves specific career paths in different areas of STEM. These findings suggest that examining changes in career plans within STEM is an essential complement to work on STEM attrition, if educators or administrators want to ensure they provide equitable supports for students to succeed in STEM career paths. Besides the gender difference in how many students reported changing plans, there were few other studentlevel factors that predicted students changing career plans besides year in school (which is not surprising, because this is a cross-sectional dataset and each year more students have an opportunity to change plans) and in some of the analyses, field of study. There were also few differences suggesting that the relation between changing career plans and STEM career motivation or certainty differed as a function of any student-level variables. As was discussed in the Introduction, much research suggests that these individual characteristics can impact students’ experiences in and attitudes about STEM careers, which in turn can affect students’ STEM career participation. However, our results suggest that the processes by which changes in career plans were associated with motivation may be somewhat robust across different types of student characteristics. This is a preliminary exploration of student characteristics, and future research should build on these findings (e.g., by examining the intersections of different student identities, by exploring different types of student characteristics, or by using methods that allow students to articulate how their identities shape their STEM career decision-making). Limitations and conclusions This study provides only a first examination of the experience of changing career plans within STEM fields, and future studies should build on the limitations of the present analysis. In particular, this analysis is cross-sectional and it is not possible to know whether having lower career motivation causes students to change plans within STEM fields, whether changing plans within STEM fields causes students to have lower career motivation, or both. We believe that theory supports a bidirectional relationship whereby students have lower motivation and career readiness both before and because of career transitions Page 16 of 18 within STEM fields, but future research should examine this possibility empirically. Only by doing so can researchers understand the best avenues for intervention for these students. Similarly, a longitudinal data analysis examining motivation at the point when students change career plans across college, rather than cross-sectional data that does not address career plan change timing directly, is a better way to examine the processes by which students make career transitions. Another limitation is that these findings were collected only at one institution. Institutions each have specific norms around how certain departments and fields of study operate (e.g., how much faculty care about equitable education, how much career preparation is required in different programs, how difficult or easy it is to obtain career training within different fields), and different types of supports for students in different years of their education (e.g., mentorship opportunities, social support, advising). Because of this, it is not possible to tease apart the role of student characteristics versus institutional norms fully in this study. We believe that given the breadth and scope of our sample and the variety of courses measured, findings are likely to be generalizable to other 4-year research-intensive U.S. universities with similar student demographics, but this would need to be confirmed at other institutions. Additionally, the same trends may not apply to all types of universities (e.g., liberal arts colleges, institutions in other countries, minority-serving institutions). Future research should explore these trends in more varied samples and explicitly measure the institutional norms and practices that might coincide with higher levels of career motivation and/or satisfaction. Going along with this aim, in more diverse samples it will be more possible to test a wider array of student-level variables that might be influential in particular learning contexts and institutions that are not the one where the present study was conducted (e.g., first-generation student status). A final limitation is in our operationalization of some constructs. In terms of motivational beliefs, we used the major constructs of situated expectancy-value theory as the lens through which we examined career motivation, but there are many other potential motivational beliefs that could be important to consider with respect to STEM career participation (e.g., autonomy, competence, and relatedness needs, intrinsic motivation, or mindset, see Miele & Wentzel, 2016, for discussion). Future research can complement this study by examining additional constructs to complement this work. We also measured career plan changes using students’ self-reports of whether they perceived themselves to have changed career plans. Although this approach helped capture a breadth of different career changes that were perceived Rosenzweig et al. International Journal of STEM Education (2024) 11:15 by students to represent a different future career, such questions are subjectively interpreted by students and we did not assess what types of different changes students made or whether all students defined career plan changes in the same ways. These avenues of investigation would provide additional nuance to the findings reported here and suggest specific types of career change pathways that might be most in need of additional supports. Acknowledging the importance of these next steps, we nonetheless believe that findings of this study contribute several novel and critical contributions to the field of STEM career education research in higher education. Most critically, results point to a critical, underexamined group of students whose experiences in college could be improved to enhance STEM workforce participation and success: those who change career plans within STEM fields. There are large numbers of students who make these shifts and thus could benefit from more precise and targeted supports to ensure that they do not ultimately experience lower career readiness than their peers. Although much research has examined STEM career participation, it often focuses heavily on STEM attrition and thus neglects to consider experiences of students who remain in STEM fields of study but shift in their career plans. Second, the gender differences observed in the frequency of shifting career plans within STEM fields points to the importance of addressing this phenomenon to ensure that women are not disproportionately undermotivated or under-prepared for success in the STEM workforce. With more attention to the experiences of students who change career plans within STEM fields, alongside attention to STEM attrition, researchers can address STEM workforce shortages and inequities more completely and comprehensively. Page 17 of 18 Acknowledgements We would like to thank the Scientists Engaged in Education Research Group at the University of Georgia for assistance in project implementation and com‑ munication, as well as all instructors who assisted in recruitment for this study. We would like to thank Mr. David Seiden for assistance with initial data analysis in the project. We would like to thank Ms. Xiying Wang and Ms. Samantha Barilla for assisting in coding of students’ career plans. Author contributions ER: conceptualization, data curation, formal analysis, investigation, meth‑ odology, project administration, resources, supervision, writing—original draft, writing—review and editing, visualization. XYC: project administration, data curation, formal analysis, investigation, methodology, validation, writ‑ ing—review and editing. YS: data curation, formal analysis, validation. Writ‑ ing—review and editing. AB, MB, MC, JD, AI, NW, JW, CW: conceptualization, investigation methodology, writing—review and editing. PPL: conceptualiza‑ tion, investigation, data curation methodology, supervision, writing—review and editing. Funding There is no funding associated with this project. Availability of data and materials The data and code from this project are available upon request to the cor‑ responding author; data are not publicly available at present due to other ongoing work with the broader project from which this data is drawn. All study questionnaires and materials analyzed in the present paper are listed in the manuscript and/or in the Additional file 1. Declarations Competing interests The authors declare that they have no competing interests. Author details 1 Department of Educational Psychology, University of Georgia, 325J Aderhold Hall, 110 Carlton Street, Athens, GA 30602, USA. 2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, USA. 3 School of Com‑ puting, University of Georgia, Athens, USA. 4 Department of Plant Biology, University of Georgia, Athens, USA. 5 Odum School of Ecology, University of Georgia, Athens, USA. 6 Department of Physics and Astronomy, University of Georgia, Athens, USA. 7 Department of Microbiology, University of Georgia, Athens, USA. Received: 12 January 2023 Accepted: 24 February 2024 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s40594-024-00475-6. Additional file 1: Table S1. Items in Career Expectancy-Value Motiva‑ tional Measure. Table S2. Results of Regressions Predicting CompetenceRelated Beliefs for Expected STEM Career Plans with Interactions by Gender and Racially Minoritized Status. Table S3. Results of Regressions Predicting Task Values for Expected STEM Career Plans with Interactions by Gender and Racially Minoritized Status. Table S4. Results of Regressions Predicting Career Satisfaction and Certainty with Interactions by Gender and Racially Minoritized Status. Table S5. Results of Regressions Predicting Competence-Related Beliefs for Expected STEM Career Plans with Interac‑ tions by Field of Study and Year in School. Table S6. Results of Regressions Predicting Task Values for Expected STEM Career Plans with Interactions by Field of Study and Year in School. Table S7. Results of Regressions Predict‑ ing Career Satisfaction and Certainty with Interactions by Field of Study and Year in School. Figure S1. Visual depiction of interactions between changing plans and year in college in predicting self-reported motiva‑ tional beliefs. Figure S2. 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https://openalex.org/W2123911773	https://europepmc.org/articles/pmc3907792?pdf=render	English	null	Breast conserving treatment for breast cancer: dosimetric comparison of different non-invasive techniques for additional boost delivery	Radiation oncology	2,014	cc-by	6,632	RESEARCH Open Access Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Background comparison of the boost techniques used in the EORTC 'boost versus no boost' trial showed no significant differ- ence between electron, photon or interstitial boost in terms of fibrosis and local control [11,12]. But it was not the primary goal of this trial to investigate different out- come with different boost techniques. Differences within several photon boost techniques have not been investi- gated. It was our objective to make a dosimetric compari- son of different non-invasive treatment techniques for additional boost delivery to offer an individualized best treatment to breast conserved patients. Postoperative irradiation after breast conserving surgery (BCS) for breast cancer has shown a gain in recurrence free and overall survival [1-9]. An additional boost to the initial tumor bed has shown an additional gain in re- currence free survival [10]. In the last decades, a lot of attention has gone to the development of new techniques to reduce side effects. In case of breast irradiation this means late side effects on skin, heart and lungs. With this evolution, several techniques to deliver a boost dose to the initial tumor bed have become available. Historically the boost dose mainly was delivered by electrons. To date, it is unclear which technique should be preferred. A © 2014 Van Parijs et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Breast conserving treatment for breast cancer: dosimetric comparison of different non-invasive techniques for additional boost delivery lde Van Parijs†, Truus Reynders†, Karina Heuninckx, Dirk Verellen, Guy Storme and Mark De Ridd Abstract Background: Today it is unclear which technique for delivery of an additional boost after whole breast radiotherapy for breast conserved patients should be state of the art. We present a dosimetric comparison of different non-invasive treatment techniques for additional boost delivery. Methods: For 10 different tumor bed localizations, 7 different non-invasive treatment plans were made. Dosimetric comparison of PTV-coverage and dose to organs at risk was performed. Results: The Vero system achieved an excellent PTV-coverage and at the same time could minimize the dose to the organs at risk with an average near-maximum-dose (D2) to the heart of 0.9 Gy and the average volume of ipsilateral lung receiving 5 Gy (V5) of 1.5%. The TomoTherapy modalities delivered an average D2 to the heart of 0.9 Gy for the rotational and of 2.3 Gy for the static modality and an average V5 to the ipsilateral lung of 7.3% and 2.9% respectively. A rotational technique offers an adequate conformity at the cost of more low dose spread and a larger build-up area. In most cases a 2-field technique showed acceptable PTV-coverage, but a bad conformity. Electrons often delivered a worse PTV-coverage than photons, with the planning requirements achieved only in 2 patients and with an average D2 to the heart of 2.8 Gy and an average V5 to the ipsilateral lung of 5.8%. Conclusions: We present advices which can be used as guidelines for the selection of the best individualized treatment. Keywords: Breast cancer, Tumor bed boost, Image guided radiation treatment (IGRT), Intensity modulated radiotherapy (IMRT), TomoTherapy, Vero © 2014 Van Parijs et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: hilde.vanparijs@uzbrussel.be †Equal contributors UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 CI ¼ TV PIV2 TVPIV TV ¼ target volume PIV ¼ prescription dose volume TVPIV ¼ overlap of TV and PIV In this setting the TV is the PTVboost-eval, the PIV is the 95% isodose of 16 Gy. Figure 1 shows the different planning techniques. For the electrons 1 beam perpendicular to the breast was used. The aperture of this beam was a rectangular shaped block surrounding the PTVboost to encompass it with the 95% isodose. The energy was chosen to reach the deepest point of the PTVboost with the 95% isodose. Available energies were in the range of 6 to 15 MeV. For the photon boost with 2 static fields either 2 tangential or 2 wedged fields could be used, depending on the localization of the PTVboost. When using the 3-field technique a perpendicular field to the breast was added to 2 tangential fields. For the planning on Vero 2 con- formal tangential fields (ring 0°) were chosen to cover the PTVboost and avoid as much as possible the ipsilateral lung, heart and contralateral breast. Afterwards 2 more beams per tangential beam were added with the same gantry angle but different ring rotation (30° and 330°). As last part more conformal beams and compensation fields were added to reach a conformal dose distribution with a low dose to OAR. The maximum amount of beams per Multiple treatment approaches were deployed for each CT set to deliver a dose of 16 Gy in 8 fractions of 2 Gy, which is the dose we prescribe in daily clinical practice in our department based on the EORTC boost versus no boost trial [12]. For each situation, all of following tech- niques were planned: electrons, a photon boost with 2 and 3 static fields and a photon boost with dynamic conformal arc using the CMS XIO planning software (Elekta AB, Stockholm, Sweden), a photon boost with the Vero® system (joint product of BrainLAB; BrainLAB AG, Feldkirchen, Germany and MHI; Mitsubishi Heavy Industries, Tokyo, Japan) [14], which has the possibility to turn the ring- gantry from 30 to 330 degrees towards the table, a photon boost with the TomoTherapy® system using rotational IMRT, as well as the static application (TomoDirect®) for tangential IMRT (Accuray Inc., Madison, USA) (Table 1). Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 The planning aims were to cover 95% of the volume of the PTVboost with at least 95% of the prescribed dose, but not more than 107%. For all OAR, except for the ipsilateral breast, the volume receiving 5 Gy should not exceed 5%. For the ipsilateral breast there were no con- straints, since today it is unclear which degree of dose spread within the breast should be considered as Ac- ceptable. As an alternative to using constraints, several measurements for conformity were used. As a measure of low dose spread the ratio of the volume of the 20% isodose to the 95% isodose (Vol20/Vol95) and to the ratio of the volume of the 50% isodose to the 95% isodose (Vol50/Vol95) were registered. As a measure of con- formity the conformity index (CI) was calculated, using the following formula [16]: localizations were equally present. On each side a tumor located in each of the 4 different quadrants and a cen- trally located tumor was chosen. Within this selection, attention was given to select small as well as larger boost volumes, small as well as larger breast volumes and deeply as well as superficially located tumors. A 'CTVboost' was drawn to include the site of the pri- mary tumor, according to pre-operative imaging of the breast and according to the visual seroma and/or fibrosis on post-operative CT, with a margin of 7 mm in all di- rections to encompass potential microscopic disease extension. When present, surgical clips were to be within the CTVboost. The CTVboost excluded the skin, pectora- lis muscle, ribs, lung and heart. PTVboost to CTVboost margin was 6 mm in all directions, but limited at the skin. The PTVboost, which could extend beyond the pectoralis major muscle/breast tissue interface, was used for deter- mining the aperture of the treatment fields. A 'PTVboost- eval' was defined as the PTVboost limited at 5 mm below the skin surface. The PTVboost-eval was used for generat- ing dose volume histograms (DVH) and comparative ana- lyses. A margin of 5 mm was chosen to minimize the contribution of the dose build-up area at the skin. As or- gans at risk (OAR) the ipsilateral lung, heart, ipsilateral breast and contralateral breast were contoured. Methods From a pool of available CT scans of early breast cancer patients treated in an earlier trial [13], 10 situations were selected. In this selection left and right breast cancer * Correspondence: hilde.vanparijs@uzbrussel.be †Equal contributors UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium Page 2 of 7 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Table 1 Used planning software Technique Planning software Type of calculation algorithm [15] Electrons CMS XIO V4.64 b Photons with 2 static fields CMS XIO Release V4.62.00.13 b Photons with 3 static fields CMS XIO Release V4.62.00.13 b Photons with dynamic arc CMS XIO Release V4.62.00.13 b Vero® iPlan RT Dose 4.1.2 for Vero® b Photons with rotational IMRT (TomoTherapy®) TomoTherapy Planning Station H-Art Version 4.0.5 b Photon boost with tangential IMRT (TomoDirect®) TomoTherapy Planning Station H-Art Version 4.0.5 b Planning software per technique and corresponding type of calculation algorithm [15]. Table 1 Used planning software Technique Table 1 Used planning software h i Page 3 of 7 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Figure 1 Dose distribution for 1 patient for all techniques. The dose distribution for 1 patient for all techniques: (a) electrons, (b) 2 tangential fields, (c) 3 fields, (d) arc, (e) Tomotherapy, (f) Tomodirect, (g) Vero. Figure 1 Dose distribution for 1 patient for all techniques. The dose distribution for 1 patient for all techniques: (a) electrons, (b) 2 tangential fields, (c) 3 fields, (d) arc, (e) Tomotherapy, (f) Tomodirect, (g) Vero. was 1.6 cm (range: 0.6 - 2.7 cm). In 3 patients the dee- pest border of the tumor was located more than 3.5 cm from the skin surface. In only 1 patient the PTVboost did not reach the skin surface. The mean PTV volume was 71.73 cc (range: 24.91 - 137.88 cc). The mean vol- ume of the ipsilateral breast was 447.92 cc (range: 108.66 - 865.74 cc). The mean PTV to ipsilateral breast ratio was 19.6% (range: 7.8% - 34.2%) with 2 patients having a ratio of more than 30%, 2 patients between 20 and 30%, 5 patients between 10 and 20% and 1 patient with a ratio of less than 10%. Not all 10 tumor localizations were clinically acceptable for electron boost delivery. In 2 patients the tumor was located too deep to cover the PTV with 15 MeV electrons. In the 3rd patient the tumor bed was located in the lateral breast fold. In practice, this patient would have been repositioned for electron boost patient was kept at 10 to keep the treatment time accept- able. A treatment with 10 beams can take up to 25 mi- nutes. TomoTherapy combines a rotational IMRT with a translational movement of the couch. Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Blocking structures and working volumes were used as was published earlier [17]. TomoDirect is the static application of TomoTherapy, where the gantry can be fixed at pre-chosen angles. Four tangential beams and 1 beam perpendicular to the breast were used to conform the dose. Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 The CI was equal to or more than 60% in 2 pa- tients with electrons, in none of the 2-field photon boost plans, in all 10 of the 3-field photon boost plans and Vero, in 9 of the dynamic arc plans, in 6 of the rotational and in 5 of the tangential TomoTherapy plans (Table 3). Today there is no literature available that gives an idea of which CI is acceptable. From this analysis, our conclusion is that a CI of 70% or more can be considered as excellent, between 60% and 70% as good, between 50% and 60% as acceptable and less than 50% as bad. The mean Vol50/ Vol95 was more than 4 with the 3-field photon technique and with the tangential TomoTherapy technique, meaning that for these 2 modalities more than 4 times the volume of the actual target received an over-dosage of 50% of the Conformity index for the different techniques, distribution per interval of 10%: (1) electrons, (2) 2 tangential fields, (3) 3 static fields, (4) arc, (5) Vero, (6) TomoTherapy, (7) TomoDirect. prescribed dose or an over-dosage of 16% to the surround- ing tissue, already treated to 50 Gy. Low dose spread was more present with dynamic arc photon boost and the TomoTherapy modalities, with a mean Vol20/Vol95 of more than 10. For the other techniques this ratio ranged between 5.8 and 9.6. The near-maximum-dose (D2) [18] to the heart for all techniques was below 5 Gy, except for 2 electron boost plans. One of these plans belonged to a patient who would, in good clinical practice, not have been a good candidate for electron boost delivery, because of the deep localization of the tumor bed. A rotational technique tends to deliver slightly more dose to the ipsilateral lung. Vero was the only technique which could keep the V5 for the ipsilateral lung below 5% in all cases. The arc technique with CMS and TomoTherapy delivered a mean V5 of 9% and 7% respect- ively, with electrons (not considering the 3 bad candidates) a mean V5 of 5% was delivered, for the other techniques the V5 ranged from 1.5 to 4% (Table 2). Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Page 4 of 7 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Table 3 conformity index (CI): distribution per interval of 10% CI 1 2 3 4 5 6 7 <50% 4 9 0 0 0 0 0 50–59% 4 1 0 1 0 4 5 60–69% 2 0 5 5 5 6 4 ≥70% 0 0 5 4 5 0 1 Conformity index for the different techniques, distribution per interval of 10%: (1) electrons, (2) 2 tangential fields, (3) 3 static fields, (4) arc, (5) Vero, (6) TomoTherapy, (7) TomoDirect. Table 3 conformity index (CI): distribution per interval of 10% CI 1 2 3 4 5 6 7 <50% 4 9 0 0 0 0 0 50–59% 4 1 0 1 0 4 5 60–69% 2 0 5 5 5 6 4 ≥70% 0 0 5 4 5 0 1 Conformity index for the different techniques, distribution per interval of 10%: (1) electrons, (2) 2 tangential fields, (3) 3 static fields, (4) arc, (5) Vero, (6) TomoTherapy, (7) TomoDirect. Table 3 conformity index (CI): distribution per interval of 10% delivery, which we could not do in this CT-based dosimet- ric comparison. For the goal of the comparison a dose dis- tribution was calculated for all 10 CT sets, but for the interpretation of the further results, we should keep in mind that there are 3 irrelevant situations present. The PTV coverage (95% of the PTV volume receiving 95% of the prescription dose) was achieved in 2 patients with electrons, in 7 with the 2-field photon boost, in 4 with the 3-field and the dynamic conformal arc photon boost and in all 10 patients with the rotational and static TomoTherapy modalities and Vero. The reason for fail- ing to reach the PTV coverage criteria for the 2-field, 3-field and rotational photon boost, often was the build- up. The mean build-up was more than 5 mm for the 2- field, 3-field and dynamic conformal arc photon technique, was slightly less than 5 mm for Vero and electrons and was close to zero for the TomoTherapy modalities (Table 2). Results Ten CT scans were selected for analysis. For both left and right side, a primary tumor location in each of the 4 quadrants was present, as well as a centrally located pri- mary tumor bed. The pathological T-stage ranged from T1b to T2. The mean maximal diameter of the tumor Discussion For this analysis all planning software used type b calculation algorithms. Today, Vero and Tomotherapy proofed state of the art. If not available, the 3-field photon technique showed the best results. The use of an arc did not deliver a sub- stantial benefit for the conformity, but did cause more low dose spread. Electrons delivered a worse PTV cover- age and more doses to the ipsilateral lung and heart. However, there are a lot of things to be considered when We concluded that Vero was the most promising tech- nique, with the best median ranking (Table 3). The sec- ond best median ranking was seen for the photon boost with 2 fields. This was caused by a better sparing of OAR, but was at the cost of a worse PTV coverage and more dose spread to the already irradiated ipsilateral breast. Assuming that the PTV coverage should be the first concern, this "second place" is unjustified. Both TomoTherapy modalities were also promising, with a median ranking of 3 and 3.5. If paying even more atten- tion to minimize the low dose spread, perhaps they could score even better. This is of course the difficult choice when using inverse planning systems. They are very dependent on the effort you are willing to invest. Once you achieve a clinical acceptable plan, do you accept it or do you try to achieve even better? The extra effort and time invested in further optimization of the dose distri- bution should be balanced to the expected clinical im- pact. In this work we compared only the boost dose of 16 Gy. Discussion We report a dosimetric comparison of different non- invasive techniques to deliver a boost dose after whole breast irradiation as part of the breast conserving treat- ment of breast cancer. We chose to select real cases for this comparison. The cases were part of a patient popu- lation of a phase III trial [13]. The target delineation was performed at the moment of the actual treatment plan- ning according to the protocol of the trial and not as preparation for this work. This decreased the risk of un- intentionally favoring a certain technique. With our se- lection we wanted to get a an idea of the different possibilities for a tumor localization in all of the four quadrants or a centrally located tumor on both sides. Within this selection we including small as well as larger breasts, small as well as larger target volumes and super- ficially as well as deeply located tumors. None of the used techniques seem significantly influ- enced by respiratory movements. Electrons probably are little affected, since they are delivered with an applicator touching the skin. Wedged techniques or field-in-field techniques seem insensitive for breathing motion, while IMRT techniques are highly sensitive to movement [21]. Tomotherapy uses IMRT, but plan delivery accuracy doesn't seem significantly affected by breathing [22]. The time per session for the patient on the treatment table differs a lot between the different techniques. Based on our clinical experience in our department, a treatment with electrons mostly takes less than 5 minutes. The deliv- ery of a photon boost with 2 or 3 fields or arc with Elekta can take about 12 minutes. A treatment with Vero or Tomotherapy on average takes 20 minutes. The difference between the electron and photon treatments for a large part is caused by the cone beam and image registration, which is performed daily for all photon techniques. A point of discussion could be the use of different planning software systems for the different techniques. Knöös reported a comparison of dose calculation algo- rithms [15]. Algorithms were divided into 2 groups: type a models, where changes in lateral transport of electrons are not modeled, and type b models, that in an approxi- mate way consider changes in lateral electron transport. According to Knöös, the use of type b calculation algo- rithms will in general reduce the uncertainty in the de- livered dose to the patient. Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Table 2 Dose comparison 1 2 3 4 5 6 7 PTV mean (sd) (Gy) 16,17 (0,27) 16,13 (0,48) 16,02 (0,44) 15,98 (0,50) 16,07 (0,35) 16,17 (0,27) 16,07 (0,25) V95% (%) 79,91 94,44 94,51 93,15 96,70 97,58 97,90 CI (%) 47 (2–63) 39 (21–57) 69 (60–84) 67 (54–73) 70 (64–78) 59 (50–66) 61 (54–82) Vol50/Vol95 3,62 2,90 4,91 3,86 3,22 3,62 4,42 Vol20/Vol95 11,90 6,42 9,59 11,95 7,97 11,90 10,93 Mean build-up (mm) 4.6 5.7 6.4 7.2 4.5 2.2 2.1 Heart mean (sd) 0,36 (0,67) 0,05 (0,10) 0,47 (0,69) 0,80 (0,68) 0,23 (0,25) 0,15 (0,20) 0,54 (0,64) D2 (Gy) 2,75 0,33 2,48 2,68 0,94 0,85 2,26 V5 (%) 1,20 0,00 0,27 0,43 0,00 0,02 0,01 Ipsilat lung mean (sd) (Gy) 0,97 (1,97) 0,47 (1,41) 1,09 (1,85) 1,62 (1,98) 0,55 (1,01) 1,44 (1,78) 1,02 (1,46) V5 (%) 5,84 2,83 4,18 9,01 1,49 7,32 2,85 V8 (%) 3,09 1,92 2,17 3,38 0,65 2,56 1,26 V15 (%) 0,12 0,25 0,18 0,11 0,01 0,09 0,07 Contralat breast mean (sd) (Gy) 0,00 (0,00) 0,02 (0,03) 0,01 (0,02) 0,31 (0,23) 0,05 (0,05) 0,04 (0,04) 0,05 (0,05) D2 (Gy) 0,00 0,10 0,07 0,81 0,25 0,15 0,20 Summary of dose comparison: (1) electrons, (2) 2 tangential fields, (3) 3 static fields, (4) arc, (5) Vero, (6) TomoTherapy, (7) TomoDirect; the build-up was evaluated in the 9 patients with PTV reaching the skin. Page 5 of 7 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 All techniques were able to spare the contralateral breast with a near-maximum dose below 1.5 Gy. Only when using electrons, zero doses were delivered in all patients (Table 2). with TomoTherapy. The use of sophisticated IMRT-IGRT linear accelerators increases the cost of radiation treatment [19]. The expected clinical benefit should also be balanced to the financial aspect. On top of that, we should keep in mind that in clinical practice, a whole breast irradiation was already performed. Even a low dose on top of 50 Gy can be of clinical importance [20] (Table 4). Discussion Because, on itself, this is a low dose, less attention could have gone to the low dose spread when planning Table 4 Ranking 1 2 3 4 5 6 7 PTV V95 7 4 6 5 1 1 1 CI 6 7 2 3 1 5 4 Vol50/Vol95 3 1 7 5 2 3 6 Vol20/Vol95 5 1 3 5 2 5 4 Build-up 4 5 6 7 3 2 1 Heart D2 7 1 4 6 3 2 4 V5 7 1 5 6 1 1 1 Ipsilat lung V5 5 2 4 7 1 6 2 V8 6 3 4 7 1 5 2 Contralat breast D2 1 3 2 7 6 4 5 TOTAL SCORE 51 28 43 58 21 34 30 MEDIAN RANKING 5.5 2.5 4 6 1.5 3.5 3 Ranking of the different techniques according to the dose comparison, techniques: (1) electrons, (2) 2 tangential fields, (3) 3 static fields, (4) arc, (5) Vero, (6) TomoTherapy, (7) TomoDirect; scoring from 1 to 7, with 1 being the best score; the build-up was evaluated in the 9 patients with PTV reaching the skin. Table 4 Ranking Page 6 of 7 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Van Parijs et al. Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 at risk for damage. In practice, even with regional lung dysfunctions measured, there were no subjective impli- cations on breathing or physical efforts in all patients. interpreting these results. In our department, when we use electrons, we choose the energy to encompass the deepest border of the target volume with the 85% iso- dose. In this analysis, we wanted to encompass the target with the 95% isodose to be able to do a comparison with photons. In daily practice, we would not give such a high dose to the OAR as was shown in this work, but the PTV coverage would be even worse. Although electrons deliver a less optimal dose distribution and despite of the fact that electrons are used for boost delivery rou- tinely, no differences in local recurrences are seen be- tween electron and photon boost techniques. In the EORTC boost versus no boost trial [11] there was no statistically significant difference in 5-year local failure rates between electron and photon boost. Another con- sideration is the localization of the target. Electrons are not used in all situations. There were 2 patients with a tumor bed located too deep to be covered with electrons with energy of 15 MeV. In our department we would choose a photon boost in these cases. One tumor bed was located laterally, where the breast formed a fold. In practice we would have repositioned the breast to have a smooth surface, which could not be done on this CT- based planning analysis, which resulted in an irrelative dose distribution. We reported the CI. We chose a formula which con- siders both the over and under dosed areas [15]. However, we do not know the ideal CI to aim for, since there is no literature on this for breast irradiation. From this analysis, our own conclusion is that a CI of 70% or more can be considered as excellent, between 60% and 70% as good, be- tween 50% and 60% as acceptable and less than 50% as bad (cfr Table 2). The used formula for CI does not give a view of the low dose spread. To have a more complete compari- son of different techniques, you should analyze both the CI and some measure of low dose spread. Table 4 Ranking We looked at the Vol20/Vol95 and to the Vol50/Vol95. The Vol20/Vol95 showed more differences for the different techniques, but the Vol50/Vol95 is perhaps of more clinical meaning. From this work we derived advices which can be used as guidelines for selection of the best individualized boost technique. If available, Vero should be used, since it was the only technique that achieved an excellent PTV- coverage and at the same time could minimize the dose to the OAR. As a second choice, the TomoTherapy modal- ities proved to be a good alternative. Special attention should go to strict constraints to the OAR. A V5 < 2% to the heart and < 15% to the ipsilateral lung and a D2 < 1 Gy for the contralateral breast and < 3 Gy for the contralateral lung should be aimed at. If no IMRT-IGRT technique is available, the best choice depends on the localization of the tumor bed. In case of a superficial tumor bed, reaching the skin surface, one should avoid using a rotational technique, unless bolus is used. In case of a deep tumor bed reaching the thoracic wall, a 3-field technique is preferable. A slightly higher dose will be given to a smaller volume of the OAR, which is preferred above a rotational technique which spreads a lower dose to al larger volume. In case the tumor bed is surrounded by breast tissue, a conformal arc can be preferred above a 3-field technique. An arc tech- nique delivers a better conformity, though there is more spread of very low dose to a larger volume of breast tissue. In most cases a 2-field technique shows an unacceptable conformity to be used in modern radiotherapy. Electrons should be reserved for a very superficially located tumor bed without contact with the thoracic wall. We strongly advice to avoid the use of an energy higher than 12 MeV. In 9 of the 10 patients, the PTV extended to the skin surface. When you want to deliver dose at the skin sur- face, it is important to choose a technique which has lit- tle build-up. We see that a rotational technique has a larger build-up area than the other techniques, except for TomoTherapy. In case it is clinically important to deliver dose at the skin surface, other techniques or the use of a bolus should be considered. Table 4 Ranking When the target is located at the thoracic wall, rotational techniques tend to spread low dose to a larger volume of the ipsilateral lung and heart. In our choice for the most ideal tech- nique, we should balance what is less damaging: a slightly higher dose to a smaller part of the OAR or a lower dose spread to a larger volume of the organ. For the heart, dose dependent regional cardiac perfusion de- fects are described [23]. Considering that part of the left ventricle already has received 50 Gy with the wide tan- gential fields, it would probably be better to minimize the volume of heart receiving extra dose by the boost delivery [20,24]. This means a technique that can avoid delivering dose in the direction of the left ventricle should be preferred. The same could be true for the ipsi- lateral lung. Verbanck et al. [25] showed that regional dysfunctions can be detected in the parts of the lung ir- radiated with the wide tangential fields. When using IMRT to minimize the dose to the lung, no changes were detected. For the boost delivery, theoretically, it is probably better to minimize the volume of ipsilateral lung receiving extra dose and thus minimize the volume Received: 22 April 2013 Accepted: 9 January 2014 Published: 27 January 2014 18. Knöös T, Wieslander E, Cozzi L, Brink C, Fogliata A, Albers D, Nystrom H, Lassen S: Comparison of dose calculation algorithms for treatment planning in external photon beam therapy for clinical situations. Phys Med Biol 2006, 51:5785–5807. References 1. Nielsen HM, Overgaard M, Grau C, Jensen AR, Overgaard J: Study of failure pattern among high-risk breast cancer patients with or without postmastectomy radiotherapy in addition to adjuvant systemic therapy: long-term results from the Danish Breast Cancer Cooperative Group DBCG 82 b and c randomized studies. J Clin Oncol 2006, 24:2268–2275. 1. Nielsen HM, Overgaard M, Grau C, Jensen AR, Overgaard J: Study of failure pattern among high-risk breast cancer patients with or without postmastectomy radiotherapy in addition to adjuvant systemic therapy: long-term results from the Danish Breast Cancer Cooperative Group DBCG 82 b and c randomized studies. J Clin Oncol 2006, 24:2268–2275. 19. 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Depuydt T, Verellen D, Haas O, Gevaert T, Linthout N, Duchateau M, Tournel K, Reynders T, Leysen K, Hoogeman M, Storme G, De Ridder M: Geometric accuracy of a novel gimbals based radiation therapy tumor tracking system. Radiother Oncol 2011, 98:365–372. HVP and TR were responsible for the conception and design of the trial. HVP, TR and KH were responsible for the acquisition of the plans and the dosimetric data. HVP and TR did the analyses, the interpretation of data and drafted the manuscript. DV, GS and MDR revised the manuscript and advised in the conception and design of the trial. All authors gave final approval of the version to be published. HVP and TR were responsible for the conception and design of the trial. HVP, TR and KH were responsible for the acquisition of the plans and the dosimetric data. HVP and TR did the analyses, the interpretation of data and drafted the manuscript. 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Radiation Oncology 2014, 9:36 http://www.ro-journal.com/content/9/1/36 Abbreviations Collette L: Impact of a higher radiation dose on local control and survival in breast-conserving therapy of early breast cancer: 10-year results of the randomized boost versus no boost EORTC 22881–10882 trial. J Clin Oncol 2007, 25:3259–3265. Collette L: Impact of a higher radiation dose on local control and survival in breast-conserving therapy of early breast cancer: 10-year results of the randomized boost versus no boost EORTC 22881–10882 trial. J Clin Oncol 2007, 25:3259–3265. BCS: Breast conserving surgery; CI: Conformity index; CT: Computer tomography; CTV: Clinical target volume; D2: Near-maximum-dose; DVH: Dose volume histogram; EORTC: European organization for research and treatment of cancer; Gy: Gray; IGRT: Image guided radiation treatment; IMRT: Intensity modulated radiotherapy; MeV: Mega electron volt; OAR: Organs at risk; PIV: Prescription dose volume; PTV: Planning target volume; TV: Target volume; TVPIV: Overlap of TV and PIV; V5: Volume receiving 5 Gy; Vol20/Vol95: Ratio of the volume of the 20% isodose to the 95% isodose. 12. 12. Poortmans P, Bartelink H, Horiot JC, Struikmans H, Van den Bogaert W, Fourquet A, Jager J, Hoogenraad W, Rodrigus P, Warlam-Rodenhuis C, Collette L, Pierart M: EORTC Radiotherapy and Breast Cancer Groups: The influence of the boost technique on local control in breast conserving treatment in the EORTC 'boost versus no boost' randomized trial. Radiother Oncol 2004, 72:25–33. 13. Van Parijs H, Miedema G, Vinh-Hung V, Verbanck S, Adriaenssens N, Kerkhove D, Reynders T, Schuermans D, Leysen K, Hanon S, Van Camp G, Vincken W, Storme G, Verellen D, De Ridder M: Short course radiotherapy with simultaneous integrated boost for stage I-II breast cancer, early toxicities of a randomized clinical trial. Radiation Oncology 2012, 7:80–90. Acknowledgements Special thanks to Antoine Delor, Université Catholique de Louvain, Brussels, Belgium, for his help with the dose calculation of the electron plans. Special thanks to Antoine Delor, Université Catholique de Louvain, Brussels, Belgium, for his help with the dose calculation of the electron plans. Special thanks to Antoine Delor, Université Catholique de Louvain, Brussels, Belgium, for his help with the dose calculation of the electron plans. 17. Grégoire V, Mackie T: State of the art on dose prescription, reporting and recording in intensity-modulated radiation therapy (ICRU report No. 83). Cancer/Radiothérapie 2011, 15:555–559. Received: 22 April 2013 Accepted: 9 January 2014 Published: 27 January 2014 Conclusions We performed a dosimetric comparison of different non- invasive techniques to deliver an additional boost after whole breast irradiation as part of the breast-conserving therapy. Guidelines were presented. Standard procedures should be replaced by an individualized treatment. The presented guidelines can help to find the best treatment technique for the individual patient. Page 7 of 7 References doi:10.1186/1748-717X-9-36 Cite this article as: Van Parijs et al.: Breast conserving treatment for breast cancer: dosimetric comparison of different non-invasive techniques for additional boost delivery. Radiation Oncology 2014 9:36. 8. Taylor ME, Haffty BG, Rabinovitch R, Arthur DW, Halberg FE, Strom EA, White JR, Cobleigh MA, Edge SB: ACR appropriateness criteria on postmastectomy radiotherapy expert panel on radiation oncology-breast. 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https://openalex.org/W2949742831	https://hal.science/hal-01341348/file/12711_2016_Article_190.pdf	English	null	A multi-trait meta-analysis with imputed sequence variants reveals twelve QTL for mammary gland morphology in Fleckvieh cattle	bioRxiv (Cold Spring Harbor Laboratory)	2,015	cc-by	8,113	To cite this version: Hubert Pausch, Reiner Emmerling, Hermann Schwarzenbacher, Ruedi Fries. A multi-trait meta- analysis with imputed sequence variants reveals twelve QTL for mammary gland morphology in Fleckvieh cattle. Genetics Selection Evolution, 2016, 48 (1), pp.14. 10.1186/s12711-016-0190-4. hal-01341348 © 2016 Pausch et al. 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HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Pausch et al. Genet Sel Evol (2016) 48:14 DOI 10.1186/s12711-016-0190-4 Ge n e t i c s Selection Evolution Ge n e t i c s Selection Evolution Open Access Abstract Background: The availability of whole-genome sequence data from key ancestors in bovine populations provides an exhaustive catalogue of polymorphic sites that segregate within and across cattle breeds. Sequence variants identified from the sequenced genome of key ancestors can be imputed into animals that have been genotyped using medium- and high-density genotyping arrays. Association analysis with imputed sequences, particularly when applied to multiple traits simultaneously, is a very powerful approach to detect candidate causal variants that underlie complex phenotypes. Results: We used whole-genome sequence data from 157 key ancestors of the German Fleckvieh cattle population to impute 20,561,798 sequence variants into 10,363 animals that had (partly imputed) genotypes based on 634,109 single nucleotide polymorphisms (SNPs). Rare variants were more frequent among the sequence-derived than the array-derived genotypes. Association studies with imputed sequence variants were performed using seven correlated udder conformation traits as response variables. The calculation of an approximate multi-trait test statistic enabled us to detect 12 quantitative trait loci (QTL) (P < 2.97 × 10−9) that affect different morphological features of the mam- mary gland. Among the tested variants, the most significant associations were found for imputed sequence variants at 11 QTL, whereas the top association signal was observed for an array-derived variant at a QTL on bovine chromo- some 14. Seven QTL were associated with multiple phenotypes. Most QTL were located in non-coding regions of the genome but in close proximity of candidate genes that could be involved in mammary gland morphology (SP5, GC, NPFFR2, CRIM1, RXFP2, TBX5, RBM19 and ADAM12). Conclusions: Using imputed sequence variants in association analyses allows the detection of QTL at maximum resolution. Multi-trait approaches can reveal QTL that are not detected in single-trait association studies. Most QTL for udder conformation traits were located in non-coding regions of the genome, which suggests that mutations in regulatory sequences are the major determinants of variation in mammary gland morphology in cattle. linkage disequilibrium (LD) that is present in bovine populations, the QTL regions are often very large, which makes the identification of underlying genes and vari- ants usually inefficient. Moreover, the current genotyping arrays interrogate only a limited number of polymorphic sites that are primarily located in non-coding regions of the genome [1].h A multi‑trait meta‑analysis with imputed sequence variants reveals twelve QTL for mammary gland morphology in Fleckvieh cattle Hubert Pausch1, Reiner Emmerling2, Hermann Schwarzenbacher3 and Ruedi Fries1 Hubert Pausch1, Reiner Emmerling2, Hermann Schwarzenbacher3 and Ruedi Fries1 Genotypes of the target population All animals were genotyped using medium- and high- density SNP arrays. The high-density dataset consisted of 3545 Fleckvieh animals that had previously been genotyped with the Illumina BovineHD Bead chip that includes 777,962 SNPs. The medium-density dataset consisted of 7073 Fleckvieh animals that had been gen- otyped with the Illumina BovineSNP50 Bead chip (ver- sion 1 and version 2) comprising approximately 54,000 SNPs. Chromosomal positions of SNPs were based on the UMD3.1 assembly of the bovine genome [15]. Mito- chondrial, X-chromosomal, Y-chromosomal SNPs and SNPs that had no known chromosomal position were not considered for further analyses. Quality controls were carried out separately for each dataset as described by Pausch et al. [16] and briefly indicated here: SNPs were retained if they had a call rate per SNP and a call rate per individual greater than 90 % and a minor allele frequency (MAF) higher than 0.5 % and if they showed no devia- tion from the Hardy–Weinberg equilibrium (P > 0.0001) and no pedigree conflicts. After quality control, the medium-density genotypes were imputed to higher den- sity using a combination of Beagle [17] and Minimac [18] as described by Pausch et al. [5]. Only SNPs with a MAF higher than 0.5 % were retained after imputation. The final array-derived dataset consisted of 10,363 animals with (partly imputed) genotypes at 634,109 autosomal SNPs. p Computationally efficient algorithms have been devel- oped to perform association studies on thousands of individuals that are genotyped at millions of polymor- phic sites (e.g., [7, 8]). In cattle, such association studies are generally performed within breeds on a trait-by-trait basis and by testing one variant at a time. Association analyses that include multiple phenotypes may be more powerful to identify QTL for complex traits, particu- larly in the case of causal variants that affect multi- ple correlated phenotypes [9]. However, a multivariate test of association for millions of sequence variants and many phenotypes is computationally challenging [9]. An approximate multi-trait test statistic allows to efficiently combine the results of multiple association studies that were performed separately and thereby increases the power to identify trait-associated variants [10]. Udder conformation traits are routinely recorded in bovine populations during the evaluation of first-crop daughters of test bulls. Phenotypes for such traits are an important source of information because mammary gland morphology is highly correlated with mastitis sus- ceptibility and productive life span [11–13]. Background Genome-wide association studies (GWAS) using dense single nucleotide polymorphisms (SNPs) have facilitated the identification of quantitative trait loci (QTL) for numerous phenotypes. However, because of the extensive Correspondence: hubert.pausch@tierzucht.tum.de 1 Lehrstuhl fuer Tierzucht, Technische Universitaet Muenchen, 85354 Freising, Germany Full list of author information is available at the end of the article Correspondence: hubert.pausch@tierzucht.tum.de 1 Lehrstuhl fuer Tierzucht, Technische Universitaet Muenchen, 85354 Freising, Germany Full list of author information is available at the end of the article The availability of whole-genome sequences has led to an exhaustive catalogue of polymorphic sites that Pausch et al. Genet Sel Evol (2016) 48:14 Page 2 of 9 breeding and reproductive measures in cattle industry. No ethical approval was required for this study. segregate within and across bovine populations [2, 3]. However, the costs of sequencing the whole genome for a large number of animals are still high, thus, using a relatively low number of sequenced key ancestors as reference animals to impute sequence variants into any animal for which dense genotyping data are available, is an interesting alternative [4, 5]. Association studies with imputed sequence variants can then pinpoint candidate causal variants that control complex trait variation [3, 6]. Generation of sequence data We used whole-genome sequence data from 263 animals that represented 10 bovine breeds, among which the Fleckvieh breed with 157 animals. Most of the sequenced animals were key ancestors for their breeds [19]. The generation and analysis of whole-genome sequence data are described by Pausch et al. [20]. SNPs, short inser- tions and deletion polymorphisms were genotyped for all 263 sequenced animals simultaneously using the multi- sample approach implemented in the mpileup module of SAMtools [21] and in a variant calling pipeline described by Jansen et al. [2]. A total of 25,426,490 polymor- phic sites were identified. The functional effects of the sequence variants were analyzed using the annotation of the UMD3.1 assembly of the bovine genome [22] and as described by Jansen et al. [2]. In this paper, we present results that are based on the imputation of whole-genome sequence variants into more than 10,000 Fleckvieh animals that were genotyped using dense SNP arrays. We performed association stud- ies with more than 16 million sequence variants using seven highly correlated udder conformation traits as response variables. The multi-trait meta-analysis enabled us to detect 12 QTL that affect different morphological features of the mammary gland in Fleckvieh cattle. Genotypes of the target population Although the definitions of udder conformation traits may vary across breeds, phenotypic records generally describe the mor- phology and placement of teats and the overall shape of the mammary gland. The heritability of such traits is rela- tively high and ranges from 0.14 to 0.42 and most traits related to mammary gland morphology are correlated with each other [11, 14]. Single‑trait genome‑wide association studies Single‑trait genome‑wide association studies We considered 16,816,809 imputed sequence vari- ants with a MAF higher than 0.5 % for the GWAS. The imputed sequence variants were tested for association with each trait in turn using a two-step variance com- ponent-based approach that accounts for population stratification and the resulting inflation of false posi- tive association signals by fitting a genomic relation- ship matrix. The EMMAX software tool [7] was used to fit the model y = μ + u + e, where y is a vector of DYD, µ is the intercept, u is a vector of additive genetic effects ~ N(0,Gσ2 a), where G is the realized genomic rela- tionship matrix that was built based on the genotypes of 634,109 autosomal SNPs for the 10,363 animals fol- lowing VanRaden’s approach [23] and σa 2 is the additive genetic variance. e is a vector of residuals ~ N(0,Iσ2 e ), where I is the identity matrix and σe 2 is the error vari- ance. In a second step, the allele substitution effect (b) was obtained from a generalized linear regression model: y = μ + xb + η, where x is a vector of expected allele dosages and η is a vector of random residual deviates with variance Gσa 2 + Iσe 2. Inflation factors were calculated Animal ethics statementi The imputation reference panel consisted of 157 sequenced Fleckvieh animals with 20,561,798 autosomal variants. Haplotypes were inferred using Beagle [17] and served as DNA of artificial insemination bulls was prepared from semen samples that were collected by approved commer- cial artificial insemination stations as part of their regular Page 3 of 9 Pausch et al. Genet Sel Evol (2016) 48:14 using the estlamdba()-function of GenABEL [24]. Sequence variants with a P value less than 2.97 × 10−9 were considered as significantly associated (5 % Bon- ferroni-corrected significance threshold for 16,816,809 independent tests). The proportion of DYD variance explained per QTL was estimated by 2p(1−p)α2 σ2 , where p and α are the frequency and substitution effect, respec- tively, of the minor QTL allele and σ2 is the DYD vari- ance. An analysis conditional on the most significantly associated variant was carried out by taking the expected allele dosages of the top variant as covariates in the linear regression model (see above). reference to impute genotypes for 20,561,798 variants into 10,363 target animals with (partly imputed) genotypes at 634,109 SNPs (see above) using Minimac [18]. Phenotypes tested for association Response variables for the association tests were daugh- ter yield deviations (DYD) with an accuracy greater than 0.7 for seven udder conformation traits (teat thickness, teat length, teat position, udder depth, central ligament, fore udder attachment and fore udder length). Depend- ing on the trait, the number of animals with phenotypes ranged from 5470 to 7159 (Table 1). In addition, asso- ciation tests with 682,047 imputed sequence variants located on BTA14 (BTA for Bos taurus chromosome) were carried out on 6838 animals using DYD for height at the sacral bone as response variable. Multi‑trait meta‑analysis located on BTA14 (BTA for Bos taurus chromos were carried out on 6838 animals using DYD for h at the sacral bone as response variable. Single‑trait genome‑wide association studies We considered 16,816,809 imputed sequence ants with a MAF higher than 0.5 % for the GWAS imputed sequence variants were tested for associ with each trait in turn using a two-step variance ponent-based approach that accounts for popul stratification and the resulting inflation of false tive association signals by fitting a genomic rela ship matrix. The EMMAX software tool [7] was to fit the model y = μ + u + e, where y is a vect DYD, µ is the intercept, u is a vector of additive ge effects ~ N(0,Gσ2 a), where G is the realized genomic tionship matrix that was built based on the geno of 634,109 autosomal SNPs for the 10,363 animal lowing VanRaden’s approach [23] and σa 2 is the ad genetic variance. e is a vector of residuals ~ N(0 where I is the identity matrix and σe 2 is the error ance. In a second step, the allele substitution effec was obtained from a generalized linear regression m y = μ + xb + η, where x is a vector of expected dosages and η is a vector of random residual dev with variance Gσa 2 + Iσe 2. Inflation factors were calcu Table 1 Characteristics of seven udder conformation Phenotype (and abbreviation) Number of animals with phenotypes Udder depth (UD) 7110 Teat thickness (TT) 7108 Teat length (TL) 7159 Teat placement (TP) 7108 Fore udder length (FUL) 7034 Central ligament (CL) 6923 Fore udder attachment (FUA) 5470 An approximate multi-trait test statistic was calcu- lated for 16,816,809 imputed sequence variants using χ2 df = n = t ′ iV−1ti, where n is the number of traits, ti is a n × 1 vector of t values (t = b se(b)) of the ith SNP and V−1 is the inverse of the n × n correlation matrix of t values [10]. The correlation matrix V was constructed from t values of 16,816,809 imputed sequence vari- ants. Sequence variants with a PMETA value less than 2.97 × 10−9 were considered as significantly associated (see above). Results More than 20 million sequence variants were imputed into 10,363 animals that had (partly imputed) array- derived genotypes for 634,109 SNPs (Fig. 1a). The distri- butions of the allele frequencies of the imputed sequence (SEQ) variants and of the array-derived variants differed. Variants from medium- (50 K) and high-density (700 K) SNP arrays were almost uniformly distributed across different MAF classes, whereas the imputed sequence variants were enriched for low-frequency MAF classes (Fig. 1b). The proportions of variants with a MAF lower than 0.05 were equal to 10.66, 9.04 and 40.52 % and the average MAF was equal to 0.249, 0.260 and 0.145 for the 50 K, 700 K and SEQ datasets, respectively. Table 1 Characteristics of seven udder conformation traits Phenotype (and abbreviation) Number of animals with phenotypes Distribution of the DYD Accuracy of the DYD Min Max Mean (±sd) Min Max Mean (±sd) Udder depth (UD) 7110 −2.22 2.75 0.11 (±0.64) 0.71 0.99 0.91 (±0.04) Teat thickness (TT) 7108 −2.32 2.17 0.04 (±0.52) 0.71 0.99 0.91 (±0.04) Teat length (TL) 7159 −2.61 2.52 −0.02 (±0.66) 0.71 0.99 0.93 (±0.04) Teat placement (TP) 7108 −1.92 2.50 0.16 (±0.55) 0.71 0.99 0.91 (±0.04) Fore udder length (FUL) 7034 −2.03 2.15 0.21 (±0.51) 0.71 0.99 0.88 (±0.04) Central ligament (CL) 6923 −2.24 2.32 0.11 (±0.57) 0.71 0.99 0.85 (±0.05) Fore udder attachment (FUA) 5470 −2.73 2.48 0.13 (±0.65) 0.71 0.99 0.87 (±0.05) Table 1 Characteristics of seven udder conformation traits Pausch et al. Genet Sel Evol (2016) 48:14 Page 4 of 9 Fig. 1 Imputation of sequence variants. a Overview of the stepwise imputation of 20,561,798 sequence variants into 10,363 Fleckvieh animals. b Distribution of allele frequencies of imputed and array-derived variants. Blue and shades of grey represent the proportion of imputed sequence (SEQ) and array-derived (50 K, 700 K) variants, respectively, for ten allele frequency classes Fig. 1 Imputation of sequence variants. a Overview of the stepwise imputation of 20,561,798 sequence variants into 10,363 Fleckvieh animals. b Distribution of allele frequencies of imputed and array-derived variants. Blue and shades of grey represent the proportion of imputed sequence (SEQ) and array-derived (50 K, 700 K) variants, respectively, for ten allele frequency classes Association studies with udder conformation traits A meta-analysis of the seven single-trait association studies revealed 11 QTL (PMETA <2.97 × 10−9) located on 11 chromosomes (Fig. 2b; Table 2). The most sig- nificantly associated variants were imputed sequence variants at ten QTL and the top association signal was found for an array-derived SNP at the QTL on BTA14. The multi-trait meta-analysis revealed a QTL on BTA2 (PMETA = 6.1 × 10−10) that was not detected in the single-trait association studies. Closer inspec- tion revealed that the QTL on BTA2 was associated with fore udder attachment (PSINGLE = 1.13 × 10−6), teat thickness (PSINGLE = 2.68 × 10−4), fore udder length (PSINGLE = 4.37x10−3) and udder depth (PSIN- GLE = 5.01 × 10−3), although the PSINGLE values were above the Bonferroni-corrected genome-wide signifi- cance threshold. Four QTL on BTA14, 21, 27 and 29 that were associated with teat placement, udder depth, teat We performed association studies between 16,816,809 imputed sequence variants with a MAF higher than 0.5 % and DYD for seven udder conformation traits (Table 1). The inflation factors of the association studies ranged from 1.04 (central ligament) to 1.08 (udder depth) with an average inflation factor of 1.06, which indicated that population stratification was under control. The number of QTL detected per trait ranged from zero (fore udder attachment) to five (fore udder length and teat place- ment) [See Additional file 1: Figure S1]. Correlation coef- ficients between the seven traits were calculated with the signed t values (i.e., allele substitution effect divided by its standard error, Fig. 2a). The highest correlations were observed between udder depth and fore udder attach- ment (r = 0.47), teat length and teat thickness (r = 0.46) and central ligament and teat placement (r = 0.37). Fig. 2 Identification of 12 QTL for mammary gland morphology. a Correlations between seven udder conformation traits. The values in the upper and lower triangles represent genomic correlations calculated with the signed t values and genetic correlations, respectively. The abbreviations of the traits are in Table 1. Shades of blue and red indicate positive and negative correlation coefficients, respectively. Genetic correlations were provided from the Institute of Animal Breeding from Bavarian State Research center for Agriculture. b Manhattan plot representing the association of 16,816,809 imputed sequence variants in the multi-trait meta-analysis. Red color represents variants with a PMETA less than 2.97 × 10−9 Fig. 2 Identification of 12 QTL for mammary gland morphology. Association studies with udder conformation traits The values in the rectangles represent the propor- tion of DYD variance explained for each QTL. The abbreviations of the traits are in Table 1. Only associations with a P less than 2.97 × 10−9 (5 % Bonferroni-corrected significance threshold for 16,816,809 tests) are shown length and udder depth, respectively, were not identi- fied in the multi-trait meta-analysis [See Additional file 1: Figure S1]. Closer inspection of these four regions in the multi-trait meta-analysis showed an association (7.91×1 0−8 < PMETA < 1.98 × 10−6), but not at the genome-wide significance. identified in the meta-analysis [See Additional file 3: Figure S3]. However, no annotated genes were present within 300-kb flanking regions of the top association sig- nal on BTA4, 13, 19 and 20, which precluded the identifi- cation of positional candidate genes. Seventy-seven variants with a PMETA value less than 1 × 10−8 were located within a 100-kb intergenic segment on BTA2 [See Additional file 3: Figure S3a]. The top asso- ciation signal (PMETA = 6.11 × 10−10) was detected for a variant (rs718110021 at 25,887,326 bp) that was located 86 kb downstream of the stop codon of the MYO3B gene (myosin IIIB) and 111 kb upstream of the translation start site of the SP5 gene (Sp5 transcription factor). i To test if a QTL was completely tagged by the top vari- ant, the most significantly associated variant was fitted as a covariate in the single-trait GWAS model and the multi-trait test statistic was re-calculated. The condi- tional analysis revealed that the associated region on BTA6 consisted of two distinct closely located QTL [See Additional file 2: Figure S2]. The QTL located at 88,723,742 bp was associated with udder depth, central ligament and fore udder length, whereas the second QTL located at 90,366,765 bp was associated with teat thick- ness (Table 2 and [See Additional file 2: Figure S2]). No significant associations were detected for any of the other QTL when the association analysis was conditioned on the respective top SNPs. A QTL region on BTA6 included 1199 variants with a PMETA value less than 1 × 10−14 that were located within a 450-kb segment (between 88,613,408 and 89,062,806 bp) [See Additional file 3: Figure S3c]. Two annotated genes reside within this interval: GC (group specific component) and NPFFR2 (neuropeptide FF receptor 2). Association studies with udder conformation traits The top variant (rs436532576 at 88,723,742 bp; PMETA = 7.44 × 10−36) was located in the first intron of GC. The most sig- nificantly associated coding variant (rs110326785 or NPFFR2: pE389 K; PMETA = 1.86 × 10−16) was consider- ably less significantly associated than the non-coding top variant. A second QTL on BTA6 was located 6 kb down- stream of the stop codon of RASSF6 (Ras association (RalGDS/AF-6) domain family member 6) [See Addi- tional file 3: Figure S3d]. Seven QTL were associated with multiple morphologi- cal features of the mammary gland (Table 2 and Fig. 3). The QTL on BTA17 was associated with four pheno- types. Two and four QTL were associated with three and two udder conformation traits, respectively. Association studies with udder conformation traits a Correlations between seven udder conformation traits. The values in the upper and lower triangles represent genomic correlations calculated with the signed t values and genetic correlations, respectively. The abbreviations of the traits are in Table 1. Shades of blue and red indicate positive and negative correlation coefficients, respectively. Genetic correlations were provided from the Institute of Animal Breeding from Bavarian State Research center for Agriculture. b Manhattan plot representing the association of 16,816,809 imputed sequence variants in the multi-trait meta-analysis. Red color represents variants with a PMETA less than 2.97 × 10−9 Fig. 2 Identification of 12 QTL for mammary gland morphology. a Correlations between seven udder conformation traits. The values in the upper and lower triangles represent genomic correlations calculated with the signed t values and genetic correlations, respectively. The abbreviations of the traits are in Table 1. Shades of blue and red indicate positive and negative correlation coefficients, respectively. Genetic correlations were provided from the Institute of Animal Breeding from Bavarian State Research center for Agriculture. b Manhattan plot representing the association of 16,816,809 imputed sequence variants in the multi-trait meta-analysis. Red color represents variants with a PMETA less than 2.97 × 10−9 Pausch et al. Association studies with udder conformation traits Genet Sel Evol (2016) 48:14 Page 5 of 9 Table 2 Chromosomal positions of 12 QTL for udder mor- phology detected in the meta-analysis The positions of the QTL correspond to the UMD3.1 assembly of the bovine genome Chr chromosome number, MAF minor allele frequency a Marks a QTL that was detected in the conditional analysis Chr Position NCBI reference ID MAF PMETA Candidate gene(s) 2 25 887 326 rs718110021 0.25 6.1 × 10−10 SP5 4 75 817 266 rs474527669 0.24 1.9 × 10−11 – 6 88 723 742 rs436532576 0.47 7.4 × 10−36 GC, NPFFR2 6a 90 366 765 rs516920231 0.21 8.5 × 10−16 RASSF6 11 18 757 907 rs384497140 0.28 7.5 × 10−13 CRIM1 12 29 248 113 rs381918313 0.03 1.6 × 10−42 RXFP2 13 22 699 039 rs109467286 0.29 2.2 × 10−19 – 14 25 015 640 rs109815800 0.12 6.2 × 10−26 PLAG1 17 62 694 032 rs109134926 0.25 2.2 × 10−57 TBX5, RBM19 19 61 493 292 rs379185255 0.35 3.4 × 10−11 – 20 27 355 591 rs378730330 0.15 1.8 × 10−11 – 26 46 058 305 rs42442597 0.34 1.3 × 10−14 ADAM12 Table 2 Chromosomal positions of 12 QTL for udder mor- phology detected in the meta-analysis Fig. 3 Effects of 12 QTL on seven udder conformation traits. Green and orange colors represent positive and negative phenotype × gen- otype associations, respectively, for 12 QTL in seven single-trait association studies. The values in the rectangles represent the propor- tion of DYD variance explained for each QTL. The abbreviations of the traits are in Table 1. Only associations with a P less than 2.97 × 10−9 (5 % Bonferroni-corrected significance threshold for 16,816,809 tests) are shown Fig. 3 Effects of 12 QTL on seven udder conformation traits. Green and orange colors represent positive and negative phenotype × gen- otype associations, respectively, for 12 QTL in seven single-trait association studies. The values in the rectangles represent the propor- tion of DYD variance explained for each QTL. The abbreviations of the traits are in Table 1. Only associations with a P less than 2.97 × 10−9 (5 % Bonferroni-corrected significance threshold for 16,816,809 tests) are shown Fig. 3 Effects of 12 QTL on seven udder conformation traits. Green and orange colors represent positive and negative phenotype × gen- otype associations, respectively, for 12 QTL in seven single-trait association studies. Genes located in the 12 QTL regions detected in the meta‑analysis None of the highly significantly associated variants were located in the cod- ing region of an annotated gene. The QTL on BTA14 was in a genomic region that con- trols growth-related traits in cattle [25, 26] and other spe- cies [27]. The top variant (rs109815800 at 25,015,640 bp, PMETA = 6.23 × 10−26) was located 6 kb upstream of the translation start site of PLAG1 (pleiomorphic adenoma gene 1) [See Additional file 3: Figure S3h]. We performed an association analysis with DYD for height at the sacral bone to test if the QTL for mammary gland morphology was also associated with stature. The association analy- sis revealed that rs109815800 was also the most signifi- cantly associated variant for height at the sacral bone (P = 1.07 × 10−52) [See Additional file 4: Figure S4]. The allele that increases body height was associated with an increased udder base. y p q To eliminate false positive association signals due to population stratification, the genomic relationship based on genome-wide SNP data was considered in the seven separate association studies. The low inflation factors obtained (1.04 to 1.08) show that this corrective measure was successful. Combining the results of the seven sepa- rate association studies by calculating an approximate multi-trait test statistic enabled us to reveal 12 QTL for mammary gland morphology. Among these, we detected a QTL on BTA2 that was not found in the single-trait studies, which showed the enhanced capacity of multi- trait approaches for detecting QTL, particularly when the phenotypes are correlated [9, 10, 31]. The QTL on BTA2 was associated with four correlated traits, although not on a genome-wide scale. Our finding that QTL, which cannot be detected at a genome-wide significance level in single-trait GWAS, can be uncovered in a multi-trait approach corroborates the findings of O’Reilly et al. [31]. The joint association analysis of multiple phenotypes might be a more powerful approach to detect QTL that underlie correlated traits than the multi-trait test statis- tic applied in our study [9]. However, phenotypic records were incomplete for some animals in this study, which precluded multivariate association testing without rely- ing on algorithms to estimate missing phenotypes. Four QTL were detected in the single-trait association studies but were not formally identified in the multi-trait meta- analysis. However, the corresponding PMETA values were only slightly above the Bonferroni-corrected genome- wide significance threshold. Genes located in the 12 QTL regions detected in the meta‑analysis To detect positional and functional candidate genes that could be involved in mammary gland morphology, we examined the gene content of 12 QTL regions that were i Eighty-one variants located within a 1.5-Mb inter- val on BTA11 (between 18,546,414 and 20,048,201 bp) had PMETA values less than 3.1 × 10−10 [See Additional Pausch et al. Genet Sel Evol (2016) 48:14 Page 6 of 9 Initially, the animals with medium-density genotypes were imputed to higher density using 3332 reference ani- mals that had been genotyped with a high-density gen- otyping array. In the second step, the (partly imputed) high-density genotypes were imputed to sequence level using sequence variants of 157 key ancestors of the Fleck- vieh population as reference animals. According to Brøn- dum et al. [28], using a multi-breed reference panel may improve imputation accuracy when the number of refer- ence animals is small. However, the imputation pipeline that we applied was expected to yield high imputation accuracy by using 157 key ancestors as reference animals [5]. Low-frequency variants occurred more often among the imputed than the array-derived variants. This agrees with previous findings in cattle [6] and other species [29]. Since the imputation of rare variants is error prone [3, 5, 28], we retained only variants with a MAF higher than 0.5 % for the association studies. Moreover, to take imputation uncertainty into account [30], we used the expected allele dosages instead of the most likely geno- types as explanatory variables in the GWAS. Thus, our association analyses should not be flawed due to inaccu- rately imputed sequence variants. file 3: Figure S3e]. This segment encompasses 12 anno- tated transcripts/genes. The top variant (rs384497140 at 18,757,907 bp; PMETA = 7.47 × 10−13) was located 77 kb upstream of the translation start site of CRIM1 (cysteine rich transmembrane BMP regulator 1). Two highly signif- icantly associated coding variants (PMETA < 1.58× 10−10) in CRIM1 (rs110451648, p.R540 K) and PRKD3 (rs380476420, p.R861H) were in high LD (r2 > 0.88) with the top non-coding variant. One hundred and twelve variants with a PMETA value less than 1 × 10−30 were located in a 4.59-Mb interval (between 25,693,051 and 30,288,956 bp) on BTA12 [See Additional file 3: Figure S3f]. The top association signal (PMETA = 1.58 × 10−42) was found for an intronic vari- ant (rs381918313 at 29,248,113 bp) in RXFP2 (relaxin/ insulin-like family peptide receptor 2). Genes located in the 12 QTL regions detected in the meta‑analysis Adjusting for multiple Eighty sequence variants with a PMETA value less than 1.87 × 10−25 were located within a 171-kb intergenic segment (between 62,667,848 and 62,838,591 bp) on BTA17 [See Additional file 3: Figure S3i]. The top variant (rs109134926 at 62,694,032 bp; PMETA = 2.23 × 10−57) was located 193 kb upstream of the translation start site of RBM19 (RNA binding motif protein 19), and 113 kb downstream of the stop codon of TBX5 (T-box 5 tran- scription factor). A QTL on BTA26 was associated with teat thick- ness. Thirty-nine variants with a PMETA value less than 2.0 × 10−13 were located in the third intron of ADAM12 (ADAM metallopeptidase domain 12) [See Addi- tional file 3: Figure S3l]. The top variant (rs42442597 at PMETA = 1.29 × 10−14) was located at 46,058,305 bp. No coding variants were significantly associated with mam- mary gland morphology. Discussion An improved functional annotation of the bovine genome [38] and multi-breed association studies with imputed sequence variants may reveal causal mutations for such QTL. 1. SP5, that is located near a QTL on BTA2: since SP5 is a downstream target gene of the Wnt signaling pathway [39], our result provides additional evidence for the crucial role of the Wnt signaling pathway for mammary gland development in cattle [36]; 2. GC and NPFFR2 that are within a QTL on BTA6 and are known to affect mastitis susceptibility in cattle [40–42]; in our study, this QTL was associated with udder depth and central ligament and since udder depth, central ligament and mastitis susceptibility are negatively correlated traits [13], this QTL may contribute to the unfavorable genetic correlation between deep udder base and udder health; 3. CRIM1 that is located close to a QTL on BTA11 and contains an insulin-like growth factor-binding domain [43]; 4. RXFP2 near a QTL on BTA12; 5. ADAM12 for which a QTL on BTA26 is located in an intronic region and which encodes the ADAM metallopeptidase domain 12 that interacts with insu- lin-like growth factor-binding proteins [44]; these findings suggest a crucial role of insulin-like growth factors and insulin-like growth factor-binding pro- teins during mammary gland development [45, 46]. Most of the QTL that we identified in this study reside in non-protein coding regions of the genome, which indicates that mutations in the regulatory regions of genes have a major role in shaping mammary gland morphology in cattle. Pinpointing causal mutations in non-coding elements is notoriously difficult since the annotation of the bovine genome is not always reliable due to assembly errors and the presence of gaps in the reference sequence [22]. Moreover, regulatory elements in the bovine genome are poorly characterized. Thus, we did not attempt to identify candidate causal variants for QTL. However, an improved functional annotation of the bovine genome is expected to facilitate a more precise characterization of regulatory QTL in the future [38]. The most significantly associated variants at 11 QTL were imputed sequence variants, which demonstrates that the mapping resolution is increased with whole- genome sequence data relative to array-derived geno- types. However, the top association signal was found for an array-derived variant (rs109815800) at a QTL on BTA14. Discussion Sequence variants were extrapolated into 10,363 ani- mals using a two-step genotype imputation approach. Page 7 of 9 Pausch et al. Genet Sel Evol (2016) 48:14 artificial insemination bulls and can be overestimated in tall animals. Thus, the association between PLAG1 and udder depth may reflect phenotypic variation in body size rather than true effects on mammary gland morphology. The fact that this association was not found when udder depth was conditioned on body height further supports this assumption [See Additional file 5: Figure S5]. testing using Bonferroni-correction assumes that the individual tests are independent from each other. Due to the small effective population size and high LD, this assumption does not hold for genome-wide association studies in livestock populations. The Bonferroni-correc- tion method is prone to over-correction, particularly in association studies that involve millions of sequence vari- ants [32, 33], which results in reduced power. i g Our association study revealed new candidate genes for mammary gland morphology in cattle, i.e.: [ , ], p Previously, association studies for udder conforma- tion traits were carried out in several cattle breeds using dense marker maps. Flury et al. [34] identified two QTL for udder length and teat diameter in the Brown Swiss cattle breed on BTA6 located at 88.92 and 90.37 Mb, respectively. We also identified two QTL on BTA6 at 88.72 and 90.37 Mb, which confirms the crucial role of both regions for mammary gland morphology in cattle. In our study, the QTL on BTA6 were associated with teat thickness and fore udder length, central ligament and udder depth. Hiendleder et al. [35] identified a QTL for udder conformation traits in the Holstein breed on BTA6 at 88 cM, which most likely corresponds to the highly significantly associated region(s) identified in our study. Another QTL that affects mammary gland morphology in cattle was reported on BTA17, close to TBX3, TBX5 and RBM19 [34, 36]. The corresponding human chromo- some segment is involved in ulnar mammary syndrome [37]. We identified a highly significantly associated QTL at that position with the top variant being only 3 kb away from the top association signal reported in the Brown Swiss breed [34], which indicates that udder traits may be under the control of a common variant in both breeds. The position of this QTL suggests that regulatory vari- ants may be involved in shaping the mammary gland. Discussion Interestingly, rs109815800 is one of the eight candidate causative trait variants that were reported for a QTL that affects bovine stature by modulating the expression of PLAG1 [25]. In our study, rs109815800 was also the top variant for body height, which suggests that it has pleiotropic effects on stature and udder traits. The allele that increased height was also associated with an increased udder base. Udder depth, i.e. the inter- space between the ankle and the udder base, is visually examined during the evaluation of first crop daughters of Conclusions Association analyses based on imputed sequence vari- ants allows the characterization of QTL at maximum Pausch et al. Genet Sel Evol (2016) 48:14 Page 8 of 9 5. Pausch H, Aigner B, Emmerling R, Edel C, Götz KU, Fries R. Imputation of high-density genotypes in the Fleckvieh cattle population. Genet Sel Evol. 2013;45:3. resolution. Variants that affect multiple correlated traits are most efficiently uncovered by their simultane- ous analysis using a multi-trait test statistic. Our study revealed 12 QTL that control different features of the mammary gland morphology in the German Fleckvieh cattle population. The positions of the QTL suggest that mutations in the regulatory elements have a major role in shaping mammary gland morphology in cattle. 6. Qanbari S, Pausch H, Jansen S, Somel M, Strom TM, Fries R, et al. Classic selective sweeps revealed by massive sequencing in cattle. PLoS Genet. 2014;10:e1004148. 7. Kang HM, Sul JH, Service SK, Zaitlen NA, Kong SY, Freimer NB, et al. Vari- ance component model to account for sample structure in genome- wide association studies. Nat Genet. 2010;42:348–54. 8. Loh PR, Tucker G, Bulik-Sullivan BK, Vilhjálmsson BJ, Finucane HK, Salem RM, et al. Efficient Bayesian mixed-model analysis increases association power in large cohorts. Nat Genet. 2015;47:284–90. Additional files 9. Zhou X, Stephens M. Efficient multivariate linear mixed model algorithms for genome-wide association studies. Nat Methods. 2014;11:407–9. Additional file 1: Figure S1. Manhattan plots representing the associa- tion between 16,816,809 imputed sequence variants and seven udder conformation traits. Additional file 2: Figure S2. Detailed view of the QTL on BTA6. Additional file 3: Figure S3. Gene content within 12 QTL regions. Additonal file 4: Figure S4. A QTL for stature on BTA14. Additional file 5: Figure S5. Detailed view of a QTL for mammary gland morphology on BTA14. 10. Bolormaa S, Pryce JE, Reverter A, Zhang Y, Barendse W, Kemper K, et al. A multi-trait, meta-analysis for detecting pleiotropic polymor- phisms for stature, fatness and reproduction in beef cattle. PLoS Genet. 2014;10:e1004198. Additional file 1: Figure S1. Manhattan plots representing the associa- tion between 16,816,809 imputed sequence variants and seven udder conformation traits. 11. Berry DP, Buckley F, Dillon P, Evans RD, Veerkamp RF. Genetic relationships among linear type traits, milk yield, body weight, fertility and somatic cell count in primiparous dairy cows. Irish J Agric Food Res. 2004;43:161–76. 12. Vollema AR, Groen AF. Genetic correlations between longevity and conformation traits in an upgrading dairy cattle population. J Dairy Sci. 1997;80:3006–14. Additional file 5: Figure S5. Detailed view of a QTL for mammary gland morphology on BTA14. 13. Pritchard T, Coffey M, Mrode R, Moore K, Wall E. Genetic parameters of udder health traits in Holstein Friesian UK dairy cattle. In: Proceedings of the 9th World Congress on Genetics applied to Livestock Production; 2010, p. 1–8. Author details 1 1 Lehrstuhl fuer Tierzucht, Technische Universitaet Muenchen, 85354 Freising, Germany. 2 Institut fuer Tierzucht, Bayerische Landesanstalt fuer Land- wirtschaft, 85586 Poing, Germany. 3 ZuchtData EDV Dienstleistungen GmbH, 1200 Vienna, Austria. 16. Pausch H, Kölle S, Wurmser C, Schwarzenbacher H, Emmerling R, Jansen S, et al. A nonsense mutation in TMEM95 encoding a nondescript transmembrane protein causes idiopathic male subfertility in cattle. PLoS Genet. 2014;10:e1004044. Authors’ contributions HP and RF conceived, designed and performed the experiments. RE and HS contributed pedigree, phenotype and genotype data. HP and RF wrote the manuscript. All authors read and approved the final manuscript. 14. Kramer M, Erbe M, Bapst B, Bieber A, Simianer H. Estimation of genetic parameters for novel functional traits in Brown Swiss cattle. J Dairy Sci. 2013;96:5954–64. 15. Zimin AV, Delcher AL, Florea L, Kelley DR, Schatz MC, Puiu D, et al. A whole-genome assembly of the domestic cow. Bos taurus. Genome Biol. 2009;10:R42. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 2 November 2015 Accepted: 26 January 2016 Received: 2 November 2015 Accepted: 26 January 2016 21. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9. 21. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9. 22. Florea L, Souvorov A, Kalbfleisch TS, Salzberg SL. Genome assembly has a major impact on gene content: a comparison of annotation in two Bos taurus assemblies. PLoS One. 2011;6:e21400. 22. Florea L, Souvorov A, Kalbfleisch TS, Salzberg SL. Genome assembly has a major impact on gene content: a comparison of annotation in two Bos taurus assemblies. PLoS One. 2011;6:e21400. Acknowledgements 17. Browning BL, Browning SR. A unified approach to genotype imputation and haplotype-phase inference for large data sets of trios and unrelated individuals. Am J Hum Genet. 2009;84:210–23. The generation of sequence data of Fleckvieh animals was funded by the German Federal Ministry of Education and Research (BMBF) within the Agro- ClustEr “Synbreed–Synergistic plant and animal breeding” (grant ids 0315527B, 0315528A). We acknowledge the Arbeitsgemeinschaft Süddeutscher Rinderzüchter e.V., the Arbeitsgemeinschaft österreichischer Fleckviehzüchter and ZuchtData EDV Dienstleistungen GmbH for providing genotype data. We thank Qualitas AG (CH-Zug) and the Swiss Cattle Breeder Association for fund- ing the sequencing of original Simmental animals. 18. Howie B, Fuchsberger C, Stephens M, Marchini J, Abecasis GR. Fast and accurate genotype imputation in genome-wide association studies through pre-phasing. Nat Genet. 2012;44:955–9. 19. Goddard ME, Hayes BJ. Genomic selection based on dense genotypes inferred from sparse genotypes. Proc Assoc Advmt Anim Breed Genet. 2009;18:26–9. 20. Pausch H, Schwarzenbacher H, Burgstaller J, Flisikowski K, Wurmser C, Jansen S, et al. Homozygous haplotype deficiency reveals deleterious mutations compromising reproductive and rearing success in cattle. BMC Genomics. 2015;16:312. References 23. VanRaden PM. Efficient methods to compute genomic predictions. J Dairy Sci. 2008;91:4414–23. References 1. Koufariotis L, Chen YPP, Bolormaa S, Hayes BJ. Regulatory and coding genome regions are enriched for trait associated variants in dairy and beef cattle. BMC Genomics. 2014;15:436. 2. Jansen S, Aigner B, Pausch H, Wysocki M, Eck S, Benet-Pagès A, et al. Assessment of the genomic variation in a cattle population by re- sequencing of key animals at low to medium coverage. BMC Genomics. 2013;14:446. 3. Daetwyler HD, Capitan A, Pausch H, Stothard P, van Binsbergen R, Brøndum RF, et al. Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle. Nat Genet. 2014;46:858–65. 4. VanRaden PM, O’Connell JR, Wiggans GR, Weigel KA. Genomic evalua- tions with many more genotypes. Genet Sel Evol. 2011;43:10. 1. Koufariotis L, Chen YPP, Bolormaa S, Hayes BJ. Regulatory and coding genome regions are enriched for trait associated variants in dairy and beef cattle. BMC Genomics. 2014;15:436. y 24. Aulchenko YS, Ripke S, Isaacs A, van Duijn CM. GenABEL: an R library for genome-wide association analysis. Bioinformatics. 2007;23:1294–6. 2. Jansen S, Aigner B, Pausch H, Wysocki M, Eck S, Benet-Pagès A, et al. Assessment of the genomic variation in a cattle population by re- sequencing of key animals at low to medium coverage. BMC Genomics 2013;14:446. 25. Karim L, Takeda H, Lin L, Druet T, Arias JAC, Baurain D, et al. Variants modu- lating the expression of a chromosome domain encompassing PLAG1 influence bovine stature. Nat Genet. 2011;43:405–13. l 26. Pausch H, Flisikowski K, Jung S, Emmerling R, Edel C, Götz KU, et al. Genome-wide association study identifies two major loci affecting calv- ing ease and growth-related traits in cattle. Genetics. 2011;187:289–97. 3. Daetwyler HD, Capitan A, Pausch H, Stothard P, van Binsbergen R, Brøndum RF, et al. Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle. Nat Genet. 2014;46:858–65. 27. Lango Allen H, Estrada K, Lettre G, Berndt SI, Weedon MN, Rivadeneira F, et al. Hundreds of variants clustered in genomic loci and biological pathways affect human height. Nature. 2010;467:832–8. 4. VanRaden PM, O’Connell JR, Wiggans GR, Weigel KA. Genomic evalua- tions with many more genotypes. Genet Sel Evol. 2011;43:10. Page 9 of 9 Pausch et al. Genet Sel Evol (2016) 48:14 28. Brøndum RF, Guldbrandtsen B, Sahana G, Lund MS, Su G. 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https://openalex.org/W4297210212	https://zenodo.org/records/7115974/files/V17I09A103.pdf	English	null	A REVIEW OF WATER QUALITY MODELLING FOR A RIVER BASIN	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	5,481	Dr. RAJESH KHERDE Professor, Department of Civil Engineering, DYPU, Ambi Pune. Email-rajesh.kherde@dyptc.edu.in Professor, Department of Civil Engineering, DYPU, Ambi Pune. Email-rajesh.kherde@dyptc.edu.in GAURI PATIL Research Scholar, Department of Civil Engineering, DYPU, Ambi Pune. Email-gauri.patil@gmail.com Abstract Quality of water and its assessment model review is here with explained in the following study. The water requirement is increases and quality is reduced due to industrialization, urbanization and population growth.in multi- scale water resources and environmental management, mangers and policy makers are significantly supported by water quality modelling. Furthermore there are many constraints and challenges in water quality modelling.it is significantly important for the model application of modelling to adhere rules and regulations. Thus water quality modelling needs to standardize, equip with the features, main area of pollutions and the existing policy relevant models this study represents summary of water quality modelling and important models frequently used for water quality assessment at water bodies scale. This study also emphases on application of different water quality models, water quality modelling, model bucketization. Water quality models are differentiated and chosen based on their application, requirement, weakness, strength. Only a few models are suitable for specific water bodies, generate water quality parameters, are not radially available, require experienced model users, and have a large data set. It is advised that when choosing acceptable models, data availability, model complexity, kind of water body, and the intended objectives to be modelled be taken into account. Herewith we observe and analyze three different stages surface water quality models are being developed. Which are in diverse models, appropriateness, precisions, and methodologies are all considered. Environmental management authorities ensure that water quality models are applied consistently for regulatory purposes. By using standardization of water quality model. Developed countries has already done conclusion over standardization of this models. Using this we can put forward sustainable majors in emerging nations for the harmonization of surface water quality models. Mainly our focus will be on MIKE 11 model which seems to be suitable for Indian River. Keywords-water quality, water quality modelling, model uncertainty, model applicability. SUCHETA SABLE/KAKDE Research Scholar, Department of Civil Engineering, DYPU, Ambi Pune. Email:suchetasable17@gamil.com Research Scholar, Department of Civil Engineering, DYPU, Ambi Pune. Email:suchetasable17@gamil.com DOI 10.5281/zenodo.7115974 DOI 10.5281/zenodo.7115974 DOI 10.5281/zenodo.7115974 DOI 10.5281/zenodo.7115974 structure, indicators The standardization of surface water quality models can be improved in emerging nations But as maintain before, it is important for the developing country environmental management agencies which don not have standardization model quality water to create their own construction, indications, processes, and frame structure of a standardized model system for surface water quality[32].Some petrochemical, hydrological, and paper-making initiatives, for example, might have a negative impact on the aquatic environment duo to water pollution. Water quality models can be used to calculate pollutant transporter in water environment. Therefore water quality models are a key tools identify water environmental pollution and behaviors of pollutants in water environment. These environmental effect are calculated by using numerical models over the construction projects. The results are very important aspects in determining pollution and its impact on environmental management agencies gets health from these data to give approval to construction projects and to provide technical assistance [34]. The model results gives scientific and reasonable approach on such decisions of authorization. Development of different water quality models and its algorithm is becoming available due to rapid upgrading computer techniques [21]. Various topography water bodies and pollutant at different space and time scale have many types of water quality models and numerous models software’s up-to- date. But it is observed that in spite of varieties of models and algorisms, there seems to be differenced in results which can head to inconsistency and thus bring difference in management decisions. INTRODUCTION Water has a major impact on life sustainability which is determine as a key element in our environment. Water found in sea and land plays an important stand in day to day life activates such as drinking, agriculture, industrial and other uses [1]. Human socio- economic growth depend upon readiness of quality water [2]. Rapid increasing in population and thus expansion in agriculture and industries has shown us how that quality water is becoming difficult to achieve [3]. [12].Models of water quality can be standardized became an important based for the development of availability of water quality in environment. By following the models in developing countries and understanding the Authentication system, status, progress, frame 1410 \| V 1 7 . I 0 9 1410 \| V 1 7 . I 0 9 The Upgrade Stage (1965 to 1995) Water quality models were divided into six systems between 1965 and 1970, and rapid progress was made based on progressive investigations on multidimensional coefficient estimate of BOD-DO models. After upgrading from a one-dimensional model to a two-dimensional model, water quality simulations were applied to lake sand gulfs [5]. The development of nonlinear system models took place between 1970 and 1975[34]. These models the study looked at the relationship between biological growth rate and nutrients, sunlight, and temperature, as well as the N and P cycles system, phytoplankton, and zooplankton systems [5, 30, 30]. Because there were nonlinear relationships, the methodologies of finite difference and finite element analysis were used to derive the results, which were done using one or two dimensional models. The hydrodynamic mode and the impact of sediments were added into water quality models at a time when three-dimensional models were still being created. The effects of sediments were handled via the models' internal interaction processes, which allowed the sediment fluxes to fluctuate depending on the input conditions. Watershed-scale non-point source pollution simulations aided in the improvement of water quality management regulations. At this stage, standard models such as QUALK, MIKE11 and WASP were designed and implemented. In the meanwhile, the USGS's one- dimensional model was used to simulate water quality. The Initial stage (1925 to 1965) Water bodies have gotten a lot more attention at this time. Water quality models at the time were primarily concerned with interactions between various components of water quality in river systems that are influenced by living and industrial point sources of pollution [14]. Sediment oxygen requirements and algae photosynthesis and respiration were treated as external inputs, similar to hydrodynamic transmission, but nonpoint source pollution was treated as a background load. The one-dimensional model was used to address pollution issues in rivers and estuaries. A basic BOD-DO bilinear system model was developed and found to be effective in predicting water quality [27, 29]. Earlier (from 1925 to 1965). Since then, several academics have continued to work on the Streeter-Phelps models. [16]For example, Thomas Jr. discovered that sediment deposition and flocculation could reduce BOD without consuming oxygen, and that the rate of reduction the to distinguish the two BOD removal processes, a flocculation coefficient was added to the steady-state S-P model. [4]Because it was related to the number of leftover BOD. [44, 51] Dobbins- Camp added two coefficients to Thomas's equation: the BOD concentrations fluctuate due to sedimentation and surface runoff, whereas DO concentrations fluctuate due to algal photosynthesis and respiration. BACKGROUND OF WATER QUALITY MODEL Since 1925, surface water quality models have generally gone through three major stag DOI 10.5281/zenodo.7115974 SCOPE OF THE PROPOSED RESEARCH OF WATER QUALITY MODEL Models of water quality are useful for simulating and forecasting the water environment. As a result, they've gotten a lot of attention in recent years. Researchers showed how to calculate DO in rivers and streams using water quality models including MIKE, SIMCAT, TOMCAT and QUAL2E. Many publications go into great detail about the models and their capabilities. However, the models were only described to a limited extent. As a result, we will discuss not only the models' capabilities but also how they might be applied to specific circumstances in this review. Eight water quality models are presented in detail in this work, including their intended purpose, development, simulation aspects, basic principle, limitations, model strengths, and application to specific scenarios. SWAT, WASP, QUALK, and MIKE 11 are among the models provided. This review offers researchers with the knowledge they need to make educated judgments about which model to use for their research. As a result, water quality should be monitored to ensure that no contaminants are present at levels that are harmful to human health. However, there has been little research on the water quality in this study region, including by regulatory authorities responsible for water quality control. In addition, pollution sources must be identified, and a map of contaminated areas must be constructed. 1411 \| V 1 7 . I 0 9 1411 \| V 1 7 . I 0 9 Now lake and gulfs issues are also simulated in summation to river estuary issues. Stage three (1972 to 1975) based on phytoplankton and animal biological chain system with nutrient nitrogen and phosphorus cyclic reaction system, a nonlinear system model is evolved this system also structures the study between sunlight and temp, biomass growth rate and nutrients. Since the intersection is nonlinear, using it can be determined using a one- dimensional or two-dimensional numerical approach in space. Stage four (since 1975) different types of After continuing investigations on the food chain in phase three, interaction systems such as those involving interactions with harmful compounds have evolved. Water resources allocation, the water quality model is used to determine water function division, water environment capacity, and other considerations. As its simulation target are becoming more and more diversified and simulated. All these has made a strong technical requirement on development of extensive water quality model QUAL. quality were discovered. These materials are also deposited on a watershed's land surface and then transported to a water body in a sequential manner. This is likewise regarded as a significant pollutant source. Atmospheric deposition, whether static or dynamic, should be linked to a specific watershed. And thus necessitates the development of an air pollution model to incorporate these processes into the model. As a result, a few to calculate the Air pollution models were merged with water quality models to account for the effect of atmospheric pollutant deposition. After the QUAL 2K model[54], the WASP model[56], the QUASAR model[25], the SWAT model and the MIKE model[6], there was the WASP model, the QUASAR model, the SWAT model, and the MIKE model[7], several In order to calculate complex water environment circumstances, different water quality models have been devised. For example, based on the effects of atmospheric nitrogen, Whitehead created a semi- distributed integrated nitrogen model (INCA) of lately there was conclusion done by Kannel that these open-source models (such as QUAL2EU, WASP7 and QUASAR) can be used to simulate dissolved oxygen in rivers and streams. The Streeter– Phelps model is a phenomenological model in which BOD is the result of a bioassay test rather than the concentration of a chemical component. The models in the second stage have a conventional chemical kinetic structure. In aerobic settings, a series of starting reactions form a cumulative component the complicated chain of activities connected with electron transfer. The Grow Stage (after 1995 to till) Powerful controlled in developed countries has led in reduction of nonpoint source contamination. However, nitrogen compounds, heavy metals, and organic molecules are found in both dry and wet atmospheric deposition [10, 20, and 24]. Increased effects on river water 1412 \| V 1 7 . I 0 9 DOI 10.5281/zenodo.7115974 quality were discovered. These materials are also deposited on a watershed's land surface and then transported to a water body in a sequential manner. This is likewise regarded as a significant pollutant source. Atmospheric deposition, whether static or dynamic, should be linked to a specific watershed. And thus necessitates the development of an air pollution model to incorporate these processes into the model. As a result, a few to calculate the Air pollution models were merged with water quality models to account for the effect of atmospheric pollutant deposition. After the QUAL 2K model[54], the WASP model[56], the QUASAR model[25], the SWAT model and the MIKE model[6], there was the WASP model, the QUASAR model, the SWAT model, and the MIKE model[7], several In order to calculate complex water environment circumstances, different water quality models have been devised. For example, based on the effects of atmospheric nitrogen, Whitehead created a semi- distributed integrated nitrogen model (INCA) of lately there was conclusion done by Kannel that these open-source models (such as QUAL2EU, WASP7 and QUASAR) can be used to simulate dissolved oxygen in rivers and streams. The Streeter– Phelps model is a phenomenological model in which BOD is the result of a bioassay test rather than the concentration of a chemical component. The models in the second stage have a conventional chemical kinetic structure. In aerobic settings, a series of starting reactions form a cumulative component the complicated chain of activities connected with electron transfer. Finally, the algae model is an ecological dynamics model that argues that phytoplankton and nutrient cycling have no linear development and perish. Because the technique for model creation was based on the incorporation of "incremental" influences by a modest amount of mechanical addition of model layers, such models have inconsistency. They also frequently lack clear operational definitions of the water quality factors at hand. Bacterial decomposition occurs in biological waste water treatment plants under well-managed conditions. The IAWPRC activated sludge model (Henze 1987) was published. One dimensional simulation methodology is considered for a river and estuary issues. Stage two (1965 to 1970) the water quality model with one dimensional calculation method is involved with a two-dimensional computation method, from the bilinear model to six linear models. This was done accordance to the advancement and application of computing technology to the study of biochemical oxygen consumption activity. DOI 10.5281/zenodo.7115974 DOI 10.5281/zenodo.7115974 DOI 10.5281/zenodo.7115974 OBJECTIVES OF WATER QUALITY MODELLING 1) To develop a river water quality model and to assess the impact of uncertainty on water quality control. 1) To develop a river water quality model and to assess the impact of uncertainty on water quality control. 2) To ensure that the wastewater permit limit is not exceeded. 3) Establish objective function-based model evaluation criteria to identify model parameters that are sensitive and require careful estimation, thereby increasing model performance. 4) Create a software package with a user-friendly interface that uses GIS data for water quality modelling. 4) Create a software package with a user-friendly interface that uses GIS data for water quality modelling. 5) To easily comprehend daily, flow event, and seasonal water quality variations, as well as the processes that underpin them. 6) Based on the researched water quality characteristics, build a water quality index (WQI) for the river basin and compare it to the current water quality standard. Finally, the algae model is an ecological dynamics model that argues that phytoplankton and nutrient cycling have no linear development and perish. Because the technique for model creation was based on the incorporation of "incremental" influences by a modest amount of mechanical addition of model layers, such models have inconsistency. They also frequently lack clear operational definitions of the water quality factors at hand. Bacterial decomposition occurs in biological waste water treatment plants under well-managed conditions. The IAWPRC activated sludge model (Henze 1987) was published. One dimensional simulation methodology is considered for a river and estuary issues. Stage two (1965 to 1970) the water quality model with one dimensional calculation method is involved with a two-dimensional computation method, from the bilinear model to six linear models. This was done accordance to the advancement and application of computing technology to the study of biochemical oxygen consumption activity. Now lake and gulfs issues are also simulated in summation to river estuary issues. Stage three (1972 to 1975) based on phytoplankton and animal biological chain system with nutrient nitrogen and phosphorus cyclic reaction system, a nonlinear system model is evolved this system also structures the study between sunlight and temp, biomass growth rate and nutrients. Since the intersection is nonlinear, using it can be determined using a one- dimensional or two-dimensional numerical approach in space. Stage four (since 1975) different types of After continuing investigations on the food chain in phase three, interaction systems such as those involving interactions with harmful compounds have evolved. Water resources allocation, the water quality model is used to determine water function division, water environment capacity, and other considerations. As its simulation target are becoming more and more diversified and simulated. All these has made a strong technical requirement on development of extensive water quality model QUAL. 1413 \| V 1 7 . I 0 9 1413 \| V 1 7 . I 0 9 MODEL CLASSIFICATION Models of water quality are categorized are based on their complexity, the type of incoming water, and the water quality metrics (dissolved oxygen, nutrients, and so on) that they would predict. The more complicated a model, such as a three-dimensional model, is, the more difficult and costly it is to apply to a given circumstance. The complexity of a model is determined by four elements. 1) The number and kind of water quality indicator parameters. The more indications that are added in a model, the more complicated it becomes. Furthermore, some indications are more difficult to forecast than others. 2) The degree of spatial realism. As the number of pollution indicators and water quality monitoring sites grows, so does the amount of data needed, and the model's size grows as well. 3) The degree of temporal precision. Long-term static averages are far easier to anticipate than short-term dynamic changes in water quality. The stochastic assumptions for the distribution Water quality parameter probabilities are frequently simpler than the score points for such measurements. The water body under investigation's intricacy. These models have a well-understood nature that has been examined more thoroughly than the other factors. At least for smaller bodies of water like rivers and lakes, basic nutritional indicators like ammonia, zinc nitrate, and phosphate concentrations may be predicted rather well. It is more difficult to accurately anticipate algae quantities, although it is done often in the eutrophication has been a serious concern in the United States and Europe for the past two decades. Toxic organic chemicals and heavy metals, on the other hand, are far more dangerous. Although these materials are used in some of the models reviewed below, their impact on the environment remains a concern. Active investigation Models can only contain a certain number of contaminants. When choosing parameters for the model, it's important to identify 1414 \| V 1 7 . I 0 9 1414 \| V 1 7 . I 0 9 INPUT REQUIREMENTS FOR WATER QUALITY MODELS All models, as shown in the table, require information on water flow and temperature. These limits get increasingly tight as the application's complexity and scale increase. Point estimates of this data are required for static, deterministic models, and they often incorporate worst-case "design flow" predictions. Simulate pollutant behavior in worst-case scenarios. High summer temperatures, which will exacerbate dissolved oxygen and algal growth difficulties, as well as limited flows, will result in high BOD levels and other pollutants, will be the worst case scenario in terms of management. Models will need base-case concentrations of essential water quality metrics in addition to hydraulic data. They serve as a platform for assessing the effectiveness of management solutions and calibrating models to current circumstances. Furthermore, the models call for pollution flows or loads from the sources in question (e.g., industrial plants). Forms of data are required for a certain project. Table 3: Input Requirements for Water Quality Models. Table 3: Input Requirements for Water Quality Models. Table 3: Input Requirements for Water Quality Models. Input Requirements Remark water discharge Simple models require average flows, while more complicated models require dynamic information. Temperatures. Simple models require average temperatures, while complicated models require precise time records. Dissolved-oxygen concentrations Generally all water quality predictive models for dissolved oxygen concentrations The effects of a different management strategy. Biochemical oxygen demand In general, all water quality models that anticipate the effects of a management option on dissolved oxygen concentrations. Organics compounds, Heavy metals, ammonia etc Generally two dimensional model predict this inputs. DOI 10.5281/zenodo.7115974 DOI 10.5281/zenodo.7115974 DOI 10.5281/zenodo.7115974 contaminants that are both a worry in and of itself, as well as representative of the larger range of compounds that can't all be included. contaminants that are both a worry in and of itself, as well as representative of the larger range of compounds that can't all be included. p Table 1: Standards for Categorization of Water Quality Models. Standards Statement Single-plant Simpler models are typically used for single-plant marginal effects. More sophisticated models are necessary for regional investigations. Static or dynamic model Stable time-varying outputs Type of receiving water Lakes and rivers are usually easier to model Application. Stochastic or deterministic Probability distributions are the outputs of stochastic models, whereas point estimates are the outputs of deterministic models. Table 2: Water quality parameters Dissolved oxygen level In most water quality models, it is used as a water quality indicator. Temperature Depends on the climatic conditions Biochemical oxygen demand Its purpose is to determine the oxygen-reducing potential of waterborne pollutants. Toxic organic compounds A wide range of chemical substances can have an impact on aquatic life and may pose a direct threat to humans. Modeling is usually very challenging. Heavy metals Its lead, mercury, cadmium, and other elements can harm the environment as well as human health. Ammonia nitrogen It lowers dissolved oxygen levels and adds nitrate to the water. Phosphates It provide nutrient for algal growth. Table 1: Standards for Categorization of Water Quality Models. Table 3: Major surface water quality models and their versions and Properties Table 3: Major surface water quality models and their versions and Properties 1415 \| V 1 7 . I 0 9 REFERENCES 1) Ajay D. Chavhan, M. P. Sharma and Renu Bhargava, “Water Quality Assessment of the Godavari River”, Hydro, Nepal, 5, 31- 35, (2009). 2) C. C. Obropta, M. Niazi, and J. S. Kardos, “Application of an environmental decision support system to a water quality trading program affected by surface water diversions,” Environmental Management, vol. 42, no. 6, pp. 946–956, 2008. 3) C.T. Hunsaker and D. A. 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Tangible knowledge of water quality modelling and standardization is required to have better understanding on how the environment is affected by water pollution.it also helps to make high-yielding decisions and directions for the management. The describe overview of water quality modelling highlighting model selection application and constraints of different water bodies. Water managers and policymakers are benefited by using the tool of water quality modelling significantly. The regular practice and application of water quality modelling is remarkably required by scholars, policy makers and designers adhering rules and regulations. Models are used to calculate different water quality characteristics and estimate water quality 1416 \| V 1 7 . I 0 9 DOI 10.5281/zenodo.7115974 17) H. M. Valett, J. A. Morrice, C. N. Dahm, and M. E. 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https://openalex.org/W4393992380	https://jurnal.staithawalib.ac.id/index.php/thawalib/article/download/289/182	Indonesian	null	Budaya Sekolah Dalam Mendorong Pembinaan Guru	Thawalib	2,024	cc-by	6,294	BUDAYA SEKOLAH DALAM MENDORONG PEMBINAAN GURU Sabariah1, Hartono2, Zairunah3, Lisda Lian4 1,2Universitas PGRI Adi Buana Surabaya, Indonesia 3,4Universitas Islam Kalimantan Muhammad Arsyad Al Banjari Banjarmasin, Indonesia 1sabariah@unipasby.ac.id, 2hartono@unipasby.ac.id, 3zai.runah1972@gmail.com, 4lisda.liana91@gmail.com Sabariah1, Hartono2, Zairunah3, Lisda Lian4 1,2Universitas PGRI Adi Buana Surabaya, Indonesia 3,4Universitas Islam Kalimantan Muhammad Arsyad Al Banjari Banjarmasin, Indonesia 1 2 3zai.runah1972@gmail.com, 4lisda.liana91@gmail.com ABSTRAK Budaya sekolah sekolah merupakan salah satu faktor yang mempengaruhi sistem manajemen sekolah dalam upaya meningkatkan kinerja guru. Tujuan penelitian untuk mengetahui dan menjelaskan budaya sekolah dalam mendorong pelaksanaan pembinaan guru. Penelitian ini menggunakan kualitatif dengan fenomenologis. Teknik pengumpulan data dilakukan melalui observasi, wawancara, dan studi dokumentasi. Keabsahan data dengan triangulasi sumber, teknik dan perpanjangan pengamatan. Hasil penelitian menunjukkan: (1) budaya sekolah: memotivasi guru untuk bekerja, disiplin guru melalui sosialisasi pertauran ssekolah dan mengecek absensi kehadiran guru, mengadakan pertemuan rutin antar guru, serta membangun komunikasi yang positif antara kepala sekolah dengan guru dan guru dengan guru. (2) dampak: kinerja guru membaik dan pengelolaan pembelajaran berjalan dengan lancar. Kesimpulan: budaya sekolah telah terimplementasi dengan baik dan teratur. Implikasi penelitian: menjadi bahan masukan dan pembelajaran bagi sekolah lain untuk membina guru. Kata Kunci: Pembinaan, Guru, Budaya Sekolah Thawalib: Jurnal Kependidikan Islam eywords: Teacher Development, Teacher, School Culture Keywords: Teacher Development, Teacher, School Culture Copyright © 2024 Sabariah; Hartono; Zairunah; Lisda Lian Thawalib: Jurnal Kependidikan Islam Volume 5 (1) (2024) 55-66 e-ISSN 2807-386X A. PENDAHULUAN Pembinaan guru merupakan serangkaian usaha yang meliputi pelayanan profesional yang disediakan oleh individu yang memiliki keahlian lebih untuk meningkatkan mutu proses dan hasil belajar, sehingga tujuan pendidikan yang telah ditetapkan dapat terwujud (Anisa & Maunah, 2022). Pembinaan guru dapat diartikan sebagai serangkaian upaya dan pengendalian profesional terhadap semua unsur dalam organisasi agar unsur-unsur tersebut berfungsi sesuai dengan yang diharapkan yang bertujuan agar rencana untuk mencapai tujuan dapat terlaksana secara efektif dan efisien (Rokimin et al., 2022). Pembinaan terhadap guru penting karena pertama perkembangan kurikulum adalah indikator kemajuan pendidikan, yang sering mengakibatkan perubahan struktur dan fungsi kurikulum, dan kedua pengembangan personal atau guru harus dilakukan secara berkelanjutan dalam suatu sekolah (Husaini, 2018). Selain itu Sardjonopriyo menjelaskan adopsi metode pengajaran yang lebih memperhatikan keunikan individu menekankan perlunya pembinaan bagi guru yang biasanya kurang berpengalaman dalam menerapkan pendekatan baru tersebut (Hafiz & Jumriadi, 2018). Pembinaan guru diharapkan dapat secara berkelanjutan untuk memperbaiki kemampuan dan profesionalisme guru, sehingga standar mutu pengelolaan pendidikan terutama dalam proses pembelajaran dapat ditingkatkan secara konsisten (Agustine et al., 2019). Hal ini dibuktikan dengan hasil penelitian bahwa pembinaan guru mempengruhi kinerja guru di sekolah (Andriani, 2018). Selain itu, pembinaan guru juga memiliki pengaruh yang positif dan signifikan tergadap komitmen guru di sekolah sebesar 50,2% (Khomarudin, 2023). Hasil penelitian lain menunjukkan bahwa pelaksanaan pembinaan guru di suatu sekolah secara tidak langsung memiliki pengaruh terhadap kepuasan kerja guru sebesar 9,48% (Ferdiyanto et al., 2020). Secara komplek hasil penelitian menunjukkan bahwa pembinaan guru dapat mempengaruhi kinerja guru lebih efisien, memanfaatkan waktu dengan optimal, peningkatan dalam pengelolaan tugas dan penjadwalan yang lebih matang bagi guru-guru (Iswanto & Prasetyo, 2019). Hasil penelitian menunjukkan bahwa pelaksanaan pembinaan guru dapat dilakukan dengan beberapa Tindakan seperti mengirim guru untuk menghadiri seminar, memperbaiki serta menyediakan fasilitas pendukung pembelajaran agar proses belajar mengajar optimal, serta meningkatkan kompetensi profesional guru untuk mendukung partisipasi penuh dalam program pendidikan profesi guru (Fauzi et al., 2023). Hasil penelitian lain menjelaskan dalam membina guru, kepala sekolah sebagai pemimpin dapat memasukkan guru-guru dalam pelatihan untuk meningkatkan pemahaman guru, memberikan arahan kepada guru dalam pelaksanaan tugas-tugas pembelajaran, menghindari praktik yang bersifat memaksa terhadap guru, dan mendorong guru untuk melakukan evaluasi terhadap hasil belajar siswa (Abu, 2014). Perbedaan penelitian sebelumnya dengan penelitian ini adalah bagaimana sekolah membudayakan atau menanamkan nilai-nilai kebaikan (perilaku positif) yang secara langsung atau tidak langsung mampu membina guru agar dapat bekerja dengan lebih baik untuk membentuk mutu guru dan pembelajaran. ABSTRACT School culture is one of the factors influencing school management systems in efforts to improve teacher performance. The research aims to determine and explain school culture in fostering teacher development. This study uses a qualitative phenomenological approach. Data collection techniques were conducted through observation, interviews, and document studies. Data validity was ensured through triangulation of sources, techniques, and prolonged observation. The results of the study indicate: (1) school culture: motivating teachers to work, disciplining teachers through the socialization of school rules and checking teacher attendance, conducting regular meetings among teachers, and building positive communication between the school principal and teachers and among teachers. (2) impacts: improved teacher performance and smooth management of learning activities. Conclusion: school culture has been implemented effectively and systematically. Research implications: serving as input and learning materials for other schools in teacher development. Keywords: Teacher Development, Teacher, School Culture Copyright © 2024 Sabariah; Hartono; Zairunah; Lisda Lian Copyright © 2024 Sabariah; Hartono; Zairunah; Lisda Lian 55 \| T h a w a l i b This work is licensed under a Creative Commons Attribution 4.0 International License. Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 This work is licensed under a Creative Commons Attribution 4.0 International License. A. PENDAHULUAN Penelitian ini bertujuan untuk mengurai dan menjelaskan bagaimana institusi pendidikan mengintegrasikan nilai-nilai serta program kerja yang bertujuan membina kualitas kinerja guru, dengan dampak positif yang dirasakan baik oleh guru itu sendiri maupun oleh proses pembelajaran yang terjadi di sekolah. Dengan memfokuskan pada pembudayaan nilai-nilai 56 \| T h a w a l i b Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 yang mendorong pertumbuhan profesionalisme dan pengembangan diri, serta implementasi program-program yang mendukung peningkatan kualitas kinerja guru, penelitian ini berupaya mengidentifikasi praktik-praktik yang efektif dalam mendukung guru untuk menjadi lebih kompeten dan berpengaruh dalam proses pendidikan. B. METODE PENELITIAN Penelitian budaya sekolah dalam mendorong pembinaan guru ini dilakukan di Madrasah Ibtidaiyah Negeri ( MIN) 4 Kota Banjarmasin dengan NPSN 60723189. Alamat Jl. Pekapuran A RT. 30 RW.VI, Desa/Kelurahan Pekapuran Raya, Kecamatan Banjarmasin Timur, Kota Banjarmasin, Provinsi Kalimantan Selatan. Penelitian dilakukan secara kualitatif dengan pendekatan fenomenologis. Kualitatif fenomenologis dilakukan untuk menjelaskan secara deskriptif gejala atau kebiasaan apa saja yang dilakukan oleh sekolah dalam mendorong pelaksanaan pembinaan guru selama bekerja di sekolah. Teknik pengumpulan data dengan wawancara kepada kepala sekolah, waka kurikulum, waka kesiswaan, guru dan siswa. Kemudian data diperdalam dengan menggali data dengan teknik observasi dan dokumentasi. Observasi dilakukan dengan. Memperhatokan kebiasaan- kebiasaan yang dilakukan sekolah untuk membina guru. Sedangkan dokumentasi dilakukan untuk mengumpulkan data berupa absen harian, foto kegiatan pembelajara, foto dan file pendukung pelaksanan guyub rukun, dan file peraturan sekolah. Penelitian dilakukan dengan tiga langkah penelitian, pertama persiapan penelitian dengan menyiapkan instrument penelitian secara garis besar disajikan dalam tabel berikut: Tabel 1. Instrumen Wawancara Fokus Penelitian Instrumen Wawancara Budaya Sekolah dalam Mendorong Pembinaan Guru 1. Apa nilai-nilai yang dominan dalam budaya sekolah di tempat Anda mengajar? 2. Apa program atau kegiatan pembinaan guru yang tersedia di sekolah Anda? 3. Siapa yang memiliki peran penting dalam membentuk budaya sekolah yang mendukung pembinaan guru? 4. Kapan budaya sekolah yang mendukung pembinaan guru mulai berkembang di sekolah Anda? 5. Di mana budaya sekolah yang mendukung pembinaan guru paling terasa atau terlihat? 6. Mengapa Anda percaya bahwa budaya sekolah di tempat Anda mengajar mendukung pembinaan guru? 7. Bagaimana budaya sekolah di tempat Anda mengajar memengaruhi interaksi antar guru dan staf sekolah? Dampak Budaya terhadap Pembinaan Guru 1. Apa dampak konkret yang telah Anda lihat dari budaya sekolah dalam mendukung pembinaan guru di lingkungan tempat Anda mengajar? 2. Apa jenis perubahan atau peningkatan yang terjadi dalam pembinaan guru sebagai akibat dari budaya sekolah yang mendukung? 3. Kapan Anda mulai melihat dampak budaya sekolah dalam mendukung pembinaan guru di sekolah Anda? 4. Di mana Anda melihat dampak budaya sekolah yang paling kuat dalam mendorong pembinaan guru? Apakah itu dalam kebijakan 2. Apa program atau kegiatan pembinaan guru yang tersedia di sekolah Anda? 3. Siapa yang memiliki peran penting dalam membentuk budaya sekolah yang mendukung pembinaan guru? 4. Kapan budaya sekolah yang mendukung pembinaan guru mulai berkembang di sekolah Anda? 5. Di mana budaya sekolah yang mendukung pembinaan guru paling terasa atau terlihat? 6. This work is licensed under a Creative Commons Attribution 4.0 International License. B. METODE PENELITIAN Mengapa Anda percaya bahwa budaya sekolah di tempat Anda mengajar mendukung pembinaan guru? 7. Bagaimana budaya sekolah di tempat Anda mengajar memengaruhi interaksi antar guru dan staf sekolah? Dampak Budaya terhadap Pembinaan Guru 1. Apa dampak konkret yang telah Anda lihat dari budaya sekolah dalam mendukung pembinaan guru di lingkungan tempat Anda mengajar? 2. Apa jenis perubahan atau peningkatan yang terjadi dalam pembinaan guru sebagai akibat dari budaya sekolah yang mendukung? 3. Kapan Anda mulai melihat dampak budaya sekolah dalam mendukung pembinaan guru di sekolah Anda? 4. Di mana Anda melihat dampak budaya sekolah yang paling kuat dalam mendorong pembinaan guru? Apakah itu dalam kebijakan 4. Di mana Anda melihat dampak budaya sekolah yang paling kuat dalam mendorong pembinaan guru? Apakah itu dalam kebijakan 57 \| T h a w a l i b 57 \| T h a w a l i b This work is licensed under a Creative Commons Attribution 4.0 International License. Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 sekolah, interaksi antar guru, atau dalam lingkungan belajar? sekolah, interaksi antar guru, atau dalam lingkungan belajar? sekolah, interaksi antar guru, atau dalam lingkungan belajar? 5. Bagaimana budaya sekolah di tempat Anda mengajar memengaruhi motivasi dan kinerja guru dalam mengikuti program pembinaan? 5. Bagaimana budaya sekolah di tempat Anda mengajar memengaruhi motivasi dan kinerja guru dalam mengikuti program pembinaan? Kedua, pelaksanaan penelitian dengan pengumpulan data penelitian melalui wawancara, observasi dan dokumentasi yang dilakukan di bulan November 2023 – Januari 2024. Ketiga pasca penelitian dengan mengolah data melalui langkah reduksi data yakni memisahkan data sesuai dengan fokuss penelitian yaitu budaya sekolah dan dampak budaya sekolah dalam mendorong pembinaan guru. Kemudian dilakukan penyajian data dalam bentuk kalimat dan tabel dan gambar untuk memudahkan peneliti mengenali dan memahami data penelitian. Dan terakhir penarikan kesimpulan untuk memutuskan dan menetapkan data yang sesuangguhkan setalah di reduksi dan disajikan. Keabsahan data dilakukan dengan kredibelitas melalui beberapa pendekatan yakni triangulasi sumber dengan mengkombinasikan hasil wawancara dari seluruh informan penelitian. Kemudian triangulasi teknik dengan mengkombinasikan hasil penelitian dari hasil wawancara, observasi dan dokumentasi untuk dicari kekonkretan data. Selanjutnya dilakukan perpanjangan pengamanatan di bulan februari 2024 selama 2 minggu untuk memastikan data yang telah diolah benar adanya. Gambar 1. Desain Penelitian Budaya Motivasi Budaya Disiplin Budaya Pertemuan Budaya Pelatihan Budaya Komunikasi Dampak Kinerja Guru dan Lingkungan Belajar Membina Guru Dampak Kinerja Guru dan Lingkungan Belajar Membina Guru Budaya Pelatihan Budaya Komunikasi Gambar 1. Desain Penelitian This work is licensed under a Creative Commons Attribution 4.0 International License. C. HASIL DAN PEMBAHASAN Guru harus memiliki beberapa indikator motivasi dalam proses pembelajaran, termasuk memiliki keinginan belajar yang tinggi, antusiasme dalam pembelajaran, kepercayaan diri, rasa ingin tahu yang besar, kemampuan untuk berkonsentrasi sepenuhnya selama pembelajaran, dan hasil yang memuaskan (Wardiana & Asroyani, 2022). Sementara itu, dalam kegiatan upacara bendera, para guru di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin diberi motivasi untuk tetap menyayangi siswa seperti anak sendiri. Hal ini penting agar para guru dapat mengajar dengan penuh kasih sayang dan perhatian, sehingga siswa merasa nyaman dan termotivasi dalam proses pembelajaran. Dengan memiliki hubungan yang baik dan hangat antara guru dan siswa, diharapkan proses belajar mengajar dapat berjalan dengan baik dan maksimal. Motivasi ini juga mengingatkan para guru akan tanggung jawab guru tidak hanya sebagai pendidik, tetapi juga sebagai figur yang memberi inspirasi dan dukungan kepada siswa dalam menjalani proses pembelajaran dan pertumbuhan guru secara menyeluruh. Dengan memiliki kesadaran diri sebagai pendorong utama, guru akan mengalami peningkatan dalam kualitas kinerja guru. Guru akan menunjukkan tingkat dedikasi yang tinggi, ketelitian yang luar biasa, dan kesabaran yang besar dalam menjalankan tugas-tugas guru, bahkan ketika itu memerlukan waktu yang cukup lama (Simarmata, 2014). Motivasi mendorong peningkatan semangat dan ketekunan dalam bekerja, di mana guru yang memiliki motivasi tinggi akan memiliki energi yang cukup untuk menyelesaikan tugas-tugas guru yang pada akhirnya dapat menghasilkan pencapaian prestasi yang lebih baik bagi guru (Wattimena et al., 2023). Motivasi adalah pendorong utama yang memacu semangat dan ketekunan dalam setiap tugas yang diemban, khususnya bagi seorang guru. Ketika seorang guru merasa termotivasi dengan tinggi terhadap pekerjaannya, guru cenderung memiliki energi ekstra dan semangat yang kuat untuk menyelesaikan tugas-tugasnya dengan penuh dedikasi. Motivasi ini tidak hanya membantu guru untuk tetap berfokus dan produktif dalam proses pengajaran, tetapi juga membawa dampak positif pada kualitas pembelajaran yang disampaikan kepada siswa. Guru yang terdorong oleh motivasi yang kuat cenderung memiliki kesiapan mental dan emosional yang lebih baik dalam mengatasi tantangan yang muncul di dalam kelas. Motivasi yang tinggi juga berdampak pada pencapaian prestasi yang lebih baik bagi guru itu sendiri. Guru yang merasa termotivasi akan lebih bersemangat dalam meningkatkan kualitas pengajaran. Guru lebih berinisiatif dalam mencari strategi pengajaran yang inovatif, meningkatkan keterampilan guru melalui pelatihan tambahan, atau bahkan terlibat dalam riset dan pengembangan pendidikan. Dengan demikian, motivasi yang tinggi tidak hanya menguntungkan guru, tetapi juga memberikan manfaat besar bagi proses pembelajaran secara keseluruhan. C. HASIL DAN PEMBAHASAN . Budaya Sekolah dalam Mendorong Pembinaan Guru 1. Budaya Sekolah dalam Mendorong Pembinaan Guru Budaya yang dilakukan oleh Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin dalam mendorong atau mendukung pembinaan guru antara lain: y g Budaya yang dilakukan oleh Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin dalam mendorong atau mendukung pembinaan guru antara lain: a. Memotivasi Guru Untuk Bekerja Secara Optimal. Pengaruh motivasi guru sangat penting dalam meningkatkan kinerja belajar siswa di sekolah, karena ini merupakan indikasi keberhasilan pendidikan ketika siswa dapat mencapai prestasi yang memadai sebagai hasil dari motivasi tersebut (Bunyamin & Faujiah, 2014). Pemberian motivasi kepada guru untuk berkerja secara optimal di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin ini dilakukan oleh kepala sekolah dan dilakukan dengan secara lisan pada rapat rutin dan upacara bendera. Dalam setiap kegiatan rapat, kepala sekolah memberikan motivasi kepada para guru untuk tetap menjaga kinerja guru dengan mengutamakan persiapan silabus dan RPP (Rencana Pelaksanaan Pembelajaran). Hal ini bertujuan agar para guru dapat memberikan layanan pembelajaran yang prima kepada siswa. Dengan memiliki silabus dan RPP yang terstruktur dan terperinci, guru dapat merencanakan pembelajaran yang efektif dan sesuai dengan kebutuhan serta kemampuan siswa. Melalui motivasi ini, para guru diharapkan dapat terus meningkatkan profesionalisme guru dalam memberikan a. Memotivasi Guru Untuk Bekerja Secara Optimal. Pengaruh motivasi guru sangat penting dalam meningkatkan kinerja belajar siswa di sekolah, karena ini merupakan indikasi keberhasilan pendidikan ketika siswa dapat mencapai prestasi yang memadai sebagai hasil dari motivasi tersebut (Bunyamin & Faujiah, 2014). Pemberian motivasi kepada guru untuk berkerja secara optimal di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin ini dilakukan oleh kepala sekolah dan dilakukan dengan secara lisan pada rapat rutin dan upacara bendera. Dalam setiap kegiatan rapat, kepala sekolah memberikan motivasi kepada para guru untuk tetap menjaga kinerja guru dengan mengutamakan persiapan silabus dan RPP (Rencana Pelaksanaan Pembelajaran). Hal ini bertujuan agar para guru dapat memberikan layanan pembelajaran yang prima kepada siswa. Dengan memiliki silabus dan RPP yang terstruktur dan terperinci, guru dapat merencanakan pembelajaran yang efektif dan sesuai dengan kebutuhan serta kemampuan siswa. Melalui motivasi ini, para guru diharapkan dapat terus meningkatkan profesionalisme guru dalam memberikan 58 \| T h a w a l i b This work is licensed under a Creative Commons Attribution 4.0 International License. Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 pendidikan yang berkualitas di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin. This work is licensed under a Creative Commons Attribution 4.0 International License. C. HASIL DAN PEMBAHASAN Kedisiplinan guru dalam tahap perencanaan pembelajaran dapat dilihat dari kegiatan penyusunan RPP (Rencana Pelaksanaan Pembelajaran), saat melaksanakan pembelajaran guru menunjukkan disiplin dengan menampilkan sikap yang baik yang bisa dijadikan contoh oleh siswa, sedangkan tahap evaluasi hasil belajar guru telah menunjukkan disiplin dengan memberikan evaluasi pada aspek kognitif, afektif maupun psikomotor (Kusumaningtyas, 2018). g y Kedua yang diambil adalah pemeriksaan absensi guru secara rutin oleh kepala sekolah. Setiap satu minggu sekali, kepala sekolah meminta data absensi kehadiran guru kepada bagian administrasi. Tindakan ini bertujuan untuk memastikan bahwa para guru di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin memiliki kedisiplinan kehadiran yang konsisten. Dengan adanya pemeriksaan rutin ini, diharapkan para guru akan lebih memperhatikan dan mematuhi jadwal kehadiran guru, sehingga dapat menciptakan lingkungan kerja yang lebih teratur dan efisien. Langkah-langkah ini merupakan bagian dari upaya sistematis untuk meningkatkan kedisiplinan dan kinerja para guru demi mencapai tujuan pendidikan yang lebih baik di madrasah tersebut. Solusi untuk mengatasi rendahnya kedisiplinan guru dapat dilakukan sebagai berikut: pertama kepala sekolah dapat melakukan penjadwalan pengawasan secara berkala dan berkelanjutan terhadap kedisiplinan guru. Dengan memantau secara teratur, kepala sekolah dapat memberikan umpan balik kepada guru dan memberikan bimbingan jika diperlukan. Kedua kepala sekolah harus menegakkan dan melaksanakan peraturan yang ada, serta mempertimbangkan pemberian reward dan punishment atau sistem poin untuk meningkatkan kedisiplinan guru. Pemberian penghargaan kepada guru yang disiplin dan penerapan sanksi yang tegas bagi pelanggaran dapat membantu menciptakan lingkungan kerja yang disiplin. Ketiga kepala sekolah dapat memberikan pelatihan pengembangan dan peningkatan kedisiplinan kepada guru. Pelatihan ini dapat mencakup strategi manajemen waktu, peningkatan kesadaran akan pentingnya kedisiplinan, dan teknik pengelolaan kelas yang efektif (Setyaningrum et al., 2021). d k G b k A G c. Mengadakan Guyub Rukun Antar Guru Kerjasama antar guru memiliki peran yang krusial dalam kemajuan sekolah agar manfaatnya dapat dirasakan dalam jangka panjang, kolaborasi antar guru perlu dipromosikan, diperkuat, dan dikelola secara berkelanjutan (Kasmawati, 2020). Di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin, para guru diwajibkan secara rutin oleh kepala sekolah untuk berkumpul minimal satu bulan sekali. Pertemuan ini bertujuan utama untuk melakukan evaluasi terhadap proses pembelajaran yang telah dilaksanakan. Selain sebagai sarana evaluasi, pertemuan ini juga dimanfaatkan sebagai forum diskusi bagi para guru untuk saling berbagi pengalaman dan memecahkan masalah yang dihadapi dalam kegiatan pembelajaran. Guru-guru dapat berdiskusi tentang berbagai hal terkait dengan pembelajaran, siswa, dan bahkan masalah pribadi yang mungkin mempengaruhi kinerja guru. C. HASIL DAN PEMBAHASAN Guru yang termotivasi secara positif akan mampu menciptakan lingkungan belajar yang dinamis dan membangun hubungan yang baik dengan siswa. Guru mampu menginspirasi dan memotivasi siswa untuk meraih kesuksesan, memperkuat ikatan antara guru dan siswa, serta menciptakan suasana kelas yang penuh semangat dan antusias. b. Menjaga kedisiplinan Guru Dalam konteks kedisiplinan kerja, dibutuhkan kemampuan untuk sepenuhnya memahami dan menginternalisasi aturan, hukum, dan tata tertib yang berlaku yang mana seseorang yang memiliki tingkat disiplin yang tinggi akan melaksanakan tugas- tugasnya dengan penuh kesadaran, mengikuti aturan dengan sungguh-sungguh, dan menganggap pekerjaannya sebagai tanggung jawab moral yang harus dipenuhi, selalu mempertahankan semangat dan dedikasi tinggi dalam menjalankan tugas-tugas, dengan tujuan untuk mendukung pencapaian tujuan organisasi atau lembaga tempat 59 \| T h a w a l i b 59 \| T h a w a l i b Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 bekerja (Baririh, 2015). Menjaga kedisiplinan guru di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin dilakukan dengan dua cara, pertama dilakukan melalui sosialisasi peraturan kedisiplinan dengan menempatkan peraturan tertulis di ruang kantor. Hal ini bertujuan untuk memudahkan seluruh guru dalam mengakses dan mengingat peraturan-peraturan yang berlaku. Dengan demikian, para guru dapat lebih memahami aturan-aturan yang harus dipatuhi dalam menjalankan tugas dan tanggung jawab guru di madrasah tersebut. Guru yang disiplin tinggi akan berdampak positif pada kinerjanya, oleh karena itu, guru perlu mengikuti jadwal yang telah ditetapkan agar dapat mencapai kinerja yang optimal (Agustina et al., 2020). Kedisiplinan guru dalam tahap perencanaan pembelajaran dapat dilihat dari kegiatan penyusunan RPP (Rencana Pelaksanaan Pembelajaran), saat melaksanakan pembelajaran guru menunjukkan disiplin dengan menampilkan sikap yang baik yang bisa dijadikan contoh oleh siswa, sedangkan tahap evaluasi hasil belajar guru telah menunjukkan disiplin dengan memberikan evaluasi pada aspek kognitif, afektif maupun psikomotor (Kusumaningtyas, 2018). bekerja (Baririh, 2015). Menjaga kedisiplinan guru di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin dilakukan dengan dua cara, pertama dilakukan melalui sosialisasi peraturan kedisiplinan dengan menempatkan peraturan tertulis di ruang kantor. Hal ini bertujuan untuk memudahkan seluruh guru dalam mengakses dan mengingat peraturan-peraturan yang berlaku. Dengan demikian, para guru dapat lebih memahami aturan-aturan yang harus dipatuhi dalam menjalankan tugas dan tanggung jawab guru di madrasah tersebut. Guru yang disiplin tinggi akan berdampak positif pada kinerjanya, oleh karena itu, guru perlu mengikuti jadwal yang telah ditetapkan agar dapat mencapai kinerja yang optimal (Agustina et al., 2020). This work is licensed under a Creative Commons Attribution 4.0 International License. C. HASIL DAN PEMBAHASAN Kerjasama antara guru kelas dan guru pembimbing khusus tercermin melalui kolaborasi dalam penyampaian materi serta pemilihan media pembelajaran (Indrianto & Rochma, 2020). 60 \| T h a w a l i b This work is licensed under a Creative Commons Attribution 4.0 International License. Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 Manfaat kolaborasi antar guru antara lain menghasilkan perubahan dalam budaya profesional yang lebih kolaboratif, serta peningkatan kepuasan kerja dan motivasi; membawa pengalaman pembelajaran yang lebih mendalam, pengawasan tugas yang lebih efektif, dan peningkatan hasil belajar; terjalinnya kerjasama yang lebih erat antar staf, peningkatan reputasi sekolah, peluang pertukaran pengalaman dengan institusi lain, dan kemitraan dengan entitas lokal serta tercapainya penyebaran informasi yang lebih baik dan hubungan yang lebih harmonis dengan komunitas sekitar (Forte & Flores, 2014). Kerjasama ini digunakan sebagai penguatan karakter yang sejalan dengan tujuan sekolah, yang diimplementasikan melalui kegiatan sekolah secara rutin (Rahmawati et al., 2020). Kerjasama antar guru atau perkumpulan guru merupakan fondasi yang penting dalam pembentukan karakter dan pencapaian tujuan sekolah. Perkumpulan antar guru para guru dapat saling berbagi pengalaman, pengetahuan, dan keterampilan, yang semuanya mendukung upaya untuk memperkuat karakter siswa. Kegiatan yang dilakukan secara rutin oleh perkumpulan guru tidak hanya menjadi wadah untuk berdiskusi tentang strategi pengajaran terbaik, tetapi juga merupakan momen untuk memperkuat semangat kolaboratif di antara staf pengajar. Melalui kegiatan ini, guru dapat merasa didukung dan terhubung dengan komunitas pendidik yang lebih besar, yang pada gilirannya meningkatkan motivasi dan semangat untuk meningkatkan diri serta memberikan yang terbaik bagi siswa. Perkumpulan antar guru menjadi sarana penting dalam memperkuat hubungan interpersonal di antara staf pengajar. Dengan berbagi pengalaman dan dukungan satu sama lain, guru dapat menciptakan lingkungan kerja yang positif dan kolaboratif. Hal ini tidak hanya menciptakan atmosfer yang kondusif untuk pengembangan pribadi dan profesional, tetapi juga memperkuat rasa saling percaya dan keterlibatan dalam mencapai visi dan misi sekolah secara bersama- sama. This work is licensed under a Creative Commons Attribution 4.0 International License. d. Pemberian Pelatihan Pelatihan bagi guru sangat penting dalam meningkatkan kualitas pendidikan. Guru adalah tulang punggung sistem pendidikan yang efektif, dan pelatihan secara teratur membantu mereka untuk terus mengembangkan keterampilan, pengetahuan, dan pendekatan baru dalam mengajar. Dengan pelatihan yang tepat, guru dapat memperbarui metode pengajaran mereka sesuai dengan perkembangan terbaru dalam kurikulum dan teknologi pendidikan. Ini membantu mereka untuk tetap relevan dan efektif dalam memberikan pembelajaran yang berkualitas kepada para siswa. Selain itu, pelatihan bagi guru juga dapat meningkatkan motivasi dan kepercayaan diri mereka. Ketika guru merasa didukung dan diberi kesempatan untuk belajar dan berkembang, mereka cenderung lebih termotivasi untuk meningkatkan kinerja mereka. Pelatihan juga memberikan ruang bagi guru untuk berkolaborasi dengan rekan-rekan mereka, berbagi pengalaman dan ide, yang secara kolektif dapat meningkatkan standar pendidikan di sekolah. Program pelatihan (Diklat) dalam pembelajaran memberikan manfaat yang besar kepada guru. Pelatihan membantu guru meningkatkan wawasan dan pengalaman guru dalam mengakses ilmu baru, yang kemudian dapat diterapkan untuk meningkatkan kualitas pengajaran, membantu guru dalam menyelesaikan permasalahan yang guru hadapi, baik itu di lingkungan sekolah maupun di luar sekolah (Djajadi, 2020). Tujuan pelatihan adalah untuk memperbaiki kualitas kinerja guru dalam proses pembelajaran, dengan fokus pada peningkatan kompetensi serta perubahan pola pikir (mindset) guru dalam perencanaan, pelaksanaan, dan evaluasi pembelajaran (Rasyid & Nurqalbiani, 2020). Pelatihan terhadap guru di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin 61 \| T h a w a l i b 61 \| T h a w a l i b This work is licensed under a Creative Commons Attribution 4.0 International License. Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 wajib dilaksanakan karena selain interuksi kepala sekolah, kegiatan ini dilakukan atas dasar kesadaran peningkatan mutu guru sendiri. Secara offline, para guru mengikuti pelatihan yang diselenggarakan oleh sekolah dan pihak eksternal seperti diklat kurikulum merdeka. Pelatihan ini memberikan kesempatan kepada para guru untuk memperdalam pemahaman tentang berbagai aspek kurikulum serta strategi pengajaran yang baru. Dengan menghadiri pelatihan offline, para guru dapat memperoleh wawasan baru dan berbagi pengalaman dengan rekan seprofesi, sehingga memperkaya dan memperluas kualitas pengajaran guru. Pelatihan dapat meningkatkan pemahaman guru dalam mengembangkan kompetensi dasar dan indikator, serta membantu guru menggunakan kata kerja operasional yang sesuai dengan kompetensi dasar serta dapat membantu guru merancang tujuan pembelajaran yang sesuai dengan kompetensi dasar dan indikator yang telah ditetapkan (Chan & Budiono, 2020). This work is licensed under a Creative Commons Attribution 4.0 International License. d. Pemberian Pelatihan Menciptakan ikatan yang baik antara pendidik dan pelajar memiliki keuntungan bagi kedua belah pihak yakni akan membentuk tanggung jawab guru untuk menjalin komunikasi yang positif dengan murid-muridnya, upaya membangun hubungan yang harmonis antara guru dan murid juga memperkuat proses pembelajaran siswa (Margijanto & Purwanti, 2021). Berikut beberapa cara yang dilakukan guru untuk membangun hubungan yang positif dengan rekan guru dan siswa: menunjukkan sikap demokratis, kerja sama, dan kepribadian yang baik, bersikap sabar, adil, dan konsisten, tanpa adanya preferensi tertentu, bersikap terbuka, suka menolong, dan ramah dalam interaksi, memiliki sisi humor, memiliki minat yang beragam, serta menguasai materi pelajaran, selalu siap membantu dan menggunakan contoh atau istilah yang sesuai, tidak memihak, tidak pilih kasih, dan tidak membedakan perlakuan terhadap siswa, menjadi contoh yang baik bagi siswa dan masyarakat sekitarnya, bersikap tegas, mampu mengelola kelas, dan mendapatkan rasa hormat dari siswa, dan berusaha untuk membuat pembelajaran menarik dan membangkitkan semangat kerja sama dengan siswa (Yusuf et al., 2023). 2. Dampak Budaya Sekolah dalam Mendorong Pembinaan Guru Pengembangan budaya sekolah di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin memiliki dampak yang pada peningkatan kualitas kinerja guru dan kualitas pembelajaran dalam kelas. Guru menjadi lebih produktif dan aktif dalam menjalankan kewajibannya yaitu sebagai pendidik, pengajar dan pelatih siswa di sekolah. Guru yang produktif dan aktif mampu menciptakan lingkungan belajar yang inspiratif dan kondusif. Guru menerapkan pendekatan yang beragam dan inovatif dalam proses pembelajaran, sehingga dapat menyesuaikan metode mengajar dengan gaya belajar siswa secara lebih efektif. Akibatnya, siswa menjadi lebih terlibat dan bersemangat dalam pembelajaran, yang pada akhirnya berdampak positif pada peningkatan prestasi akademik dan pengembangan kemampuan guru secara menyeluruh. Budaya sekolah memainkan peran penting dalam meningkatkan kinerja guru, yang tercermin dalam perilaku guru mengelola pembelajaran dan memanfaatkan media serta sumber belajar secara optimal, mentransfer nilai-nilai kepada siswa melalui keteladanan sikap, menunjukkan budaya kerja yang baik melalui kedisiplinan kerja dan pendampingan siswa yang maksimal, komunikasi yang ramah, penampilan yang mencerminkan kualitas pelayanan, kesediaan untuk mengembangkan kompetensi diri, loyalitas terhadap lembaga, dan penghargaan terhadap etos kerja juga menjadi bagian dari budaya sekolah yang dijunjung tinggi (Oktaviani, 2015). Selain itu, budaya sekolah memiliki pengaruh yang signifikan terhadap kualitas proses pembelajaran yang menandakan bahwa kualitas pembelajaran dipengaruhi oleh adanya etika pembelajaran, sikap sopan santun, kondisi fisik sekolah, budaya literasi, dan budaya kebersihan (Saputra et al., 2021). p Budaya sekolah memiliki dampak yang signifikan dalam mendorong pembinaan guru. d. Pemberian Pelatihan Pelatihan antar guru memungkinkan pertukaran masalah yang dihadapi beserta solusinya terkait dengan kegiatan pembelajaran, serta penilaian terhadap sarana dan prasarana yang digunakan dalam kegiatan pembelajaran (Pratama & Lestari, 2020). Pelatihan yang dilakukan secara online oleh guru di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin atas inisiatif dan kemauan para guru. Contohnya, guru mengikuti workshop tentang penyusunan RPP secara online, serta workshop tentang pendekatan parenting siswa dan topik lainnya yang relevan. Pelatihan online ini memberikan fleksibilitas kepada para guru untuk memilih topik yang ingin guru tingkatkan dan mengikuti pelatihan sesuai dengan jadwal yang lebih fleksibel. Dengan menggabungkan kedua pelatihan ini, Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin menunjukkan komitmen sekolah dalam memberikan pelatihan yang komprehensif dan berkelanjutan bagi para guru, sehingga dapat terus meningkatkan mutu pendidikan di sekolah. Pelatihan memberikan kesempatan bagi guru untuk memperoleh pengetahuan baru atau menyegarkan kembali pengetahuan yang telah dimiliki sebelumnya (Rahmawati et al., 2017). Pelatihan guru melalui Program Pendidikan dan Latihan Profesi Guru (PLPG) telah terbukti secara efektif dalam meningkatkan kompetensi guru (Sabon, 2018). Selain itu, secara parsial pelatihan yang dilakukan telah terbukti memiliki pengaruh positif dan signifikan terhadap profesionalisme guru (Rakib et al., 2016). e. Membangun Hubungan Yang Positif Membangun hubungan yang positif di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin dibuktikan dengan adanya kesopanan dan keterbukaan yang terjalin antara guru dengan kepala sekolah, sesama guru, dan siswa menjadi bukti nyata akan komitmen untuk menciptakan lingkungan belajar yang harmonis dan produktif. Dengan terbentuknya hubungan yang harmonis, diharapkan tercipta suasana yang memungkinkan adanya dialog terbuka, penerimaan masukan, serta kritikan yang konstruktif. Ini memungkinkan guru untuk terus meningkatkan kualitas diri, pembelajaran, dan pengelolaan sekolah guna memberikan layanan pendidikan yang lebih baik kepada siswa. Guru perlu melakukan beberapa cara untuk membangun hubungan yang positif dengan siswa, termasuk memahami kemampuan individual siswa, menghargai masukan dan pendapat guru, memfasilitasi kerja sama antar siswa, serta memperlakukan siswa secara adil (Kurniawati & Basuki, 2023). p Tujuan dari terjalinnya hubungan yang positif di Madrasah Ibtidaiyah Negeri 4 Kota Banjarmasin ini adalah untuk menciptakan dinamika interaksi yang saling menguntungkan antara semua pihak yang terlibat dalam proses pendidikan. Dengan adanya kesempatan untuk memberikan masukan dan kritikan, baik guru maupun kepala sekolah dapat memahami kebutuhan serta harapan siswa secara lebih baik. Sebaliknya, siswa juga merasa lebih nyaman dalam mengemukakan pendapat dan 62 \| T h a w a l i b Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 permasalahan yang guru hadapi di sekolah. This work is licensed under a Creative Commons Attribution 4.0 International License. d. Pemberian Pelatihan Budaya sekolah yang mendukung pertumbuhan profesional dan kolaborasi antar guru akan menciptakan lingkungan yang kondusif bagi pengembangan keterampilan dan pengetahuan guru. Pertama, budaya sekolah yang mendorong pembinaan guru akan menyediakan sarana dan program pelatihan yang relevan dengan kebutuhan guru. Ini termasuk workshop, seminar, dan pelatihan yang dirancang untuk meningkatkan keterampilan mengajar, kepemimpinan, dan penggunaan teknologi pendidikan. Kedua, budaya sekolah yang menekankan pentingnya refleksi dan umpan balik akan memfasilitasi pembinaan guru secara terus-menerus. Guru akan didorong untuk merefleksikan praktik mengajar guru dan menerima umpan balik dari sesama guru, pengawas, atau ahli pendidikan. Hal ini akan membantu guru untuk terus meningkatkan kualitas pengajaran guru dan menyesuaikan metode pembelajaran sesuai dengan perkembangan terbaru dalam pendidikan. Ketiga, budaya sekolah yang mempromosikan 63 \| T h a w a l i b 63 \| T h a w a l i b Thawalib: Jurnal Kependidikan Islam, 2024, 5(1), Pages 55-66 kolaborasi dan pertukaran pengalaman antar guru akan memperkaya pengalaman pembelajaran. Melalui diskusi kelompok, pengembangan kurikulum bersama, atau proyek kolaboratif, guru dapat belajar dari satu sama lain dan mengembangkan praktik terbaik yang dapat diterapkan dalam kelas. kolaborasi dan pertukaran pengalaman antar guru akan memperkaya pengalaman pembelajaran. Melalui diskusi kelompok, pengembangan kurikulum bersama, atau proyek kolaboratif, guru dapat belajar dari satu sama lain dan mengembangkan praktik terbaik yang dapat diterapkan dalam kelas. D. SIMPULAN Pelaksanaan pbudaya atau penanaman nilai – nilai di Madrasah Ibtidaiyah Negeri ( MIN) 4 Kota Banjarmasin telah berjalan dengan baik baik oleh kepala sekolah, guru dan tenaga kependidikannya. Budaya yang dilakukan sekolah untuk mendorong pembinaan guru di Madrasah Ibtidaiyah Negeri ( MIN) 4 Kota Banjarmasin antara lain: Budaya sekolah yang berorientasi pada memotivasi guru untuk bekerja, menerapkan disiplin melalui sosialisasi aturan sekolah, dan memastikan kehadiran guru dengan mengawasi absensi merupakan fondasi yang kuat untuk menciptakan lingkungan belajar yang produktif. Dengan mengadakan pertemuan rutin antar guru, tidak hanya tercipta saling pengertian antar sesama staf pengajar, tetapi juga memungkinkan pertukaran ide dan pengalaman yang dapat memperkaya metode pengajaran. 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Retrieved from https://ojs3.unpatti.ac.id/index.php/jmp/article/view/7328 Yususf, O. Y. H., Andrianti, D., Endriani, L., Taunar, I., Salmia, Rusiana, & Rustia. (2023). Perilaku Guru yang Menumbuhkan Hubungan Positif Antara Guru dan Siswa. JETISH: Journal of Education Technology Information Social Sciences and Health, 02(01), 587 – 591. This work is licensed under a Creative Commons Attribution 4.0 International License. Bulian. Jurnal Abdi https://doi.org/10.33369/abdipendidikan.1.2.76-81 66 \| T h a w a l i b
https://openalex.org/W4302440601	https://dergipark.org.tr/tr/download/article-file/385582	Turkish	null	Modeling of Heating-Drying System Assisted Solar Energy and Heat Pump	DergiPark (Istanbul University)	2,011	cc-by-sa	4,592	ABSTRACT In this study, heating and drying process assisted heat pump and solar energy was modeled. In this system designing, energy requirements were supplied by solar energy and heat pump. Less energy input and more quality dried product can be provided by using proportional integral derivation (PID) control designing. Energy analysis of this system designing was made. Key Words: Solar energy, Heat pump, Energy, Heating, Drying. 1. GİRİŞ (INTRODUCTION) 2009 yılında Türkiye’de tahıl ürünlerinde 33 Key Words: Solar energy, Heat pump, Energy, Heating, Drying. 1. GİRİŞ (INTRODUCTION) 200 2009 yılında Türkiye’de tahıl ürünlerinde 33.6 milyon ton, sebzelerde 26.8 milyon ton ve meyvelerde 16.6 milyon ton üretim gerçekleşmiştir. Türkiye, kuru- tulmuş sebze konusunda ihracatçı bir ülke konumunda- dır. Dünyada ticareti yapılan kurutulmuş sebzelerin % 97-98’i kontrollü şartlarda sıcak hava ile kurutulmakta- dır. Isıtma veya kurutma yapılacak mahalde uygun bir sıcaklık elde edebilmek için belli bir miktarda enerji ge- reklidir. Gerekli enerji; genelde elektrik, fosil yakıtlar ve yenilenebilir enerji kaynaklarından elde edilir. Ku- rutma sistemlerinde tüketilen enerji miktarı göz önüne alındığında, günümüzde enerjinin kısıtlı ve pahalı olma- sından dolayı araştırmacılar daha az enerji girdisi olan sistemler yapabilmek için çeşitli yöntemlere başvur- muştur. Bu yöntemlerden bir tanesi de güneş destekli ısı pompası sistemlerinin (GDIP) kullanılmasıdır. GDIP sisteminin kurutma şartlarını (sıcaklık, bağıl nem ve hız) sağlamasıyla kurutma esnasında harcanan enerji miktarı düşmektedir. Bu çalışmada, enerji verimliliği dikkate alınarak güneş enerjisi ve ısı pompası destekli bir sis- tem tasarımı ve tasarımın enerji analizi yapılmıştır. Günümüzde, enerjinin verimli kullanılması ko- nusunun gün geçtikçe önem kazanması ile enerji arz gü- venliği ve sürdürülebilirliğinin sağlanması için gelenek- sel enerji kaynakları olan fosil yakıtların yerini temiz ve yenilenebilir enerji kaynakları almaya başlamıştır. Dünya enerji talebinin yaklaşık yüzde 80’lik kısmı fosil yakıtlardan karşılanmaktadır. Buna paralel olarak ener- jisini fosil yakıtlardan karşılayan Türkiye’nin enerjide dışa bağımlılığı % 80’e yaklaşmıştır. Bu bağlamda Tür- kiye enerji verimliliğine, yerli, temiz ve yenilenebilir enerji kaynaklarına yönelerek dışa bağımlılığını azalt- ması en önemli konulardan birisidir. Enerjinin verimli kullanılabilmesi için bilgi ve teknolojiden yararlanıl- ması gerekmektedir. Bu bilgi ve teknoloji sayesinde ha- yat daha yaşanılabilir ve gelecek nesillere de iyi bir ya- şam ortamı sağlanabilir. Türkiye’de enerjinin yaklaşık % 30’u, toplam elektrik tüketiminin ise yaklaşık % 40’ı binalarda kulla- nılmaktadır. Toplam enerji tüketiminin üçte birinden daha fazlası binaların ısıtılması, soğutulması ve aydın- latılması için harcanmaktadır. Hane halkları da toplam enerji tüketiminin % 60’ını ısıtma için harcamaktadır. Türkiye’de binaların yıllık enerji maliyeti 14 milyar dolar’ı aşmaktadır. Politeknik Dergisi Cilt:14 Sayı: 1 s. 85-91, 2011 Politeknik Dergisi Cilt:14 Sayı: 1 s. 85-91, 2011 Journal of Polytechnic Vol: 14 No: 1 pp. 85-91, 2011 ABSTRACT Türkiye’de yaklaşık 18 milyon ko- nut olduğu ve büyük kısmında yalıtımın yetersiz olması sebebiyle konutlarda ısıtma için harcanan enerji miktarı oldukça fazladır. * Sorumlu Yazar (Corresponding Author) e-posta: hdogan@gazi.edu.tr Digital Object Identifier (DOI) : 10.2339/2011.14.1, 85-91 ÖZET Bu çalışmada, ısı pompası ve güneş enerjisi destekli ısıtma ve kurutma prosesi modellenmiştir. Bu sistem tasarımında enerji ihtiyacı güneş enerjisi ve ısı pompası ile sağlanmıştır. Daha az enerji girdisi ile ve daha kaliteli olarak kurutulmuş ürün, oransal-integral-türevsel (PID) kontrol tasarımı kullanılarak sağlanabilecektir. Bu sistem dizaynının enerji analizi yapılmıştır. Anahtar Kelimeler: Güneş enerjisi Isı pompası Enerji Isıtma Kurutma Güneş Enerjisi ve Isı Pompası Destekli Isıtma- Kurutma Sisteminin Modellenmesi Seyfi ŞEVİK1, Hikmet DOĞAN2, Mustafa AKTAŞ2 G.Ü. Fen Bilimleri Enstitüsü , Ankara ** Gazi Üniversitesi Teknoloji Fakültesi, Ankara, Geliş/Received : 05.08.2011, Kabul/Accepted : 12.10.2011 2. LİTERATÜR TARAMASI (LITERATURE REVIEW) Güneş enerjisi ve ısı pompası sistemlerinin tekil olarak kullanılması yerine birlikte kullanılması ile sis- tem performans katsayısının artması nedeniyle bu tür sistemler performans arttırmak amacı ile kullanılmakta- dır. Güneş kolektöründe ısıtılan suyun ısı pompasının ısı kaynağı olarak kullanıldığı tipik güneş destekli ısı pom- pası (GDIP) ve güneş kolektörünün ısı pompasının bu- harlaştırıcısı olarak kullanıldığı direkt genleşmeli ısı 85 Seyfi ŞEVİK, Hikmet DOĞAN, Mustafa AKTAŞ / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1 2011 pompası (D-GDIP) olmak üzere iki tasarım şekli mev- cuttur. Literatürde çalışmalar, GDIP yerine ilk olarak 1955 yılında Batı Virginia’da Sporn ve Ambrose [1] ta- rafından ortaya konmuş olan D-GDIP sistem tasarımla- rının geliştirilmesi yönünde yoğunlaşmıştır. Ayrıca, GDIP konusunda da pek çok çalışma mevcuttur. Tara- fımızdan hazırlanan modellemede de GDIP sistemi tasa- rıma uyarlanmıştır. çalışabilmesi için Ocak-Mart, Kasım ve Aralık ayları için ek ısıtıcıya ihtiyaç duyacağı belirlenmiştir. COPIP değeri 3.1’dir [12]. Trillat-Berdal ve ark. (2007), tara- fından yapılan jeotermal enerji ve GDIP’nın birlikte kullanıldığı sistem ile sıcak su ve ısıtma-soğutma gibi binanın enerji ihtiyacı karşılanmıştır. 11 ay sonunda, je- otermal enerji gücünden 39.5 W/m ve 40.3 W/m orta- lama güç değerleri elde edilmiştir. Isıtma modunda COPIP değeri ortalama 3.75 bulunmuştur [13]. Dikici ve Akbulut (2008) tarafından, düzlemsel kolektörlü GDIP sistemi evsel ısıtmada kullanılabilirliği araştırılmıştır. Isıtma sezonunda, sistemin COP’si 3.08 olarak bulun- muştur [14]. Han ve ark. (2008), GDTKIP ısıtma sis- temi ile gizli ısı depolama tankı (GIDT) araştırılmış ve sistemin matematiksel modeli geliştirilmiştir. Isıtma pe- riyodunda sistemin COP değeri 3.28 ve en yüksek COP değeri 5.95 olmuştur. [15]. Bakırcı ve ark. (2009) tara- fından, ısıtma amaçlı GDIP sisteminin analizi için, ısıtma sezonunda ısı pompasının ve tüm sistemin orta- lama performans katsayıları sırasıyla 4.2 ve 3.6, ekserji verimleri ise ortalama % 44.3 ve % 6.8 olarak bulun- muştur [16]. Çomaklı ve ark. (1992), Türkiye’nin Karadeniz Bölgesi’nde evsel ısıtma için enerji depolu GDIP siste- minin performansını incelemiştir. Kolektör verimi, ısı pompası performans katsayısı (COP), sistem COP’u ve depolama veriminin aritmetik ortalama değerleri, sıra- sıyla % 70, % 4.5, % 4 ve % 60 olarak bulunmuştur [2]. Kurun (1994) çalışmasında, GDIP ısıtma sistemi ile elde edilecek sıcaklık seviyesinin yaklaşık 50 °C civarında olduğunu, klasik radyatörlü ısıtmaya uygun olmadığını ancak yerden ısıtma sistemi için ideal olduğunu belirt- miştir [3]. Best ve ark. (1996), pirinç kurutma için GDIP kurutucusu performansını araştırmış ve kurutucunun COP değeri 5,3 olarak bildirilmiştir [4]. Achariyaviriya ve ark. 3. ISITMA ve KURUTMA SİSTEMİ (HEATING AND DRYING SYSTEM) Isıtma işlemlerinde; havanın sıcaklığı, havanın hızı, mahalin kullanım amacı gibi faktörler önemlidir. Isıtılmak istenen mahal eğer bir konut ise ortama verilen ısı ortamdan olan ısı kayıplarını karşılamak zorundadır. Türkiye’de toplam enerji tüketiminin yaklaşık üçte biri binaların ısıtılmasında kullanılmaktadır. Enerji tasarrufu düşünülerek yapılan binalarda, özellikle ekonomik ısıtma sistemlerinin kullanılması ile binaların enerji tü- ketimi belirgin ölçüde azaltılabilir. Güneş enerjisi veya ısı pompası sistemlerinin ayrı ayrı veya birlikte kulla- nılmalarıyla, mevcut enerji kaynaklarının tüketimine ve çevrenin korunmasına önemli katkılar sağlanabilir. Ma- hal ısıtmasında güneş enerjisi veya GDIP sistemlerinin kullanılması durumunda yüksek bir enerji tasarruf po- tansiyeli bulunmaktadır. Son yıllarda GDIP sistemleri sıcak su üreterek ısıtma amacıyla kullanılmasına yönelik çalışmalar yapılmış ve piyasada bazı uygulama ör- nekleri mevcuttur. Özgener ve Hepbaşlı (2005), güneş enerjisi des- tekli düşey toprak kaynaklı ısı pompası (GDTKIP) ile sera ısıtmasında TKIP’nın ve hibrid sistemin; 10.93 C referans sıcaklığında, ekserji verimini sırasıyla; % 71.8 ve % 67.7 olarak elde etmiştir. TKIP’nın modellenmesi ve COP’lerinin değerlendirilmesinde, dikey ve yatay tip TKIP’nın COP değerleri sırasıyla; 3.64 ve 3.12 olarak bulunmuştur. Güneş enerjisi ile birlikte çalışan dikey ve yatay tip TKIP’nın COP değeri ise 3.43 ve 2.72’dir [7]. Aktaş ve ark. (2005), ısı pompalı endüstriyel fındık ku- rutma fırınının modellemesini yapmışlardır. Yapılan modelleme ile güneşli günlerde güneş enerjisinden fay- dalanılarak daha az enerji sarfiyatı olurken, diğer za- manlarda kurutma işleminin devamlılığının ısı pompası yardımı ile sağlanabileceğini belirtmişlerdir [8]. Ceylan ve ark. ( 2009), ısı pompalı bir kurutma fırınınında elma kurutmuşlardır [9]. Aktaş ve ark. ( 2009), güneş enerjili kurutucu ve ısı pompalı kurutucuda elma kurutarak ku- ruma karakteristiklerini belirlemişlerdir [10]. Hawlader ve Jahangeer (2006), GDIP kurutucusunun ve su ısıtıcı- sının performansı araştırmak için bir benzetim programı geliştirmişlerdir. Yükün 20 kg ve kompresör hızının 1200 rpm olması durumunda özgül nem çekme oranı (SMER) değeri 0.65 olarak saptanmıştır. Çalışmada, kompresör hızı arttığında SMER ve COP değerinin azaldığı gözlemlenmiştir [11]. Gündüz (2007), Bile- cik’te GDIP sistemi ile bir dairenin ısıtılması için ze- minden ısıtmalı sistem kurmuştur. Isı pompası sistemi- nin, Bilecik için 7 aylık süre boyunca verimli bir şekilde Bu çalışmada, düşük enerji tüketimi ile ısıtma ve kurutma amaçlı sıcak hava üretimi ve sıcak su üretimi yapabilen GDIP su ısıtıcısı sistemi tasarımı yapılmıştır. Bu tasarımda üretilen havanın sıcaklık kontrolü yapıl- maktadır. Bu tasarımda amaç, güneş enerjisini kullana- rak ısı enerjisi üretmek aynı zamanda da elde edilen bu enerjinin depolanmasını, sonrasında ise mahal ısıtması, ürün kurutması ve sera ısıtması gibi farklı alanlarda kullanılmasını sağlamaktır. 2. LİTERATÜR TARAMASI (LITERATURE REVIEW) (2000), ısı pompalı kurutucuda papaya ürününü 50 oC’de % 40.4 (kuru baz) nem değerinden % 23.2 (kuru baz) nem değerine kadar kurutmuşlardır. Maksimum özgül nem çekme oranı (SMER) 0.091 kg/kWh olarak hesaplanmıştır [5]. Kaygusuz (2000), seri bağlı güneş kollektörleri ile, enerji depolu ısı pompası sistemlerinin Karadeniz Bölgesi’ndeki ko- nutların ısıtılması için önerilebileceği sonucuna varmış- tır [6]. GÜNEŞ ENERJİSİ VE ISI POMPASI DESTEKLİ ISITMA VE KURUTMA AMAÇLI… / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1, 2011 İklime bağlı kalmadan, insan karar faktörünü en aza indirerek veya devre dışı bırakarak bütün yıl bo- yunca ekonomik olarak ısıtma ve/veya ürün kurutulma- sına imkân sağlayan bir sistem tasarımlanmıştır. Ancak sistemin hangi amaca hizmet edeceği önceden belirlen- melidir. Çünkü ısıtma veya kurutma için sistemde küçük değişiklikler yapılabilir. Sistem, hazırlanan bir bilgisa- yar programı yardımıyla farklı senaryolar eşliğinde PLC (programlanabilir lojik kontrol) veya PID kontrollü ola- rak ortam sıcaklığı, nem oranı, ürün ağırlığı vb değerle- rin izlenip uygun şartlar oluşturularak verim artışı ve kaliteli ürün almayı olanaklı kılacak şekilde modellen- miştir. Tasarlanan ve analizi yapılan sistemde güneş enerjisi ve ısı pompası gibi iki farklı sistemden faydala- nılarak ısı üretilmesi ile enerji tasarrufu sağlanabileceği düşünülmektedir. sistemin normal çalışma kurallarına uygun olarak kulla- nılmak üzere, diğer 2 buharlaştırıcı ise ilk buharlaştırı- cının iş yapamadığı durumlarda devreye girerek, dış ortamdan veya ısı depolama tankından ısı çekerek, sis- teme devamlılık sağlanması amacıyla kullanılmaktadır. Sistemde enerji üretimi; seri olarak bağlanan 3 adet güneş kolektöründen veya ısı pompasından sağlanmaktadır. Güneş enerjisi devresinde akışkanın devir daimi, dolaşım pompasıyla gerçekleştirilir. Bu sayede akışkan, güneşten aldığı ısı enerjini ihtiyaç yerine veya enerji deposuna bırakarak tekrar güneş kolektörlerine döner. Güneş kolektöründen gelen sıcak çalışma akışkanı kullanım yerinde kullanılır, ısı ihtiyacı olmadığı durumlarda ise ısı depolama tankına yönlendirilir. Isı depolama tankı içerisinde bulunan serpantinden geçerken enerjisini bırakır. Depodan çıkan düşük enerjili çalışma akışkanı, pompa yardımıyla güneş kolektörüne pompalanarak sistemde dolaşımı sağlanır. Tank içerisindeki ısı depolama akışkanı olarak su kullanılmaktadır. Ancak yüksek ısı depolama kapasitesine sahip endüstri yağının da performans deneyleri yapılarak ısı depolama akışkanı olarak kullanılabilir. Şekil 1’de tasarım modeli verilen sistem farklı fonksiyonlarda çalışabilme yeteneğine sahiptir. Mo- delde bulunan iki sistemin ayrı ayrı veya birlikte kulla- nılabilmesine olanak sağlanmıştır. Sistemde, güneş enerjisinden elde edilen ısının yetersiz olması duru- munda ısı pompası devreye girecek, yeterli olması ha- linde devreden çıkacaktır. Güneş enerjisinden elde edi- len ısı enerjisi, invertörlü olan ve sıcaklığa göre hız Şekil 1. GDIP sisteminin tasarım modeli. Şekil 1. GDIP sisteminin tasarım modeli. kontrolü yapılabilen bir fan yardımıyla mahale veril- mektedir. Isı pompası sistemi, ısı gereksinimine göre hareket eden bir devri ayarlanabilen bir kompresör ve 2 adet ilave buharlaştırıcıya sahiptir. Birinci buharlaştırıcı kontrolü yapılabilen bir fan yardımıyla mahale veril- mektedir. Isı pompası sistemi, ısı gereksinimine göre hareket eden bir devri ayarlanabilen bir kompresör ve 2 adet ilave buharlaştırıcıya sahiptir. 3. ISITMA ve KURUTMA SİSTEMİ (HEATING AND DRYING SYSTEM) Tasarım, ısı pompası ve gü- neş kolektörleri birbirinden ayrı iki kapalı sistem ha- linde düzenlenmiştir. Güneş kolektöründe ısıtılan su ısı pompasının ısı kaynağı olarak kullanılmaktadır. Dolayı- sıyla, ısı pompası için gerekli olan ısı kaynağının sıcak- lığı arttırılmıştır. Böylece, hava kaynaklı ısı pompası sistemlerindeki yoğuşturuculardan atmosfere atılan ısı, tasarımda değerlendirmeye alınmıştır. 86 GÜNEŞ ENERJİSİ VE ISI POMPASI DESTEKLİ ISITMA VE KURUTMA AMAÇLI… / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1, 2011 GÜNEŞ ENERJİSİ VE ISI POMPASI DESTEKLİ ISITMA VE KURUTMA AMAÇLI… / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1, 2011 Birinci buharlaştırıcı Tamamen taze hava ile çalışan sistemlerde ku- rutma havası ürün üzerinden geçirildikten sonra egzoz- dan atıldığından bu tür sistemlerde enerji tüketimi çok 87 Seyfi ŞEVİK, Hikmet DOĞAN, Mustafa AKTAŞ / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1 2011 Şekil 2. Sistemde enerji akışı. Şekil 3. Isı pompası sisteminin Log P-h diyagramı. fazla olmaktadır. Tarafımızdan modellenen sistemde havanın izlediği yol; güneş kolektörlerinden gelen ısı, ihtiyaca göre ya sadece hat üzerindeki serpantin üzerin- den ya da ısı pompasının da devreye girmesiyle yoğuşturucu ve serpantin üzerinden bir fan vasıtasıyla mahal içerisine doğru olmaktadır. Sisteme gerektiğinde bir miktar taze hava alınabilir. Şekil 2. Sistemde enerji akışı. Birçok uygulamada, havanın kurutma sisteminde yeniden dolaştırılması ısıl verimi arttırır. Fazla dolaşım ile oluşan düşük ısı kaybıyla, az dolaşımla oluşan yük- sek kuruma hızı, dolaştırılan hava oranı en uygun du- ruma getirilerek giderilebilir. Kurutma havasının nem içeriği, yeniden çevrimden etkilendiği için, istenilen hava neminin korunması amacıyla zaman zaman analiz edilmelidir [17]. Isı depolama; kullanılan toplayıcı miktarına, yani toplam kolektör alanına göre tayin edilen ve toplayıcı- lardan elde edilen sıcak suyun toplandığı kapta olur. Güneş enerjili sıcak su sisteminde, genellikle toplayı- cıya göre 1m2 toplayıcı yüzey alanı için 70-100 litre hacminde bir tank seçilir. Havanın uzun süre kapalı ola- bileceği veya uzun süre soğuk olabileceği ve gece kul- lanılabilmesi gibi durumlar gözetilerek depo hacmi daha büyük seçilebilir. Şekil 2. Sistemde enerji akışı. Şekil 3. Isı pompası sisteminin Log P-h diyagramı. Şekil 3. Isı pompası sisteminin Log P-h diyagramı. Kontrol işlevi; PID kontrolü, devri ayarlanabilen fan, devri ayarlanabilen kompresör, veri kaydedici, selenoid valfler, invertör, higrostat, sistemin yönlendi- rilmesinde kullanılan ölçü aletleri ile senkronize çalışan kontrol kartları, yazılım ve benzeri sistem ve sistem elemanlarının birlikte birbiriyle uyumlu kullanıldığı bir dizi elektro-mekanik kontrol elemanları tarafından ya- pılmaktadır. Şekil 3. Isı pompası sisteminin Log P-h diyagramı. göre çevrim 1-2-3-4 şeklini almıştır. Sistemde kullanılacak soğutucu akışkan debisi; 4. ENERJİ ANALİZİ (ENERGY ANALYSIS) Sistemde havanın kazandığı enerji: T c m q   . . 1 1 1 (8) T c m Q   . . (20) (20) T c m q   . . 1 1 1 (8) Üflenen havanın ısıtılması için gerekli enerji: Üflenen havanın ısıtılması için gerekli enerji: ile bulunur. Isı pompası sisteminin güneş kolektörü destekli buharlaştırıcının (Gke) verimi için; T c v q   . . . 2 2  (9) (9) 100 I Q TOP Gg KV   (21) (21) Mahalden veya fırından çevre havasına olan kayıpları karşılamak için harcanan enerji: Z A K q T T T d iç k            2 ) ( . . 3 (10) eşitliğinden faydalanılır. Bunun için kolektör yüzeyine gelen enerji (Itop) ya karmaşık formüllerle hesaplanır ya da bir solarimetre ile anlık olarak takip edilebilir. (10) Bir kolektörün topladığı faydalı enerji: Bir kolektörün topladığı faydalı enerji: (10) nolu eşitlikteki ısı geçirgenlik katsayısı: (10) nolu eşitlikteki ısı geçirgenlik katsayısı:  t e t f I A Q . .  (22) (22) (11)      n n dıı iç d d d K       ...... 1 1 1 2 2 1 1 (11)  t e t f I A Q . .  (22) eşitliği ile hesaplanır Anlık toplayıcı verimi:      n n dıı iç d d d K       ...... 1 1 1 2 2 1 1 (11) eşitliği ile hesaplanır. Anlık toplayıcı verimi: Fırın içerisindeki ürünlerin ısıtılması için gerekli enerji: Fırın içerisindeki ürünlerin ısıtılması için gerekli enerji:            I T T F e ç tg e t t K. ) ( .   (23) (23) k f f T c m q   . . 4 (12) (12) (12) Üründeki nemin buharlaştırılması için gerekli enerji: ifadesiyle bulunabilmektedir. Eşitlikte yerine yazılırsa; a a q S q 5 5 .  (13) (13) (13) a a q S q 5 5 .  (24) ] .( . . ) .( [. T T K F I F A Q tç tg t e e t t f     (24) kg başına verilen ısı; ifadesi elde edilir. 4. ENERJİ ANALİZİ (ENERGY ANALYSIS) Termodinamiğin I. Kanunu enerjinin korunumu ilkesine göre; ısı pompasının enerji denklemi olarak bu- harlaştırıcıdan sisteme alınan ısı (QBuh) ile kompresörde harcanan enerji (Wkomp) toplamı yoğuşturucudan ku- rutma havasına atılan ısıya (QYoğ.) eşittir. Bu nedenle yoğuşturucudan havaya aktarılan ısı; ) ( 3 ' 2 h h m QK    (2) (2) eşitliği ile hesaplanır. Yoğuşturucunun ısıtma veya kurutma havasına verdiği ısı; h k ve S Q Q .  (3) (3) QYoğ.= QBuh+ QKomp (1) şeklinde ifade edilir. (1) eşitliği yardımıyla hesaplanmaktadır. Isı pompası çalışma süresi ( Sh ) ile sistem çalışma süresi farklı olacaktır. Çünkü sistemde gerekli ısının sağlanması neticesinde ısı pompası çalışmayacaktır. Isı pompası sisteminde kompresör gücü; Şekil 2’de GDIP sistemi ve Şekil 3’de de ısı pompası sisteminin logP-h diyagramı görülmektedir. Gerekli toplam ısı miktarını ısı pompası yoğuşturucu ünitesi ve güneş enerjisi kolektörlerinden gelen sıcak su hattı üzerindeki ısı değiştiricisi sağlamaktadır. Şekil 2’de sistemde enerji akışı gösterilmektedir. Burada, ısı çekiminde kolektör çıkış sıcaklığı 20-25 ºC ile sınırlıdır. Şekil 3’de ise, IP çevriminin logP-h diyagramı verilmiştir. Bu diyagram kullanılarak gerekli buharlaştırıcı, yoğuşturucu ve kompresör kapasiteleri hesaplanabilir. Kullanılan soğutucu akışkanın log P-h diyagramında; (h1-h2) kompresör giriş çıkışını, (h2-h3) yoğuşturucu giriş çıkışını, (h3=h4) ise kılcal boru giriş çıkışını ifade eder. Tasarlanan sistem ile diyagram farklılaştırılmıştır. Buna ) ( ' 1 ' 2 h h m QC    (4) (4) eşitliği ile hesaplanır. Sistemde buharlaştırıcı kapasitesi ise; ) ( 4 ' 1 h h m QE    (5) (5) eşitliği ile hesaplanır. Mahalin yıllık ısıtma enerjisi ihtiyacı TS 825’de verilen hesap metoduyla kWh/m3 olarak hesaplanır. Mahal ısıtması için gerekli olan toplam ısı miktarı; 88 ERJİSİ VE ISI POMPASI DESTEKLİ ISITMA VE KURUTMA AMAÇLI… / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1, 201 h k IDve F Q Q .  (18) 4 q (6) h k IDve F Q Q .  (18) h k IDve F Q Q .  4 3 2 1 q q q q QVTOP     (18) (6) Kurutma için gerekli olan toplam ısı miktarı; Kurutma için gerekli olan toplam ısı miktarı; Sistemin güneş kolektöründen (Gg) fazladan kazandığı enerji; 5 4 3 2 1 q q q q q QVTOP      (7) (7) E TOP Gg Q Q Q   (19) (19) olarak harcanan veya kaybolan ısıların toplanması ile bulunur. Mahal veya fırın duvarlarının ısıtılması için gerekli enerji: eşitliğinden yararlanılır. 4. ENERJİ ANALİZİ (ENERGY ANALYSIS) Isı depolama tankındaki suyu ısıtmak için gerekli enerji; q 5a = [ '' ss h - ' sh ] (14) (14) T c m Q su   . . (25) (25) eşitliği ile bulunur [8]. SMER sd, üründen 1kg su çekebilmek için güneşli kurutucuda kullanılan enerjiyi tanımlar ve aşağıdaki eşitlik ile tarif edilir; eşitliği ile bulunur ifadesiyle bulunabilir. Isı depolama tankının enerji balansı ise; stl Ls s st st q q q dt dT V cp    ) . . ( (26) F sd sd W m SMER    (15) (15) (26) Isı pompası sistemindeki buharlaştırıcı, güneş kolektöründen gelen sıcak su hattı üzerindeki ikinci ısı değiştiricisinden ısı çekmektedir. Bu şekilde tasarlanması ile sisteme fazladan bir enerji kazandırılmaktadır. Güneş kolektöründen ısı sağlanmasıyla buharlaştırının çektiği toplam ısı; formülüyle hesaplanır. Bu formül için yardımcı eşitlikler ise; T cp m q L Ls   . . (27) T A U q st st stl   . . (28) 3 / 2 st st V ) D / h 2 ( 845 ,1 A   (29) (29) QTOP = m( h 1-h 4) (16) (16) ile ifade edilir. Sirkülâsyon pompasının enerji analizi; eşitliği ile bulunur. Sistemdeki birinci ısı değiştiricisinden kazanılan enerji: ) .( g ç p p h h m W   (30) (30) ) ( ç g ID h h m Q    ile hesapl ) ( ç g ID h h m Q    (17) ) ( ç g ID h h m Q    ile hesaplanır. Isı pompasının etkinlik katsayısı; (17) Komp Kond IP W Q COP  (31) (31) eşitliği ile hesaplanır. Birinci ısı değiştiricisinin ısıtma veya kurutma havasına verdiği ısı; 89 Seyfi ŞEVİK, Hikmet DOĞAN, Mustafa AKTAŞ / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1 2011 yardımıyla elde edilir. Tüm sistemin etkinlik katsayısı ise; yardımıyla elde edilir. Tüm sistemin etkinlik katsayısı ise; 5q Üründeki nemin buharlaştırılması için gerekli enerji (kJ) pomp Fan Komp ID Kond sist sist sist W W W Q Q W Q COP      (32) a q5 Ürün içerisindeki suyun buharlaştırılması için kg başına verilmesi gerekli enerji (kJ/kg) (32) a q5 Ürün içerisindeki suyun buharlaştırılması için kg başına verilmesi gerekli enerji (kJ/kg) eşitliğinden yararlanılarak elde edilir. eşitliğinden yararlanılarak elde edilir. a S Ürün içerisindeki toplam su miktarı (kg) a S Ürün içerisindeki toplam su miktarı (kg) 5. SONUÇ VE ÖNERİLER (RESULT AND SUGGESTIONS C Q Kompresör gücü (kW) C Q Kompresör gücü (kW) C Q Kompresör gücü (kW) E Q Buharlaştırıcı gücü (kW) E Q Buharlaştırıcı gücü (kW) ve Q Yoğuşturucunun ısıtma veya kurutma havasına verdiği ısı (kJ) TOP Q Güneş kolektöründen ısı sağlanmasıyla buharlaştırıcının çektiği toplam ısı (kJ) Oluşturulan bu model, sera ısıtmasında kullanılabileceği gibi havalandırma ve iklimlendirme sistemlerine de entegre edilerek çalıştırılabilir ve uygulanacak sistem ile enerji giderleri azaltılabilir. KV  Isı pompası sisteminin güneş kolektörü destekli buharlaştırıcının verimi 5. SONUÇ VE ÖNERİLER (RESULT AND SUGGESTIONS SMER sd 1 kg su kaldırmak için güneşli kurutucuda kullanılan enerji (kg/kWh) SMER sd 1 kg su kaldırmak için güneşli kurutucuda kullanılan enerji (kg/kWh) Enerji verimliliğinin son derece önemli olduğu günümüzde yenilenebilir bir enerji kaynağı olan güneş enerjisi ve bir ısı kaynağı olarak verimli olarak çalıştırılabilen ısı pompası sistemi birlikte düşünülüp bir sıcak hava üretim ve kurutma sistemi tasarlanmıştır. Buna ek olarak, enerji giderleri az olan ve verimli bir şekilde çalışacak olan bu ısı enerjisi üretim sisteminin enerji analizi yapılmıştır. VTOP Q Kurutma için gerekli toplam enerji miktarı (kJ) Gg Q Sistemin güneş kolektöründen fazladan kazandığı enerji (kJ) Gg Q Sistemin güneş kolektöründen fazladan kazandığı enerji (kJ) K Q Yoğuşturucu gücü (kW) ID Q Isı değiştiricisi gücü (kW) Q IDve Isı değiştiricisinin ısıtma veya kurutma havasına verdiği ısı (kJ) C Q Kompresör gücü (kW) E Q Buharlaştırıcı gücü (kW) ve Q Yoğuşturucunun ısıtma veya kurutma havasına verdiği ısı (kJ) TOP Q Güneş kolektöründen ısı sağlanmasıyla buharlaştırıcının çektiği toplam ısı (kJ) KV  Isı pompası sisteminin güneş kolektörü destekli buharlaştırıcının verimi kt Kurutma fırınının işletme sıcaklığı (oC) içt Kurutmaya başlamadan önceki iç hava sıcaklığı (oC) dt Dış hava sıcaklığı (oC) v Fırın hacmi içerisindeki toplam hava miktarı (m3) αiç İç yüzey ısı taşınım katsayısı (W/m2K) αdış Dış yüzey ısı taşınım katsayısı (W/m2K)  Duvarları oluşturan her bir katmanın ısı iletim katsayısı (W/mK) kT  Fırın işletme sıcaklığı ile ürün sıcaklığı arasındaki fark (oC) T  Fırın işletme sıcaklığı ile iç ortam sıcaklığı arasındaki fark (oC)  Yoğunluk (kg/m3) Z Isıtma süresi (saat) At Kolektör yüzey alanı (m²) Ie Birim eğik yüzeye düşen anlık ışınım gücü (W/m2) ηt Anlık kolektör verimi Ft Kolektör ısı kazancı faktörü (τ)e Efektif yutma katsayısı Ttg Suyun kolektöre giriş sıcaklığı ( ºC). Ttç Çevre sıcaklığı ( ºC). m Kütle (g) ID Q Isı değiştiricisi gücü (kW) Q IDve Isı değiştiricisinin ısıtma veya kurutma havasına verdiği ısı (kJ) Bu çalışmada, GDIP ile bir alanın ısıtma ve bir kurutma fırınının kurutma ısı ihtiyacını karşılayan bir sistem tasarımı üzerine hazırlanmış model çalışma incelenmiştir. Modeli oluşturulan GDIP’nın ısıtma ve kurutma amaçlı kullanılmasının Türkiye şartlarında verimli ve kullanılabilir olduğunun başarılı sonuçlarla desteklenmesi sonucunda bu tür çalışmalar sektörde uygulamaya konulabilir. WFan Fan gücü (kW) 11) Hawlader, M.N.A., Jahangeer, K. A., “Solar heat pump drying and water heating in the tropics”, Solar Energy, 80(5): 492-499, 2006. KAYNAKLAR (REFERENCES) SEMBOLLER (NOMENCLATURE) SEMBOLLER (NOMENCLATURE) A Toplam mahal veya fırın duvar yüzey alanı (m2) c1 Duvar malzemelerinin özgül ısısı (kJ/kg K) c2 Havanın özgül ısısı (kJ/kg K) d Duvarları oluşturan her bir katmanın kalınlığı (m) Sh Sistem çalışma süresi (saat) h Soğutucu akışkanın entalpisi (kJ/kg) ' s h Suyun ısı miktarı (Entalpi) (kJ/kg) '' ss h Suyun doymuş buhar halindeyken ısı miktarı (Entalpi) (kJ/kg) K Isı geçirgenlik katsayısı (W/m2 K) m Soğutucu akışkan debisi (kg/s) 1 m Duvar malzemelerinin toplam kütlesi (kg) f m Kurutulacak ürünün kütlesi (kg) fc Kurutulacak ürünün özgül ısısı (kJ/kgK) 1q Duvarların ısıtılması için gerekli enerji miktarı (kJ) 2 q Üflenen havanın ısıtılması için gerekli enerji miktarı (kJ) 3q Mahalden veya fırından çevre havasına olan ısı kayıpları (kJ), 4 q Fırın içindeki ürünlerin ısıtılması için gerekli enerji (kJ) kt Kurutma fırınının işletme sıcaklığı (oC) A Toplam mahal veya fırın duvar yüzey alanı (m2) m Kütle (g) 90 GÜNEŞ ENERJİSİ VE ISI POMPASI DESTEKLİ ISITMA VE KURUTMA AMAÇLI… / POLİTEKNİK DERGİSİ, CİLT 14, SAYI 1, 2011 Vst Depolama tankı hacmi (m3) qs Yararlı güneş ısı yükü (W) qls Güneş enerjisi tarafından toplanan yük (W) qstl Depolama tankının ısı kaybı yükü (W) st A Isı depolama tankının yüzey alanı (m2) st U Tankın ısı kaybı yükü katsayısı (W/m2 C) D Tank çapı (m) h Tank yüksekliği (m) WFan Fan gücü (kW) Vst Depolama tankı hacmi (m3) qs Yararlı güneş ısı yükü (W) qls Güneş enerjisi tarafından toplanan yük (W) qstl Depolama tankının ısı kaybı yükü (W) st A Isı depolama tankının yüzey alanı (m2) st U Tankın ısı kaybı yükü katsayısı (W/m2 C) D Tank çapı (m) h Tank yüksekliği (m) WFan Fan gücü (kW) 8) Aktaş, M., Ceylan İ., Doğan, H., “Isı pompalı endüstriyel fındık kurutma fırınının modellenmesi”, Gazi Üniversitesi Teknik Eğitim Fakültesi, Politeknik Dergisi, 8(4): 329-336, 2005. 8) Aktaş, M., Ceylan İ., Doğan, H., “Isı pompalı endüstriyel fındık kurutma fırınının modellenmesi”, Gazi Üniversitesi Teknik Eğitim Fakültesi, Politeknik Dergisi, 8(4): 329-336, 2005. 9) Ceylan, İ., Aktaş, M., Doğan H., “Isı pompalı kurutma odasında elma kurutulması”, Isı Bilimi ve Tekniği Dergisi, 25(2): 9-14, 2005. 10) [Aktaş, M., Ceylan, İ., Yılmaz, S., “Determination of drying characteristics of apples in a heat pump and solar dryer”, Desalination, 239:266-275, 2009. D Tank çapı (m) h Tank yüksekliği (m) KAYNAKLAR (REFERENCES) 1) Sporn, P., Ambrose, E.R., “The heat pump and solar energy”, In: Proceedings of the World Symposium on Applied Solar Energy, Phoenix, Ariz, November 1-5, 1955. 1) Sporn, P., Ambrose, E.R., “The heat pump and solar energy”, In: Proceedings of the World Symposium on Applied Solar Energy, Phoenix, Ariz, November 1-5, 1955. 12) Gündüz, A.B., “Bilecik ilinde güneş enerjisi destekli ısı pompasıyla sıcak su üretiminin performansı”, Yüksek Lisans Tezi, Eskişehir Osmangazi Üniversitesi Fen Bilimleri Enstitüsü, Eskişehir, 2007. 2) Çomaklı, Ö., Kaygusuz, K., Ayhan, T., “Solar- assisted heat pump and energy storage for residental heating”, Solar Energy, 51 (5): 357-366, 1992. 13) [Trillat-Berdal, V., Souyri, B., Achard, G., “Coupling of geothermal heat pumps with thermal solar collectors”, Applied Thermal Engineering, 27:1750-1755, 2007. 3) Kurun, E., “Güneş toplacından faydalanmanın bilgisayarla hesabı”, Osmangazi Üniversitesi Fen Bilimleri Enstitüsü, 84 s., 1994. 14) Dikici, A., Akbulut, A., “Performance Characteristics and Energy-Exergy Analysis of Solar-Assisted Heat Pump System”, Building ang Environment, 43 (11), 1961-1972, 2008. 4) Best, R., Cruz, J.M., Gutierrez, J., Soto, W., “Experimental results of a solar assisted heat pump rice drying system”, Renew Energy 9:690-694, 1996. 15) Han, Z., Zheng, M., Kong, F., Wang, F., Li, Z., Bai, T., “Numerical simulation of solar assisted ground-source heat pump heating system with latent heat energy storage in severely cold area”, Applied Thermal Engineering, 28:1427-1436, 2008. 5) Achariyaviriya, S., Sopanronnarit, S., Terdyothin A., “Mathematical model development and simulation of heat pump fruit dryer”, Drying Technology, 18(1-2): 479-491, 2000. 6) Kaygusuz, K., “Experimental and theoretical investigation of a solar heating system with heat pump”, Renewable energy, 21: 79-102, 2000. 16) Bakırcı, K., Çomaklı, K., Özyurt, Ö., Yılmaz, M., “Güneş kaynaklı ısı pompasının enerji ve ekserji analizi”, Mühendis ve Makina, cilt 50, sayı 590, 2009. 7) Özgener, Ö., Hepbaşlı, A., “Performance Analysis of a Solar Assisted Ground Source Heat Pump System for Greenhouse Heating: an Experimental Study”, Building and Environment, 40:1040-1050, 2005. 17) Günerkan, H., “Endüstriyel kurutma sistemleri”, Türk Tesisat Mühendisleri Derneği Dergisi, 36(13): 1-10, Mart-Nisan 2005. 91
https://openalex.org/W2947302528	https://vestnik.mgimo.ru/jour/article/download/930/840	Kirghiz, Kyrgyz	null	«Dragons» and «Chimeras» of Hybrid Wars	Vestnik MGIMO-universiteta	2,019	cc-by	2,835	A. Dimitrovska A. Dimitrovska Book Reviews Вестник МГИМО-Университета. 2019. 2(65). С. 196-201 DOI 10.24833/2071-8160-2019-2-65-196-201 УДК 327.8 Поступила в редакцию 01.04.2019 г. Принята к публикации 10.04.2019 г. «DRAGONS» AND «CHIMERAS» OF HYBRID WARS A. Dimitrovska Institute of European Studies, Belgrade Abstract: Review of the book by Egorchenkov D.A., Danyuk N.S. Hybrid Dragon and Chimera of War. Мoscow, UNITY-DANA Publ., 2019. 367 p. (in Russian) Abstract: Review of the book by Egorchenkov D.A., Danyuk N.S. Hybrid Dragon and Chimera of War. Мoscow, UNITY-DANA Publ., 2019. 367 p. (in Russian) Keywords: hybrid warfare, hybrid threats, subversion, unconventional forms of con frontation, countermeasures, USSR, Russia, the West, the Middle East, Central Asia «Hybridization» is one of the main characteristics of the changes taking place in world politics. The «ideal storm» of the world political organization, the likelihood of a new Cold War, the rapid growth of local conflicts and the destruction of nation-states are global trends that are under scrutiny of research [1]. Under the increasing inter national tensions, the term «hybridization» is increasingly used to characterize the processes of «reorganization of social spaces and the emergence of mixed forms of co operation and trans-local cultures» [1, p. 7]. In social and scientific discourse, «hybrid ity» is increasingly being blocked with tools and mechanisms of «hard power», shaping the phenomenon of «hybrid wars». Initially a systematic study of this phenomenon appeared in the United States and, up to a certain point, it was a prerogative of the mili tary. Currently, the issue of «hybrid wars» is becoming relevant and in demand in the Russian political science. It is indicative that young researchers have shown particular interest in the study of methods and practices of conducting hybrid wars, as well as its concomitant «fakes» and «chimeras». The book by D.A. Egorchenkov and N.S. Danyuk is an attempt to comprehensively analyze such phenomena as «hybrid warfare» and «hybrid threat». As its known, hy brid warfare (GW) implies «the convergence of the physical and psychological, kinetic and non-kinetic, combatants and non-combatants» [2, p. 34]; «The use of military and non-military means in an «integrated campaign» aimed at achieving a surprise effect, MGIMO REVIEW OF INTERNATIONAL RELATIONS • 2 • 2019 196 Ана Димитровска Рецензии at seizing the initiative and obtaining both psychological and physical advantages us ing diplomatic means»1.h The authors are supporters of the «hybrid warfare» concept that is combining unconventional forms of confrontation, the purpose of which is to undermine the enemy-state from the inside, without using any military force, at least not at the first stage. «DRAGONS» AND «CHIMERAS» OF HYBRID WARS In their opinion, the GW has a global, transboundary nature and is carried out in the form of a complex external pressure in order to stimulate instability within the targeted state. Under GW conditions, «strikes» are directed against all external and internal areas of the vital activities of States, including the political, socio-economic and spiritual spheres. As tools and mechanisms of hybrid warfare, the authors are con sidering political destructive technologies, «color revolutions» as «modern practices of state coups» [4, p. 27], ideological attacks, economic and sanction pressure, infor mation wars, the use of terrorism and extremism. In the world we live in, «war is a dragon ... war is Chimera, whose modern image is far from the canonical description, because the head and neck of the lion is personified in terrorism and extremism, the goat’s body has been turned into a financial and economic pressure, the tail of the snake are «color revolutions», and the fire erupting from the mouth became a cultural- informational aggression» (p. 10). Young scientists – representatives of the Institute of Strategic Studies and Forecasts (ICIF) of RUDN – thoroughly analyze the aforemen tioned manifestations of hybrid wars using specific examples. y gi «An integral part of the hybrid war … is the information warfare; in a broader context – information-psychological warfare. Its main task is to suppress the enemy’s (a geopolitical and geo-economic competitor) will to resist and force them undertake actions in favor of the information-psychological aggressor» [3, p. 5-6]. It is not by chance that information-psychological operations (IPO) are being actively used by the world politics leading actors in order to achieve their economic and political goals. According to the authors, information warfare is a complex «tool of asserting con trolled socio-psychological impact on the society as a whole and on each individual separately in the enemy country in order to change psychological characteristics in the desired direction for the influencing party (views, opinions, worldview, values, attitudes, stereotypes)» (p. 124). A key feature of such warfare is the hidden nature of the attacks. Informational attacks are a serious threat, since they contribute to internal division and weaken the morale of the population, as a result of which, in the case of a direct external threat, the population is not capable of mobilizing and implementing a consolidated resistance. 1 The Military Balance 2015. IISS. URL: https://www.iiss.org/publications/the-military-balance/the-military-balance-2015 (accessed: 29.03.2019). «DRAGONS» AND «CHIMERAS» OF HYBRID WARS IPO can lead to irreversible consequences: complete disrup tion of the State governance system and informational chaos. As a case of informational-psychological aggression directed from the outside, the scientists are exploring the political crisis in Ukraine in the 2013-2014. The book high lights three stages of the information campaign during the implementation of the coup 197 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 2 • 2019 A. Dimitrovska Book Reviews A. Dimitrovska d’état – the «color revolution» scenario. The first stage refers to the highly intensive financial injections into the so-called «independent» media, as a result of which a me dia pool is formed, ready to fulfill directives to discredit the government and stimulate protest moods. The second stage – the corruptly hired media reinforce in the public mind the «chimera» about the rightness of their actions manifested in destructive and aggressive protests. The third stage is the legitimization of the results of the coup d’etat. Considering the widespread practice of «color revolutions», it can be argued that in the context of further hybridization of world politics, the focus of actions will increas ingly shift towards IPO, allowing for the interested party to achieve its goals without d’état – the «color revolution» scenario. The first stage refers to the highly intensive financial injections into the so-called «independent» media, as a result of which a me dia pool is formed, ready to fulfill directives to discredit the government and stimulate protest moods. The second stage – the corruptly hired media reinforce in the public mind the «chimera» about the rightness of their actions manifested in destructive and aggressive protests. The third stage is the legitimization of the results of the coup d’etat. Considering the widespread practice of «color revolutions», it can be argued that in the context of further hybridization of world politics, the focus of actions will increas ingly shift towards IPO, allowing for the interested party to achieve its goals without launching the «hot» conflict phase. At the same time, it should not be forgotten that not every IPO allows solving all the tasks set by its initiators and sponsors. In this case, the IPO is an «advanced cognitive preparation work prior to open armed conflict» [3, p. 5]. p The authors are convinced that in the conditions of the information war launched by the West against Russia, Moscow should perceive information attacks as a threat coming during a conventional war. MGIMO REVIEW OF INTERNATIONAL RELATIONS • 2 • 2019 «DRAGONS» AND «CHIMERAS» OF HYBRID WARS On the other hand, this process was initiated by a part of the Soviet elite, who were perusing their own tasks.l As for the collapse of the socialist bloc under outside influence, the book provides an analysis of the whole complex of subversive activities. These include the creation of anti-communist, anti-soviet and russophobic bourgeois-nationalist groups that acted both within Eastern European countries and worked with diasporas in Western states; extensive studies in closed research institutes of psychology and social expectations of citizens of the target country; the creation of vast agent networks in each country; use of economic sanctions, etc.i Today we are witnessing the intensification of «all new destructive scenarios in which sometimes insignificant events (anti-constitutional actions of small groups, sabotage and subversive activities of an extremist and terrorist nature, etc.) with the assistance of political agents on the ground (for example, middle and upper echelons of power) and heated media-protest masses are transformed artificially into events of a nationwide scale» (p. 69). i Sanctions and various kinds of financial restrictions — the «chimeras» of hybrid warfare — have always been used as coercive methods. Restrictions may apply to eco nomic, political, diplomatic and other fields of activity. The authors analyzed in detail the economic pressure that was put on Russia, Yugoslavia, Iran and North Korea, and concluded that financial and economic pressure is not effective by itself. Of course, with the help of sanctions, the economy of a state or region can be weakened, but achieving one’s goals without using the most destructive political technologies that are very conveniently superimposed on a particular country experiencing unfavorable social situation is virtually impossible. For example, the use of sanctions as a coercive instrument on Iran since 1979 after the Islamic revolution has not yielded the desired result for the US. Iran has not changed its political discourse. The same outcome was achieved as a result of the use of sanctions as a coercive instrument against the USSR, and then against Russia. Therefore, a comprehensive toolkit of subversive actions had to be utilized. It should be noted that «the overtness in formulating the task aimed to preserve global domination with the use of all available tools has to rely on the established ideo logical basis» (p. 61). Therefore, for effective countermeasures to sanctions, it is nec essary to act not only in the field of foreign policy, but also domestically. «DRAGONS» AND «CHIMERAS» OF HYBRID WARS Therefore, it is necessary to actively develop mech anisms of counteraction to destructive information and misinformation, which is ag gressively imposed from the outside both on Russian society within the country and on the Russian diaspora beyond its borders, which is a direct threat to its national security and sovereignty. Of all the tools of a hybrid war, international terrorism and extremism are the most horrific «dragons». The book thoroughly describes how terrorism has already become part of a hybrid war, analyzing the process of how the leading actors of world politics are using international Islamist terrorist organizations, starting with Al-Qaida and the Taliban, created with the direct involvement of the CIA to fight the Soviet Union in Afghanistan, and ending with the «Islamic State». The West prefers to call ISIS, the Front of En-Nusra and other terrorist Islamist organizations «the democratic moderate opposition», as long as they realize its goals. In Syria, Afghanistan, Libya and other states, terrorist groups of extremists are used to destabilize large spaces, to eliminate specific political regimes, while terrorists in the world media are represented as «freedom fighters». i No less interesting is the section devoted to the analysis of implementation of de structive political technologies used to change political regimes. It is noteworthy that authors have revealed a close relationship not only between the «color» revolutions of the last 20 years, but also with the events of the Cold War period. The events in ques tion are Hungary 1956, Czechoslovakia 1968 and Poland 1981. The comparison of «color» and «velvet» revolutions is relevant both from a scientific and practical point of view. The method is the same, the technic of execution is different. What messengers can do today was carried out by word of mouth in the late 1980s. The main conclusion of the authors is as follows: the so-called democratization of the countries of Central and Eastern Europe was not an objective historical process, but, on the one hand, it was the result of the planned activities of the Western intelligence services to destroy 198 MGIMO REVIEW OF INTERNATIONAL RELATIONS • 2 • 2019 Ана Димитровска Рецензии the socialist bloc. nologii i metody protivodeistviya [Contempo rary informational and psychological operations: technologies and methods of counteraction]. Obosrevatel’ – Observer. 2019, no. 2 (349), pp. 5-17. (in Russian). About the author: Ana Dimitrovska – Visiting Fellow to the Institute of European Studies. Nikola Pasic’s Square 11, Belgrade, Serbia, 11000. E-mail: ana_afro05@hotmail.com Ana Dimitrovska – Visiting Fellow to the Institute of European Studies. Nikola Pasic’s Square 11, Belgrade, Serbia, 11000. E-mail: ana_afro05@hotmail.com «DRAGONS» AND «CHIMERAS» OF HYBRID WARS The authors emphasized social and economic measures, but especially some necessary efforts in education – the creation of «active patriotism» within the society. In today’s postmodern world Fernand Braudel’s words that «capitalism only tri umphs when it becomes identified with the state» best explain, according to the au thors, the US policy of promotion the idea of «a world without borders», to undermine the international legal system and facilitate the practice of «humanitarian interven tions» and the implementation of other aggressive actions (p. 54). Only strong states can resist such a hegemonic policy. Authors are optimistic about the future. They are convinced that, having gone through trial and error, and external threats and provoca 199 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 2 • 2019 Book Reviews A. Dimitrovska A. Dimitrovska A. Dimitrovska tions, the Russian Federation has embarked on a sustainable trajectory of national de velopment. Despite the unfavorable influence of the «aggressively obedient majority»2, which still supports Washington, Russia will be able to protect itself and its allies from the «dragons» and «chimeras» of hybrid wars.hi g y The book under review is not only a serious scientific study, but also a sharply polemical text, which, of course, will cause both positive responses and disagreement. The book itself should be considered as a vivid example of the Russia’s political and information resistance against the West. The authors should be praised as selfless war riors fighting dragons and chimeras in a brave new world of hybrid warfare. References: 1. Gibridizaciya mirovoy i vneshney politiki v svete sociologii mezhdunarodnyh otnosheniy [Hybridi zation of world and foreign policy in the light of sociology of international relations] Ed. P.A. Cygankov. Moscow. Goryachaya liniya – Tel ekom. 2017. 356 p. (in Russian). 1. Gibridizaciya mirovoy i vneshney politiki v svete sociologii mezhdunarodnyh otnosheniy [Hybridi zation of world and foreign policy in the light of sociology of international relations] Ed. P.A. Cygankov. Moscow. Goryachaya liniya – Tel ekom. 2017. 356 p. (in Russian). 4. Ponomareva E.G. Chto takoe «cvetnye revoly ucii» i kak s nimi borot’sya? [What «colour revolutions» are and how to deal with them?]. Representative power – 21st century, 2016, no. 1-2 (144-145), pp. 26-38. (in Russian). 2. Hoffman F. Hybrid Warfare and Challenges. Joint Force Quarterly. 2009, no. 52, pp. 34-39. 3. Manoylo A.V., Ponomareva E.G. Sovremennye informacionno-psihologicheskie operacii: tekh Ана Димитровска DOI 10.24833/2071-8160-2019-2-65-196-201 Ана Димитровска Д р DOI 10.24833/2071-8160-2019-2-65-196-201 Ponomareva E.G. Chto takoe «cvetnye revoly ucii» i kak s nimi borot’sya? [What «colour revolutions» are and how to deal with them?]. Representative power – 21st century, 2016, no. 1-2 (144-145), pp. 26-38. (in Russian). Ana Dimitrovska – Visiting Fellow to the Institute of European Studies. Nikola Pasic’s Square 11, Belgrade, Serbia, 11000. E-mail: ana_afro05@hotmail.com 2 A term, used by Yuriy Afanaseyev, USSR Deputy, member of the Interregional Deputies Group, addressing the delegates voting for the Presidium at the First Congress of People’s Deputies of the USSR in 1989. Институт европейских исследований, Белград Институт европейских исследований, Белград Рецензия на книгу: Егорченков Д.А., Данюк Н.С. Гибридный дракон и химера войны. М.: ЮНИТИ-ДАНА, 2019. 367 с. Ключевые слова: гибридная война, гибридные угрозы, диверсионные действия, не типичные формы конфронтации, контрмеры, СССР, Россия, Запад, Ближний Восток, Центральная Азия. 2 A term, used by Yuriy Afanaseyev, USSR Deputy, member of the Interregional Deputies Group, addressing the delegates voting for the Presidium at the First Congress of People’s Deputies of the USSR in 1989. 200 MGIMO REVIEW OF INTERNATIONAL RELATIONS • 2 • 2019 Ана Димитровска Рецензии Список литературы: С. 5-17. 3. Пономарева Е.Г. Что такое Цветные революции и как с ними бороться? // Представительная власть. 2016. №1-2. С. 26-38. 4. Hoffman F. Hybrid Warfare and Chal lenges // Joint Force Quarterly. 2009. No. 52. Pp. 34-39. 1. Гибридизация мировой и внешней по литики в свете социологии междуна родных отношений / под ред. П.А. Цы ганкова. М.: Горячая линия – Телеком. 2017. 356 с. 3. Пономарева Е.Г. Что такое Цветные революции и как с ними бороться? // Представительная власть. 2016. №1-2. С. 26-38.f 2. Манойло А.В., Пономарева Е.Г. Со временные информационно-психоло гические операции: технологии и ме тоды // Обозреватель. 2019. № 2(349). 4. Hoffman F. Hybrid Warfare and Chal lenges // Joint Force Quarterly. 2009. No. 52. Pp. 34-39. ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 2 • 2019 Об авторе: Ана Димитровска – приглашенный научный сотрудник Института европейских исследований, Сербия, г. Белград. Nikola Pasic’s Square 11, Belgrade, Serbia, 11000. E-mail: ana_afro05@hotmail.com. Ана Димитровска – приглашенный научный сотрудник Института европейских исследований, Сербия, г. Белград. Nikola Pasic’s Square 11, Belgrade, Serbia, 11000. E-mail: ana_afro05@hotmail.com. ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 2 • 2019 201
https://openalex.org/W2771900456	http://iapress.org/index.php/soic/article/download/soic.171203/312	English	null	Optimal control of linear time-varying systems with state and input delays by Chebyshev wavelets	Statistics, optimization & information computing	2,017	cc-by	18,139	AMS 2010 subject classiﬁcations 90C20, 49N10 DOI: 10.19139/soic.v5i4.341 DOI: 10.19139/soic.v5i4.341 DOI: 10.19139/soic.v5i4.341 STATISTICS, OPTIMIZATION AND INFORMATION COMPUTING Stat., Optim. Inf. Comput., Vol. 5, December 2017, pp 302–324. Published online in International Academic Press (www.IAPress.org) STATISTICS, OPTIMIZATION AND INFORMATION COMPUTING Stat., Optim. Inf. Comput., Vol. 5, December 2017, pp 302–324. Published online in International Academic Press (www.IAPress.org) STATISTICS, OPTIMIZATION AND INFORMATION COMPUTING Stat., Optim. Inf. Comput., Vol. 5, December 2017, pp 302–324. Published online in International Academic Press (www.IAPress.org) (Received: 13 August 2017; Accepted: 18 October 2017) Abstract This paper presents a novel method for ﬁnding the optimal control, state and cost of linear time-delay systems with quadratic performance indices. The basic idea here is to convert a time-delay optimal control problem into a quadratic programming one which can be easily solved using MATLAB®. To implement this idea we choose a state and control parameterization method by using Chebyshev wavelets. The inverse time operational matrix of Chebyshev wavelets is introduced and applied for parameterizing state and control terms containing inverse time. The method is also applicable to linear quadratic time-delay systems with combined constraints. Illustrative examples demonstrate the validity and applicability of the approach which new expansions for initial vector functions of state and control variables are deﬁned. Keywords time-delay system, optimal control, inverse time operational matrix of Chebyshev wavelets, multiple delays, constrained lag system. ∗Correspondence to: Iman Malmir (iman.malmir@outlook.com). ISSN 2310-5070 (online) ISSN 2311-004X (print) Copyright c⃝2017 International Academic Press Optimal control of linear time-varying systems with state and input delays by Chebyshev wavelets Iman Malmir ∗ Department of Aerospace Engineering, MUT University of Technology, Tehran, Iran (Received: 13 August 2017; Accepted: 18 October 2017) (Received: 13 August 2017; Accepted: 18 October 2017) 1. Introduction A time-delay (TD) system is a system in which time delays occur between the application of the delayed variables to the system and their resulting effect on it. The TD system can be constructed either with inherent delays in the components or with a deliberate application of time delays into the system for some purposes [1]. Delays occur frequently in chemical processes, electronic, aerospace and mechanical systems, transmission lines and industrial processes. A mathematical model of a system such as population growth, epidemic growth, economic growth and neural networks results in a delay differential equation involving the variable time [2]. The extension of Pontryagin Maximum Principle on optimal control system with delays, as described by Kharatishvili in [3], constitute a two- point boundary value problem which its solution is usually very difﬁcult due to the coupled nature of the solutions. In some papers [8, 9, 10, 17], orthogonal functions and polynomials, such that block-pulse and Walsh functions, Chebyshev and Legendre polynomials, were applied to ﬁnd the optimal control of TD systems. We can see the properties of Chebyshev polynomials in [12] produce an approximating function which minimizes error in its application. Chebyshev polynomials form a special class of polynomials especially suited for approximation theory. These polynomials as scaled Mother functions, are basis of Chebyshev wavelets [13]. In this paper, we introduce a direct method to solve the linear quadratic optimal control problems with delays and inverse time terms. The method consists of transforming the original optimal control problem to a quadratic programming (QP) one by using Chebyshev scaling functions and then solving the converted problem. We do not need a special program to solve the QP problem and many numerical methods are available to solve it. We can use 303 IMAN MALMIR the quadprog function provided by the optimization toolbox in MATLAB. This toolbox can solve constrained and unconstrained optimization problems. The proposed method can be applied to the system regardless of the stability and dimension of the system, the lack of smoothness of the input, the number of the delays, and the type of initial functions. Moreover, this is a powerful method for the problem with very large dimension and in the proposed procedure, we can easily change the time delays, the state, input, and weighting matrices of the system and impose the mixed constraints. 1. Introduction It is worth remarking that in the presented formulation for solving the optimal control of linear TD system, without changing many of the model parameters, we will be able to see the effect of changing the state and/or input delays on the system. The outline of this paper is as follows. In section 2 we introduce the basic formulation of Chebyshev wavelets required for the direct collocation method. Section 3 is devoted to the formulation of the TD optimal control problem containing inverse time. In Section 4, various numerical examples are solved to demonstrate applicability and effectiveness of the proposed method. 2.1. Deﬁnition and Function approximation Chebyshev polynomial of the ﬁrst kind Tm(x) deﬁned by Tm(x) = cos(m arccos(x)) is a polynomial in x of degree m which satisﬁes the equation (1 −x2)T ′′ m −xT ′ m + m2Tm = 0. Chebyshev polynomials form an orthogonal polynomial set on the interval [−1, 1] with respect to the weight w(x) = (1 −x2)−1/2. Some of the useful relations are R 1 −1 Tm(x)Tm′(x)w(x)dx =    π, m = m′ = 0 π 2 , m = m′ ̸= 0 0, m ̸= m′, Tm(x)Tm′(x) = 1 2 (Tm+m′(x) + Tm−m′(x)) , T m(x) = Tm(x), R 1 −1 Tm(x)Tm′(x)w(x)dx =    π, m = m′ = 0 π 2 , m = m′ ̸= 0 0, m ̸= m′, Tm(x)Tm′(x) = 1 2 (Tm+m′(x) + Tm−m′(x)) , T−m(x) = Tm(x), Tm(−x) = (−1)mTm(x). T−m(x) Tm(x), Tm(−x) = (−1)mTm(x). m( ) m( ), Tm(−x) = (−1)mTm(x). Chebyshev wavelets of the ﬁrst kind ψnm deﬁned on the interval [0, 1], have four arguments: k ∈N>1 speciﬁes the number of subintervals, n = 1, 2, . . . , 2k−1 refers to the segment number, m = 0, 1, . . . , M −1 is the degree of Tm(x) and t is as an independent variable denotes the time; Chebyshev wavelets of the ﬁrst kind ψnm deﬁned on the interval [0, 1], have four arguments: k ∈N>1 speciﬁes the number of subintervals, n = 1, 2, . . . , 2k−1 refers to the segment number, m = 0, 1, . . . , M −1 is the degree of Tm(x) and t is as an independent variable denotes the time; ψnm(t) = ( 2 k 2 ℘mTm(2kt −2n + 1) , n−1 2k−1 ≤t ≤ n 2k−1 0 , t < n−1 2k−1 , t > n 2k−1 , (1) (1) where where here ℘m = ( 1 √π, m = 0 q 2 π, m = 1, 2, 3, . . . , M −1. (2) ℘m = ( 1 √π, m = 0 q 2 π, m = 1, 2, 3, . . . , M −1. (2) (2) They form an orthogonal basis with respect to the weight function wn(t), where wn(t) = w(2kt −2n + 1). 2.1. Deﬁnition and Function approximation A function f(t) deﬁned over the interval [0, 1] can be approximated by Chebyshev wavelets as They form an orthogonal basis with respect to the weight function wn(t), where wn(t) = w(2kt −2n + 1). A function f(t) deﬁned over the interval [0, 1] can be approximated by Chebyshev wavelets as f(t) ∼= fk,M(t) = 2k−1 X n=1 M−1 X m=0 fnmψnm(t) = fΨ(t), (3) (3) where f⊤and Ψ(t) are 2k−1M × 1 column vectors, ⊤denotes transposition and where f⊤and Ψ(t) are 2k−1M × 1 column vectors, ⊤denotes transposition and f = [f10, f11, . . . , f1M−1, f20, . . . , f2M−1, . . . , f2k−10, . . . , f2k−1M−1], (4) Ψ(t) = [ψ10(t), ψ11(t), . . . , ψ1M−1(t), ψ20(t), . . . , ψ2M−1(t), . . . , ψ2k−10(t), . . . , ψ2k−1M−1(t)]⊤. (5) (4) Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 304 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS We need to to ﬁnd f; when t ∈[ n−1 2k−1 , n 2k−1 ], we have f(t) = PM−1 m=0 fnmψnm(t). With multiplying this equation by ψn′m′(t)wn′(t) and then integrating both sides from n−1 2k−1 to n 2k−1 , we ﬁnd We need to to ﬁnd f; when t ∈[ n−1 2k−1 , n 2k−1 ], we have f(t) = PM−1 m=0 fnmψnm(t). With multiplying this equation by ψn′m′(t)wn′(t) and then integrating both sides from n−1 2k−1 to n 2k−1 , we ﬁnd Z n 2k−1 n−1 2k−1 f(t)ψn′m′(t)wn′(t)dt = M−1 X m=0 fnm Z n 2k−1 n−1 2k−1 ψnm(t)ψn′m′(t)wn′(t)dt, Z n 2k−1 n−1 2k−1 f(t)ψn′m′(t)wn′(t)dt = M−1 X m=0 fnm Z n 2k−1 n−1 2k−1 ψnm(t)ψn′m′(t)wn′(t)dt, where m′ = 0, 1, . . . , M −1, n′ = 1, 2, . . . , 2k−1; the deﬁnition of Chebyshev wavelets in (1) demands that where m′ = 0, 1, . . . , M −1, n′ = 1, 2, . . . 2.1. Deﬁnition and Function approximation , 2k−1; the deﬁnition of Chebyshev wavelets in (1) demands that ψnm(t)ψn′m′(t) = 0 when n ̸= n′, (6) ψnm(t)ψn′m′(t) = 0 when n ̸= n′, (6) ψnm(t)ψn′m′(t) = 0 when n ̸= n′, (6) (6) t −2n + 1 by x in the right side for n′ = n we obtain thus by replacing 2kt −2n + 1 by x in the right side for n′ = n we obtain Z n 2k−1 n−1 2k−1 f(t)ψnm′(t)wn(t)dt = M−1 X m=0 fnm Z n 2k−1 n−1 2k−1 ψnm(t)ψnm′(t)wn(t)dt = M−1 X m=0 fnm℘m℘m′ Z 1 −1 Tm(x)Tm′(x)(1 −x2)−1/2dx = fnm℘2 m    π, m = m′ = 0 π 2 , m = m′ ̸= 0 0, m ̸= m′, so so fnm = Z n 2k−1 n−1 2k−1 f(t) ψnm(t)wn(t) dt. (7) fnm = Z n 2k−1 n−1 2k−1 f(t) ψnm(t)wn(t) dt. (7) (7) Theorem 1 Assume that a function f(t) is a δ-Lipschitz function on [0, 1], then f(t) can be expanded as f(t) = P∞ n=1 P∞ m=0 fnmψnm(t), where this series converges toward the function f(t). Theorem 1 Assume that a function f(t) is a δ-Lipschitz function on [0, 1], then f(t) can be expanded as f(t) = P∞ n=1 P∞ m=0 fnmψnm(t), where this series converges toward the function f(t). k 1 Theorem 2 (Error bound) Let f(t) be a δ-Lipschitz function on [0, 1], where f(t) = P2k−1 n=1 P∞ m=0 fnmψnm(t). Then for M > 1, we have the following error bound Theorem 2 (Error bound) Let f(t) be a δ-Lipschitz function on [0, 1], where f(t) = P2k−1 n=1 P∞ m=0 fnmψnm(t). Then for M > 1, we have the following error bound ∥f(t) −fk,M(t)∥2 wn≤π3δ2 192 . Proof: Because f(t) is a Lipschitz function, ∀t, ζ ∈[0, 1], \|f(t + ζ) −f(t)\| ≤δ\|ζ\|; we may write \| f(t+ζ)−f(t) ζ \| ≤δ, thus \|f ′(t)\| ≤δ. We know n−1 2k−1 ≤t ≤ n 2k−1 , this yields −1 ≤2kt −2n + 1 ≤1. Hence by substituting Proof: Because f(t) is a Lipschitz function, ∀t, ζ ∈[0, 1], \|f(t + ζ) −f(t)\| ≤δ\|ζ\|; we may write \| f(t+ζ)−f(t) ζ \| ≤δ, thus \|f ′(t)\| ≤δ. We know n−1 2k−1 ≤t ≤ n 2k−1 , this yields −1 ≤2kt −2n + 1 ≤1. 2.1. Deﬁnition and Function approximation Hence by substituting cos θ = 2kt −2n + 1 (8) (8) into formula (7) fnm = ℘m √ 2k Z π 0 f(cos θ + 2n −1 2k ) cos mθ dθ. (9) (9) o, after integration by parts for m ≥1 and some ζ, we get So, after integration by parts for m ≥1 and some ζ, we get \|fnm\| = ℘m m √ 2k f(cos θ + 2n −1 2k ) sin mθ π 0 + ℘m m √ 23k Z π 0 f ′(cos θ + 2n −1 2k ) sin mθ sin θ dθ ≤ ℘m m √ 23k π \|f ′(ζ)\| ≤ δ√π m √ 23k−1 . By using the fact that k ≥2 and the norm deﬁnition of a function, we can write By using the fact that k ≥2 and the norm deﬁnition of a function, we can write Stat., Optim. Inf. Comput. Vol. 5, December 2017 305 IMAN MALMIR ∥f(t) −fk,M(t)∥2 wn = Z 1 0 wn(t) f(t) − 2k−1 X n=1 M−1 X m=0 fnmψnm(t) !2 dt = Z 1 0 wn(t) 2k−1 X n=1 ∞ X m=0 fnmψnm(t) − 2k−1 X n=1 M−1 X m=0 fnmψnm(t) !2 dt = Z 1 0 wn(t) 2k−1 X n=1 ∞ X m=M fnmψnm(t) !2 dt = 2k−1 X n=1 ∞ X m=M f 2 nm ≤ 2k−1 X n=1 ∞ X m=M δ√π m √ 23k−1 2 ≤πδ2 32 ∞ X m=1 1 m2 = π3δ2 192 . 2.2. The product operational matrix of Chebyshev wavelets Let f be a row vector deﬁned in (4). Then the product operational matrix of two Chebyshev wavelet vectors ˜f is obtained from: Let f be a row vector deﬁned in (4). Then the product operational matrix of two Chebyshev wavelet vectors ˜f is obtained from: ˜ obtained from: fΨ(t)Ψ⊤(t) ∼= Ψ⊤(t)˜f (10) fΨ(t)Ψ⊤(t) ∼= Ψ⊤(t)˜f (10) fΨ(t)Ψ⊤(t) ∼= Ψ⊤(t)˜f fΨ(t)Ψ⊤(t) ∼= Ψ⊤(t)˜f (10) as a 2k−1M × 2k−1M matrix and can be found by the compact support property (6) in the form of a block diagonal matrix as rix and can be found by the compact support property (6) in the form of a block diagonal ˜f = blkdiag(˜f1,˜f2, . . . ,˜f2k−1). (11) ˜f = blkdiag(˜f1,˜f2, . . . ,˜f2k−1). (11) Assume ˜fn = [ ˜fnab] where a = m + 1 and b = m′ + 1. The M × M matrices ˜fn are determined by using (1) and the recurrence relation of Chebyshev polynomials on the left side of (10), and then equating coefﬁcients of same wavelet on both sides of (10). So from ψn0(t)ψnm′(t) = 2k/2 √π ψnm′(t) and the following ﬁndings: Assume ˜fn = [ ˜fnab] where a = m + 1 and b = m′ + 1. The M × M matrices ˜fn are determined by using (1) and the recurrence relation of Chebyshev polynomials on the left side of (10), and then equating coefﬁcients of same k/2 th sides of (10). So from ψn0(t)ψnm′(t) = 2k/2 √π ψnm′(t) and the following ﬁndings: ψnm(t) ψnm′(t) = 2k+1 π Tm(2kt −2n + 1)Tm′(2kt −2n + 1) = 2k/2 √π [ 2(k+1)/2 √ 2π Tm+m′(2kt −2n + 1) + 2(k+1)/2 √ 2π Tm−m′(2kt −2n + 1)] = 2k/2 √π 1 √ 2(ψnm+m′(t) + ψnm−m′(t)) when m, m′ ̸= 0, m + m′ ≤M −1 ψnm(t)ψnm′(t) = 2k/2 √π ( 1 √ 2ψn m+m′(t) + ψn0(t)) when m = m′, m + m′ ≤M −1 ψnm(t) ψnm′(t) ≈2k/2 √π ( 1 √ 2ψnm−m′(t)) when m, m′ ̸= 0, m + m′ ≥M we get g ˜fn = q 2k π   fn0 fn1 ··· ··· ··· ··· fnM−2 fnM−1 fn1 fn0+ 1 √ 2 fn2 ··· ··· ··· ··· 1 √ 2 (fnM−3+fnM−1) 1 √ 2 fnM−2 fn2 1 √ 2 (fn1+fn3) ··· ··· ··· ··· 1 √ 2 fnM−4 1 √ 2 fnM−3 ... ... ... ... ... ... ... ... 2.3. The delay operational matrix of Chebyshev wavelets 0 0 · · · 0 0 0 0 0 0     ndM columns (2k−1 −nd)M rows 0 0 · · · 0 1 0 0 · · · 0 0 0 · · · 0 0 1 0 · · · 0 0 0 · · · 0 0 0 1 · · · 0 ... ... ... ... ... ... ... ... ... 0 0 · · · 0 0 0 0 · · · 1 0 0 · · · 0 0 0 0 · · · 0 ... ... ... ... ... ... ... ... ... 0 0 · · · 0 0 0 0 0 0     ndM columns (2k−1 −nd)M rows thus this matrix can be written compactly as thus this matrix can be written compactly as D = 02k−1M×ndM I(2k−1−nd)M 0ndM×(2k−1−nd)M . (15) D = 02k−1M×ndM I(2k−1−nd)M 0ndM×(2k−1−nd)M . (15) (15) Throughout this paper we use 0 and I to denote the zero and identity matrices, respectively. Throughout this paper we use 0 and I to denote the zero and identity matrices, respectively. Throughout this paper we use 0 and I to denote the zero and identity matrices, respectively. 2.2. The product operational matrix of Chebyshev wavelets fnM−1−ν ··· ··· fn0+ 1 √ 2 fnµ 1 √ 2 (fn1+fnξ) ··· 1 √ 2 fnv−1 1 √ 2 fnv fnM−ν ··· ··· 1 √ 2 (fn1+fnξ) fn0 ··· ··· 1 √ 2 fnv−1 ··· ··· ··· 1 √ 2 fn2 1 √ 2 fn1 ··· ··· ··· ... ... ... ... ... ... ... ... fnM−2 1 √ 2 (fnM−3+fnM−1) ··· ··· ··· ··· fn0 1 √ 2 fn1 fnM−1 1 √ 2 fnM−2 ··· ··· ··· ··· 1 √ 2 fn1 fn0   , (12) Stat., Optim. Inf. Comput. Vol. 5, December 2017 ··· fnM−2 fnM−1 ··· 1 √ 2 (fnM−3+fnM−1) 1 √ 2 fnM−2 ··· 1 √ 2 fnM−4 1 √ 2 fnM−3 ... ... ... +fnξ) ··· 1 √ 2 fnv−1 1 √ 2 fnv ··· ··· 1 √ 2 fnv−1 1 ··· ··· ··· ... ... ... ··· fn0 1 √ 2 fn1 ··· 1 √ 2 fn1 fn0   , (12) Stat., Optim. Inf. Comput. Vol. 5, December 2017 (12) fn0 1 √ 2 fn1 Stat., Optim. Inf. Comput. Vol. 5, December 2017 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS 306 where where µ = M −2, M even M −1, M odd, v = M/2, M even (M −1)/2, M odd, and fnξ = fnM−1, M even 0, M odd. 2.3. The delay operational matrix of Chebyshev wavelets The delay Chebyshev scaling function Ψ(t −td) is the shifted function of Ψ(t) along the t-axis and it is found to be as The delay Chebyshev scaling function Ψ(t −td) is the shifted function of Ψ(t) along the t-axis be as The delay Chebyshev scaling function Ψ(t −td) is the shifted function of Ψ(t) along the t-axis and it is found to be as Ψ(t −td) = 0, 0 ≤t < td DΨ(t), td ≤t ≤1, (13) (13) where D is the 2k−1M × 2k−1M delay operational matrix of Chebyshev wavelets. We use (1) to compute D; assuming 2k−1td ∈N, we deﬁne nd by k 1 1M × 2k−1M delay operational matrix of Chebyshev wavelets. We use (1) to compute D; N, we deﬁne nd by (14) nd = 2k−1td. (14) Note that if tf ̸= 1 then we let t/tf →t. From (1) it follows that when td ≤t ≤1, for ψnm(t −td) we have n−1 2k−1 ≤t −td ≤ n 2k−1 . Substituting td = nd 2k−1 into this inequality, yields nd+n−1 2k−1 ≤t ≤nd+n 2k−1 . Therefore, Note that if tf ̸= 1 then we let t/tf →t. From (1) it follows that when td ≤t ≤1, for ψnm(t −td) we have n−1 2k−1 ≤t −td ≤ n 2k−1 . Substituting td = nd 2k−1 into this inequality, yields nd+n−1 2k−1 ≤t ≤nd+n 2k−1 . Therefore, ψnm(t −td) = 2 k 2 ℘mTm(2k(t −td) −2n + 1) = 2 k 2 ℘mTm(2kt −2(nd + n) + 1) = ψnd+nm(t). If n > 2k−1 −nd, then ψnm(t −td) = 0. Consequently D is a sparse matrix and has the following structure: If n > 2k−1 −nd, then ψnm(t −td) = 0. Consequently D is a sparse matrix and has the following structure: If n > 2k−1 −nd, then ψnm(t −td) = 0. Consequently D is a sparse matrix and has the following structure: 0 0 · · · 0 1 0 0 · · · 0 0 0 · · · 0 0 1 0 · · · 0 0 0 · · · 0 0 0 1 · · · 0 ... ... ... ... ... ... ... ... ... 0 0 · · · 0 0 0 0 · · · 1 0 0 · · · 0 0 0 0 · · · 0 ... ... ... ... ... ... ... ... ... 2.4. The inverse time operational matrix of Chebyshev wavelets Some linear time-varying systems comprise the expressions x(tf −t) and/or u(tf −t) in their state equations. For expanding these terms by Chebyshev scaling functions we can write (by setting t/tf →t) Ψ(1 −t) = ΥΨ(t) , 0 ≤t ≤1 (16) Ψ(1 −t) = ΥΨ(t) , 0 ≤t ≤1 (16) Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 IMAN MALMIR 307 307 IMAN MALMIR where we denote the inverse time operational matrix of Chebyshev wavelets by Υ. To ﬁnd this matrix, we can write ψnm(1 −t) = 2 k 2 ℘mTm(2k(1 −t) −2n + 1) , n−1 2k−1 ≤1 −t ≤ n 2k−1 . where we denote the inverse time operational matrix of Chebyshev wavelets by Υ. To ﬁnd this where we denote the inverse time operational matrix of Chebyshev wavelets by Υ. To ﬁnd this matrix, we can write ( ) k ( k( ) ) n 1 n e inverse time operational matrix of Chebyshev wavelets by Υ. To ﬁnd this matrix, we can write ψnm(1 −t) = 2 k 2 ℘mTm(2k(1 −t) −2n + 1) , n−1 2k−1 ≤1 −t ≤ n 2k−1 . ψnm(1 −t) = 2 k 2 ℘mTm(2k(1 −t) −2n + 1) , n−1 2k−1 ≤1 −t ≤ n 2k−1 . Now by deﬁning Now by deﬁning ni = 2k−1 −n + 1, from n−1 2k−1 ≤1 −t ≤ n 2k−1 we see that ni−1 2k−1 ≤t ≤ ni 2k−1 . Hence ψnm(1 −t) = 2 k 2 ℘mTm(−2kt + 2k −2n + 1) = 2 k 2 ℘mTm(−2kt + 2ni −1) = 2 k 2 ℘mTm(−[2kt −2ni + 1]) = (−1)mψnim(t). In other words, when n = 1, 2, . . . , 2k−1 we have ni = 2k−1, 2k−1 −1, . . . , 1. So the 2k−1M × 2k−1M matrix Υ is a block anti-diagonal matrix of the form In other words, when n = 1, 2, . . . , 2k−1 we have ni = 2k−1, 2k−1 −1, . . . , 1. So the 2k−1M × 2k−1M matrix Υ is a block anti-diagonal matrix of the form Υ =   0 · · · 0 Z 0 · · · Z 0 ... ... ... ... Z · · · 0 0  , (17) Υ =   0 · · · 0 Z 0 · · · Z 0 ... ... ... ... Z · · · 0 0  , (17) (17) in which Z is a M × M diagonal matrix as Z =   1 0 ··· 0 0 0 −1 ··· 0 0 ... ... ... ... ... 0 0 ··· (−1)M−2 0 0 0 ··· 0 (−1)M−1  . (18) Z =   1 0 ··· 0 0 0 −1 ··· 0 0 ... ... ... ... ... 0 0 ··· (−1)M−2 0 0 0 ··· 0 (−1)M−1  . (18) (18) 2.5. The operational matrix of Chebyshev wavelets for integration 2.5. The operational matrix of Chebyshev wavelets for integration 2.5. The operational matrix of Chebyshev wavelets for integration 2.5. The operational matrix of Chebyshev wavelets for integration The integration of the Chebyshev wavelet vector on the interval [0, t] can be obtained as Z t 0 Ψ(ι)dι ∼= PΨ(t). (19) Z t 0 Ψ(ι)dι ∼= PΨ(t). (19) (19) The matrix P is called the operational matrix of Chebyshev wavelets for integration, where p y g P = 1 2k   P1 P2 ... P2k−1  , Pn = 0 0 · · · 0 \| {z } (n−1) times Y J J · · · J \| {z } (2k−1−n) times . (20) Y and J are M × M matrices given by Y =   1 1 √ 2 0 0 ··· 0 0 − √ 2 4 0 1 4 0 ··· 0 0 − √ 2 3 −1 2 0 1 6 ··· 0 0 √ 2 8 0 −1 4 0 ··· 0 0 ... ... ... ... ... ... ... (−1)M−1√ 2 (M−2)2−1 0 0 0 ··· 0 1 2(M−1) (−1)M√ 2 (M−1)2−1 0 0 0 ··· − 1 2(M−2) 0   , J =   2 0 0 ··· 0 0 0 0 ··· 0 −2 √ 2 3 0 0 ··· 0 0 0 0 ··· 0 ... ... ... ... ... −(1+(−1)m) √ 2 m2−1 0 0 ··· 0 ... ... ... ... ... −(1+(−1)M−1) √ 2 (M−1)2−1 0 0 ··· 0   . (21) Stat., Optim. Inf. Comput. Vol. 5, December 2017 P = 1 2k   P1 P2 ... P2k−1  , Pn = 0 0 · · · 0 \| {z } (n−1) times Y J J · · · J \| {z } (2k−1−n) times . (20) (20) Y and J are M × M matrices given by Y =   1 1 √ 2 0 0 ··· 0 0 − √ 2 4 0 1 4 0 ··· 0 0 − √ 2 3 −1 2 0 1 6 ··· 0 0 √ 2 8 0 −1 4 0 ··· 0 0 ... ... ... ... ... ... ... 2.6. The integration matrix of the product operational matrix of Chebyshev wavelets We need to ﬁnd the integration matrix of the product of two Chebyshev scaling function vectors on the time interval [0, 1]. By deﬁning 1 L = Z 1 0 Ψ(t)Ψ⊤(t)dt (22) (22) and using the properties of Chebyshev polynomials, we deduce that L is a 2k−1M × 2k−1M symmetric matrix and can be obtained by integrating the entries of Ψ(t)Ψ⊤(t) from 0 to 1. This may be done by introducing the auxiliary row vector φn(t) = [ψn0(t), ψn1(t), . . . , ψnM−1(t)] and using (1) as follows, L = Z 1 2k−1 0   φ⊤ 1 (t)φ1(t) 0 ··· 0 0 0 ··· 0 ... ... ... ... 0 0 ··· 0  dt + Z 2 2k−1 1 2k−1   0 0 ··· 0 0 φ⊤ 2 (t)φ2(t) ··· 0 ... ... ... ... 0 0 ··· 0  dt + · · · + Z 1 2k−1−1 2k−1   0 0 ··· 0 0 0 ··· 0 ... ... ... ... 0 0 ··· φ⊤ 2k−1 (t)φ2k−1 (t)  dt. This yields This yields 2.5. The operational matrix of Chebyshev wavelets for integration (−1)M−1√ 2 (M−2)2−1 0 0 0 ··· 0 1 2(M−1) (−1)M√ 2 (M−1)2−1 0 0 0 ··· − 1 2(M−2) 0   Y =   1 1 √ 2 0 0 ··· 0 0 − √ 2 4 0 1 4 0 ··· 0 0 − √ 2 3 −1 2 0 1 6 ··· 0 0 √ 2 8 0 −1 4 0 ··· 0 0 ... ... ... ... ... ... ... (−1)M−1√ 2 (M−2)2−1 0 0 0 ··· 0 1 2(M−1) (−1)M√ 2 (M−1)2−1 0 0 0 ··· − 1 2(M−2) 0   , J =   2 0 0 ··· 0 0 0 0 ··· 0 −2 √ 2 3 0 0 ··· 0 0 0 0 ··· 0 ... ... ... ... ... −(1+(−1)m) √ 2 m2−1 0 0 ··· 0 ... ... ... ... ... −(1+(−1)M−1) √ 2 (M−1)2−1 0 0 ··· 0   . (21) (21) Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS 308 L = blkdiag(L1, L2, . . . , L2k−1). L = blkdiag(L1, L2, . . . , L2k−1). We let Ln = [lnab]M×M, where a and b are the same as before. When m = m′ = 0, we simply have l11 = 2 π; otherwise, from (8) we see that lnab = Z n 2k−1 n−1 2k−1 ψnm(t)ψnm′(t)dt = λmm′ Z π 0 cos mθ cos m′θ sin θ dθ, lnab = Z n 2k−1 n−1 2k−1 ψnm(t)ψnm′(t)dt = λmm′ Z π 0 cos mθ cos m′θ sin θ dθ, where where where λmm′ = 2 π, m, m′ ̸= 0 √ 2 π , m = 0 or m′ = 0. When m + m′ = 1, then we get lnab = 0; when \|m −m′\| = 1, lnab = 0. Otherwise, \|m ± m′\| ̸= 1 and we can write λ [ 1 1 ( ′ ) 1 1 where λmm′ = 2 π, m, m′ ̸= 0 √ 2 π , m = 0 or m′ = 0. When m + m′ = 1, then we get lnab = 0; when \|m −m′\| = 1, lnab = 0. Otherwise, \|m ± m′\| ̸= 1 and we can write lnab = λmm′ 4 [ 1 m+m′+1 − 1 m+m′−1 cos(mπ + m′π) + 1 m+m′+1 − 1 m+m′−1 + 1 m−m′+1 − 1 m−m′−1 cos(mπ + m′π) + 1 m−m′+1 − 1 m−m′−1]. λmm′ = 2 π, m, m′ ̸= 0 √ 2 π , m = 0 or m′ = 0. λmm′ = 2 π, m, m′ ̸= 0 √ 2 π , m = 0 or m′ = 0. = 0; when \|m −m′\| = 1, lnab = 0. Otherwise, \|m ± m′\| ̸= 1 and we can write λmm′ = 2 π, m, m′ ̸= 0 √ 2 π , m = 0 or m′ = 0. π , m = 0 or m = 0. When m + m′ = 1, then we get lnab = 0; when \|m −m′\| = 1, lnab = 0. Otherwise, \|m ± m′\| ̸= 1 and we can write π When m + m′ = 1, then we get lnab = 0; when \|m −m′\| = 1, lnab = 0. Otherwise, \|m ± m′\| ̸= 1 and we can write 1, then we get lnab = 0; when \|m −m′\| = 1, lnab = 0. 3.1. Problem statement and Approximation process In this section we use the above-mentioned results to model a linear quadratic TD control problem as a QP one. Consider a linear TD system t) + F(t)x(t −tdx) + G(t)u(t) + H(t)u(t −tdu) + V(t)x(tf −t) + W(t)u(tf −t), 0 ≤t ≤tf (2 ˙x(t) = E(t)x(t) + F(t)x(t −tdx) + G(t)u(t) + H(t)u(t −tdu) + V(t)x(tf −t) + W(t)u(tf − (26) x(0) = x0, (27) x(t) = α(t), −tdx ≤t ≤0 u(t) = β(t), −tdu ≤t ≤0 (28) x(t) = α(t), −tdx ≤t ≤0 u(t) = β(t), −tdu ≤t ≤0 (28) (28) with a quadratic performance index (PI) or cost functional uadratic performance index (PI) or cost functional with a quadratic performance index (PI) or cost functional with a quadratic performance index (PI) or cost functional J = 1 2x⊤(tf)Tx(tf) + 1 2 Z tf 0 x⊤(t) Q(t) x(t) + u⊤(t) R(t) u(t) dt, (29) (29) where x(t) ∈Rq and u(t) ∈Rr are state and control vectors, E(t), F(t), G(t), H(t), V(t) and W(t) are piecewise- continuous matrices of compatible dimensions, tdx and tdu represent state and control delays, respectively, x0 is an initial condition vector, α(t) and β(t) are, respectively, q- and r-dimensional initial state and initial control vector functions, T and Q(t) are positive semi-deﬁnite matrices and R(t) is a positive deﬁnite matrix. The problem is to ﬁnd u∗(t), x∗(t) and J∗such that the PI in (29) is minimized while satisfying Eqs. (26)–(28), where ∗indicates optimal value. ﬁnite m (29) p First we approximate the system dynamics. Since Chebyshev wavelets are deﬁned on [0, 1], it makes sense to deﬁne a new variable ϱ by ϱ = t/tf; replacing t by ϱ, the state equation (26) becomes ˙x(ϱ) = tf(E(ϱ)x(ϱ) + F(ϱ)x(ϱ −ϱx) + G(ϱ)u(ϱ) + H(ϱ)u(ϱ −ϱu) + V(ϱ)x(1 −ϱ) + W(ϱ)u(1 −ϱ)), 0 ≤ϱ ≤1 (30) where ϱx = tdx/tf and ϱu = tdu/tf. By integrating (30) from 0 to ϱ and rearranging, we get x(ϱ) −x0 = tf Z ϱ 0 {E(ι)x(ι) + G(ι)u(ι) + F(ι)x(ι −ϱx) + H(ι)u(ι −ϱu) + V(ι)x(1 −ι) + W(ι)u(1 −ι)} dι. 3. QP representation of an optimal control problem 3.1. Problem statement and Approximation process L = blkdiag(L1, L2, . . . , L2k−1). Otherwise, \|m ± m′\| ̸= 1 and we can wri lnab = λmm′ 4 [ 1 m+m′+1 − 1 m+m′−1 cos(mπ + m′π) + 1 m+m′+1 − 1 m+m′−1 + 1 m−m′+1 − 1 m−m′−1 cos(mπ + m′π) + 1 m−m′+1 − 1 m−m′−1]. lnab = λmm′ 4 [ 1 m+m′+1 − 1 m+m′−1 cos(mπ + m′π) + 1 m+m′+1 − 1 m+m′−1 + 1 m−m′+1 − 1 m−m′−1 cos(mπ + m′π) + 1 m−m′+1 − 1 m−m′−1]. Clearly one can see that when m + m′ is odd, then lnab = 0. It is apparent the values of the entries lnab of Ln depend not on n but only on m and m′, thus L1 = L2 = L3 = · · · = L2k−1. We take Ln = LM = [lab]. Hence the integration matrix of the product of two Chebyshev wavelet vectors takes the form L = blkdiag(LM, LM, . . . , LM \| {z } 2k−1 times ), (23) L = blkdiag(LM, LM, . . . , LM \| {z } 2k−1 times ), (23) (23) where lab = ( λ (1−(a−1)2−(b−1)2) ((a−b)2−1)((a+b−2)2−1), (a + b) even 0, (a + b) odd, and λ =    2 π, a = b = 1 2 √ 2 π , a = 1 or b = 1 4 π, a, b ̸= 1. (24) (24) For instance if M = 8, then For instance if M = 8, then For instance if M = 8, then (25) Stat., Optim. Inf. Comput. Vol. 5, December 2017 309 IMAN MALMIR 309 IMAN MALMIR 3.1. Problem statement and Approximation process (31) ˙x(ϱ) = tf(E(ϱ)x(ϱ) + F(ϱ)x(ϱ −ϱx) + G(ϱ)u(ϱ) + H(ϱ)u(ϱ −ϱu) + V(ϱ)x(1 −ϱ) + W(ϱ)u(1 −ϱ)), 0 ≤ϱ ≤1 (30) h / d / B i i (30) f 0 d i (30) (31) ( ) In the approximation process we will use the symbolsˆand¯placed on top of a matrix to denote Kronecker product of the matrix and Iq and Kronecker product of the matrix and Ir, respectively. We parameterize the state and control vectors as x(ϱ) ∼= ˆΨ⊤(ϱ)X, (32) u(ϱ) ∼= ¯Ψ⊤(ϱ)U, (33) (32) (33) where X and U are 2k−1qM × 1 and 2k−1rM × 1 column vectors of unknown parameters and represented by X = [X1 10, . . . , Xq 10, . . . , X1 1M−1, . . . , Xq 1M−1, . . . , X1 2k−1M−1, . . . , Xq 2k−1M−1]⊤, (34) U [U 1 U r U 1 U r U 1 U r ]⊤ (35) where X and U are 2k−1qM × 1 and 2k−1rM × 1 column vectors of unknown parameters and represented by X = [X1 10, . . . , Xq 10, . . . , X1 1M−1, . . . , Xq 1M 1, . . . , X1 2k−1M 1, . . . , Xq 2k−1M 1]⊤, (34) where X and U are 2k−1qM × 1 and 2k−1rM × 1 column vectors of unknown parameters and represented by re 2k−1qM × 1 and 2k−1rM × 1 column vectors of unknown parameters and represented by where X and U are 2k 1qM × 1 and 2k 1rM × 1 column vectors of unknown parameters and represented by X = [X1 10, . . . , Xq 10, . . . , X1 1M−1, . . . , Xq 1M−1, . . . , X1 2k−1M−1, . . . , Xq 2k−1M−1]⊤, (34) U = [U 1 10, . . . , U r 10, . . . , U 1 1M−1, . . . , U r 1M−1, . . . , U 1 2k−1M−1, . . . , U r 2k−1M−1]⊤. (35) (34) (35) oal is to ﬁnd X and U from the optimization. Using (3), the initial state can be expanded in term l t Our immediate goal is to ﬁnd X and U from the optimization. 3.1. Problem statement and Approximation process Using (3), the initial state can be expanded in terms of Chebyshev wavelets as of Chebyshev wavelets as x0 = ˆΨ⊤(ϱ)X0, x0 = ˆΨ⊤(ϱ)X0, (36) (36) where we have where we have X0 = [ 2k−1 times z }\| { x0, x0, · · · , x0]⊤, x0 = √π 2k/2 [x1(0), x2(0), . . . , xq(0), q(M−1) z }\| { 0, 0, . . . , 0]. (37) (37) Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS 310 Also for the matrices E(ϱ) and G(ϱ) we can write Also for the matrices E(ϱ) and G(ϱ) we can write E(ϱ) = [E10, E11, . . . , E1M−1, E20, . . . , E2M−1, . . . , E2k−10, . . . , E2k−1M−1] ˆΨ(ϱ) = E ˆΨ(ϱ), E(ϱ) = [E10, E11, . . . , E1M−1, E20, . . . , E2M−1, . . . , E2k−10, . . . , E2k−1M−1] ˆΨ(ϱ) = E ˆΨ(ϱ), G(ϱ) = [G10, G11, . . . , G1M−1, G20, . . . , G2M−1, . . . , G2k−10, . . . , G2k−1M−1] ¯Ψ(ϱ) = G ¯Ψ(ϱ) = EΨ(ϱ), G(ϱ) = [G10, G11, . . . , G1M−1, G20, . . . , G2M−1, . . . , G2k−10, . . . , G2k−1M−1] ¯Ψ(ϱ) = G ¯Ψ(ϱ), G(ϱ) = [G10, G11, . . . , G1M−1, G20, . . . , G2M−1, . . . , G2k−10, . . . , G2k−1M−1] ¯Ψ(ϱ) = G ¯Ψ(ϱ), where [Enm] and [Gnm] are q × 2k−1qM and q × 2k−1rM matrices of constants and we take E = [Enm] and G = [Gnm]. To illustrate, suppose   where [Enm] and [Gnm] are q × 2k−1qM and q × 2k−1rM matrices of constants and we take E = [Enm] and G = [Gnm]. To illustrate, suppose   [ ] [ ] G = [Gnm]. To illustrate, suppose [ nm] , pp E(ϱ) =   e11(ϱ) e12(ϱ) ··· e1q(ϱ) e21(ϱ) e22(ϱ) ··· e2q(ϱ) ... ... ... ... eq1(ϱ) eq2(ϱ) ··· eqq(ϱ)  . By (9), we ﬁnd Enm =   e11nm e12nm ··· e1qnm e21nm e22nm ··· e2qnm ... ... ... ... 3.1. Problem statement and Approximation process eq1nm eq2nm ··· eqqnm  ; R π θ+2 1 [ ] E(ϱ) =   e11(ϱ) e12(ϱ) ··· e1q(ϱ) e21(ϱ) e22(ϱ) ··· e2q(ϱ) ... ... ... ... eq1(ϱ) eq2(ϱ) ··· eqq(ϱ)  . By (9), we ﬁnd Enm =   e11nm e12nm ··· e1qnm e21nm e22nm ··· e2qnm ... ... ... ... eq1nm eq2nm ··· eqqnm  ; in terms of the symbolism,   for E(ϱ) = [eij(ϱ)] we have Enm = [eijnm] where eijnm = ℘m √ 2k R π 0 eij( cos θ+2n−1 2k ) cos mθ dθ, i, j = 1, 2, . . . , q. Similarly, for G(ϱ) = [giκ(ϱ)] we get Gnm = [giκnm] where giκnm = ℘m √ 2k R π 0 giκ( cos θ+2n−1 2k ) cos mθ dθ, κ = 1, 2, . . . , r. In like manner we take F(ϱ) = [Fnm] ˆΨ(ϱ) = F ˆΨ(ϱ), V(ϱ) = [Vnm] ˆΨ(ϱ) = V ˆΨ(ϱ), H(ϱ) = [Hnm] ¯Ψ(ϱ) = H ¯Ψ(ϱ), W(ϱ) = [Wnm] ¯Ψ(ϱ) = W ¯Ψ(ϱ), where F, V, H and W are constant matrices of dimensions q × 2k−1qM, q × 2k−1qM, q × 2k−1rM and q × 2k−1rM, respectively. Now using (10) and (19), we see that where F, V, H and W are constant matrices of dimensions q × 2k−1qM, q × 2k−1qM, q × 2k−1rM and q × 2k−1rM, respectively. Now using (10) and (19), we see that Z ϱ 0 E(ι)x(ι)dι = Z ϱ 0 E ˆΨ(ι) ˆΨ⊤(ι)Xdι = Z ϱ 0 ˆΨ⊤(ι)˜EXdι = ˆΨ⊤(ϱ)ˆP ⊤˜EX. (38) (38) Likewise, by also knowing G ¯Ψ(ϱ) ¯Ψ⊤(ϱ)U = IqG(Ψ(ϱ) ⊗Ir)(Ψ⊤(ϱ) ⊗Ir)U = (Ψ⊤(ϱ) ⊗Iq) ˜GU, where ⊗ denotes Kronecker product, we ﬁnd Likewise, by also knowing G ¯Ψ(ϱ) ¯Ψ⊤(ϱ)U = IqG(Ψ(ϱ) ⊗Ir)(Ψ⊤(ϱ) ⊗Ir)U = (Ψ⊤(ϱ) ⊗Iq) ˜GU, where ⊗ denotes Kronecker product, we ﬁnd G(ι)u(ι)dι = Z ϱ 0 G ¯Ψ(ι) ¯Ψ⊤(ι)Udι = Z ϱ 0 ˆΨ⊤(ι) ˜GUdι = ˆΨ⊤(ϱ)ˆP ⊤˜GU. (39) (39) Assume that α(ϱ) = [α1(ϱ), α2(ϱ), . . . , αq(ϱ)] and β(ϱ) = [β1(ϱ), β2(ϱ), . . . , βr(ϱ)]. By (13) we can expand x(ϱ −ϱx) and u(ϱ −ϱu) in terms of Chebyshev scaling function. Doing this gives, in turn, Assume that α(ϱ) = [α1(ϱ), α2(ϱ), . . . , αq(ϱ)] and β(ϱ) = [β1(ϱ), β2(ϱ), . . . , βr(ϱ)]. 3.1. Problem statement and Approximation process By (13) we can expand x(ϱ −ϱx) and u(ϱ −ϱu) in terms of Chebyshev scaling function. Doing this gives, in turn, x(ϱ −ϱx) = ( α(ϱ −ϱx), 0 ≤ϱ ≤ϱx ˆΨ⊤(ϱ)ˆD ⊤ x X, ϱx ≤ϱ ≤1 = ( ˆΨ⊤(ϱ)A, 0 ≤ϱ ≤ϱx ˆΨ⊤(ϱ)ˆD ⊤ x X, ϱx ≤ϱ ≤1, (40) u(ϱ −ϱu) = β(ϱ −ϱu), 0 ≤ϱ ≤ϱu ¯Ψ⊤(ϱ)¯D ⊤ u U, ϱu ≤ϱ ≤1 = ¯Ψ⊤(ϱ)B, 0 ≤ϱ ≤ϱu ¯Ψ⊤(ϱ)¯D ⊤ u U, ϱu ≤t ≤1. (41) (40) (41) A and B are 2k−1qM × 1 and 2k−1rM × 1 column vectors of constants; here we deﬁne them as follows A = [A1 10, . . . , Aq 10, . . . , A1 1M−1, . . . , Aq 1M−1, . . . , A1 ndxM−1, . . . , Aq ndxM−1, (2 −ndx)qM z }\| { 0, 0, 0, . . . , 0 ]⊤, (42) B = [B1 10, . . . , Br 10, . . . , B1 1M−1, . . . , Br 1M−1, . . . , B1 nduM−1, . . . , Br nduM−1, (2k−1−ndu)rM z }\| { 0, 0, 0, . . . , 0 ]⊤, (43) (42) B = [B1 10, . . . , Br 10, . . . , B1 1M−1, . . . , Br 1M−1, . . . , B1 nduM−1, . . . , Br nduM−1, z }\| { 0, 0, 0, . . . , 0 ]⊤ (43) in which Ai n′m = ℘m √ 2k R π 0 αi( cos θ+2n′−1 2k −ϱx) cos mθ dθ, Bκ n′′m = ℘m √ 2k R π 0 βκ( cos θ+2n′′−1 2k −ϱu) cos mθ dθ for n′ = 1, 2, . . . , ndx, n′′ = 1, 2, . . . , ndu, and ndx and ndu are obtained by (14) as ndx = 2k−1ϱx and ndu = 2k−1ϱu. According to (42) and (43) we can rewrite (40) and (41), in turn, as x(ϱ −ϱx) = ˆΨ⊤(ϱ)A + ˆΨ⊤(ϱ)ˆD ⊤ x X and Stat., Optim. Inf. Comput. Vol. 3.1. Problem statement and Approximation process 5, December 2017 IMAN MALMIR 311 311 IMAN MALMIR u(ϱ −ϱu) = ¯Ψ⊤(ϱ)B + ¯Ψ⊤(ϱ)¯D ⊤ u U; so by (10) and (19) Z ϱ 0 F(ι)x(ι −ϱx)dι = Z ϱ 0 F ˆΨ(ι) ˆΨ⊤(ι)A + F ˆΨ(ι) ˆΨ⊤(ι)ˆD ⊤ x X dι = ˆΨ⊤(ϱ)ˆP ⊤˜FA + ˆΨ⊤(ϱ)ˆP ⊤˜FˆD ⊤ x X, (44) Z ϱ 0 H(ι)u(ι −ϱu)dι = Z ϱ 0 H ¯Ψ(ι) ¯Ψ⊤(ι)B + H ¯Ψ(ι) ¯Ψ⊤(ι)¯D ⊤ u U dι = ˆΨ⊤(ϱ)ˆP ⊤˜HB + ˆΨ⊤(ϱ)ˆP ⊤˜H¯D ⊤ u U. (45) (45) (45) Z 0 Z 0 (45) Finally, using (16), (10) and (19) for the inverse time terms yields (45) Finally, using (16), (10) and (19) for the inverse time terms yields Finally, using (16), (10) and (19) for the inverse time terms yields Z ϱ 0 V(ι)x(1 −ι)dι = Z ϱ 0 V ˆΨ(ι) ˆΨ⊤(ι) ˆΥ⊤Xdι = ˆΨ⊤(ϱ)ˆP ⊤˜V ˆΥ⊤X, (46) Z ϱ 0 W(ι)u(1 −ι)dι = Z ϱ 0 W ¯Ψ(ι) ¯Ψ⊤(ι) ¯Υ⊤Udι = ˆΨ⊤(ϱ)ˆP ⊤˜W ¯Υ⊤U. (47) (46) (47) Substituting these ﬁndings into (31) gives us ˆΨ⊤(ϱ)X −ˆΨ⊤(ϱ)X0 = tf( ˆΨ⊤(ϱ)ˆP ⊤˜EX + ˆΨ⊤(ϱ)ˆP ⊤˜GU + ˆΨ⊤(ϱ)ˆP ⊤˜FA + ˆΨ⊤(ϱ)ˆP ⊤˜FˆD ⊤ x X + ˆΨ⊤(ϱ)ˆP ⊤˜HB + ˆΨ⊤(ϱ)ˆP ⊤˜H¯D ⊤ u U + ˆΨ⊤(ϱ)ˆP ⊤˜V ˆΥ⊤X + ˆΨ⊤(ϱ)ˆP ⊤˜W ¯Υ⊤U). (48) As a result −X0 −tf ˆP ⊤˜FA −tf ˆP ⊤˜HB = tf(ˆP ⊤˜E + ˆP ⊤˜FˆD ⊤ x + ˆP ⊤˜V ˆΥ⊤) −Iqs X + tf( ˆP ⊤˜G + ˆP ⊤˜H¯D ⊤ u + ˆP ⊤˜W ¯Υ⊤)U, (49) −X0 −tf ˆP ⊤˜FA −tf ˆP ⊤˜HB = tf(ˆP ⊤˜E + ˆP ⊤˜FˆD ⊤ x + ˆP ⊤˜V ˆΥ⊤) −Iqs X + tf( ˆP ⊤˜G + ˆP ⊤˜H¯D ⊤ u + ˆP ⊤˜W ¯Υ⊤)U, (49) (49) where s = 2k−1M and ˜E, ˜F, ˜V, ˜G, ˜H and ˜W are of dimensions qs × qs, qs × qs, qs × qs, qs × rs, qs × rs and qs × rs, respectively and can be obtained from (11) and (12). For expample, ˜E = blkdiag(˜E1, ˜E2, ..., ˜E2k−1), where E E (49) where s = 2k−1M and ˜E, ˜F, ˜V, ˜G, ˜H and ˜W are of dimensions qs × qs, qs × qs, qs × qs, qs × rs, qs × rs and qs × rs, respectively and can be obtained from (11) and (12). 3.1. Problem statement and Approximation process For expample, ˜E = blkdiag(˜E1, ˜E2, ..., ˜E2k−1), where ˜En = q 2k π   En0 En1 ··· En1 En0+ 1 √ 2 En2 ··· ... ... ...  . ˜En = q 2k π   En0 En1 ··· En1 En0+ 1 √ 2 En2 ··· ... ... ...  . . . Now we approximate the terminal cost function in addition to the integral cost function (Bolza type) by writing the weighting matrices as Q(ϱ) = Q ˆΨ(ϱ) and R(ϱ) = R ¯Ψ(ϱ) like E(ϱ) and G(ϱ) and using(10): J = 1 2X⊤ˆΨ(1)T ˆΨ⊤(1)X + 1 2tf Z 1 0 n X⊤ˆΨ(ϱ)Q ˆΨ(ϱ) ˆΨ⊤(ϱ)X + U⊤¯Ψ(ϱ)R ¯Ψ(ϱ) ¯Ψ⊤(ϱ)U o dϱ = 1 2X⊤(Ψ(1)Ψ⊤(1) ⊗T)X + 1 2tf Z 1 0 n X⊤(Ψ(ϱ)Ψ⊤(ϱ) ⊗Iq) ˜QX + U⊤(Ψ(ϱ)Ψ⊤(ϱ) ⊗Ir)˜RU o dϱ, (50) (50) in which ˜Q and ˜R are obtained by using (11) and (12) similarly to ˜E. Finally, form (22), it follows that J = 1 2 X U ⊤ tf ˆL ˜Q + (Ψ(1)Ψ⊤(1) ⊗T) 0 0 tf ¯L˜R X U . J = 1 2 X U ⊤ tf ˆL ˜Q + (Ψ(1)Ψ⊤(1) ⊗T) 0 0 tf ¯L˜R X U . (51) (51) 3.1.1. Compatibility constraint The proposed method follows a procedure in which the interval [0, 1] is divided into 2k−1 subintervals. To ensure the continuity of the obtained state functions on the segment interfaces (boundary points) which are given by ti = i 2k−1 , i = 1, 2, . . . , 2k−1 −1, we enforce the following compatibility constraint: 3.1.1. Compatibility constraint The proposed method follows a procedure in which the interval [0, 1] is divided into 2k−1 subintervals. To ensure the continuity of the obtained state functions on the segment interfaces (boundary points) which are given by ti = i 2k−1 , i = 1, 2, . . . , 2k−1 −1, we enforce the following compatibility constraint: (Ψc ⊗Iq)X = 0(2k−1−1)q×1, (52) (52) where where Ψc =   φ1(t1) −φ2(t1) 01×M ··· 01×M 01×M φ2(t2) −φ3(t2) ··· 01×M 01×M 01×M φ3(t3) ··· 01×M ... ... ... ... ... 01×M 01×M 01×M ··· −φ2k−1(t2k−1−1)  . (53) Ψc =   φ1(t1) −φ2(t1) 01×M ··· 01×M 01×M φ2(t2) −φ3(t2) ··· 01×M 01×M 01×M φ3(t3) ··· 01×M ... ... ... ... ... 01×M 01×M 01×M ··· −φ2k−1(t2k−1−1)  . (53) (53) Stat., Optim. 3.3. Remark 1 In a linear time-invariant TD system, E(t), F(t), G(t), H(t), V(t) and W(t) are matrices of constants, that is, ˙x(t) = Ex(t) + Fx(t −tdx) + Gu(t) + Hu(t −tdu) + Vx(tf −t) + Wu(tf −t), we simply have In a linear time-invariant TD system, E(t), F(t), G(t), H(t), V(t) and W(t) are matrices of constants, that is, ˙x(t) = Ex(t) + Fx(t −tdx) + Gu(t) + Hu(t −tdu) + Vx(tf −t) + Wu(tf −t), we simply have In a linear time-invariant TD system, E(t), F(t), G(t), H(t), V(t) and W(t) are matrices of constants, that is, ˙x(t) = Ex(t) + Fx(t −tdx) + Gu(t) + Hu(t −tdu) + Vx(tf −t) + Wu(tf −t), we simply have In a linear time-invariant TD system, E(t), F(t), G(t), H(t), V(t) and W(t) are matrices of constants, that is, ˙x(t) = Ex(t) + Fx(t −tdx) + Gu(t) + Hu(t −tdu) + Vx(tf −t) + Wu(tf −t), we simply have Λ = tf P⊤⊗E+(P⊤⊗F)ˆD ⊤ x +(P⊤⊗V) ˆΥ⊤ −Iqs tf(P⊤⊗G+(P⊤⊗H)¯D⊤ u +(P⊤⊗W) ¯Υ⊤) ˆΨc 0(2k−1−1)q×rs , (59) Λ = tf P⊤⊗E+(P⊤⊗F)ˆD ⊤ x +(P⊤⊗V) ˆΥ⊤ −Iqs tf(P⊤⊗G+(P⊤⊗H)¯D⊤ u +(P⊤⊗W) ¯Υ⊤) ˆΨc 0(2k−1−1)q×rs , (59) (59) b = − h X0+tf((P⊤⊗F)A+(P⊤⊗H)B) 0(2k−1−1)q×1 i . (60) (60) By using the relations of Chebyshev polynomials we ﬁnd By using the relations of Chebyshev polynomials we ﬁnd φn(ti) = q 2k π , q 2k+1 π , q 2k+1 π , . . . , q 2k+1 π , −φn(ti) = − q 2k π , q 2k+1 π , − q 2k+1 π , . . . , (−1)M q 2k+1 π . (54) (54) 3.4. Remark 2 If 2k−1td /∈N, we may set nd = [2k−1td] + 1, where [ ] denotes greatest integer value (see [17]) or we can use some approximations produced by numerical manipulations. If 2k−1td /∈N, we may set nd = [2k−1td] + 1, where [ ] denotes greatest integer value (see [17]) or we can use some approximations produced by numerical manipulations. 3.2. TD optimal control problem reformulation Now we are in a position to generalize the preceding method for the optimization of linear TD systems; taken together, Eqs. (51), (49) and (52) form the following QP problem which can be easily solved by standard numerical methods such as the quadprog function in MATLAB: minimize 1 2z⊤Ξz subject to Λz = b, where where z⊤= [ X U ], (55) Ξ = tf ˆL ˜Q + (Ψ(1)Ψ⊤(1) ⊗T) 0qs×rs 0rs×qs tf ¯L˜R , (56) Λ = " tf ˆP ⊤˜E + ˆP ⊤˜FˆD ⊤ x + ˆP ⊤˜V ˆΥ⊤ −Iqs tf ˆP ⊤˜G + ˆP ⊤˜H¯D ⊤ u + ˆP ⊤˜W ¯Υ⊤ ˆΨc 0(2k−1−1)q×rs # , (57) b = − " X0 + tf ˆP ⊤˜FA + ˆP ⊤˜HB 0(2k−1−1)q×1 # . (58) z⊤= [ X U ], (55) z⊤= [ X U ], (55) Ξ = tf ˆL ˜Q + (Ψ(1)Ψ⊤(1) ⊗T) 0qs×rs 0rs×qs tf ¯L˜R , (56) z⊤= [ X U ], (55) Ξ = tf ˆL ˜Q + (Ψ(1)Ψ⊤(1) ⊗T) 0qs×rs 0rs×qs tf ¯L˜R , (56) Λ " tf ˆP ⊤˜E + ˆP ⊤˜FˆD ⊤ x + ˆP ⊤˜V ˆΥ⊤ −Iqs tf ˆP ⊤˜G + ˆP ⊤˜H¯D ⊤ u + ˆP ⊤˜W ¯Υ⊤ # (57) (55) Ξ = tf ˆL ˜Q + (Ψ(1)Ψ⊤(1) ⊗T) 0qs×rs 0rs×qs tf ¯L˜R , (56) (56) Ξ = f Q ( ( ) ( ) ) qs×rs 0rs×qs tf ¯L˜R , (56) Λ = " tf ˆP ⊤˜E + ˆP ⊤˜FˆD ⊤ x + ˆP ⊤˜V ˆΥ⊤ −Iqs tf ˆP ⊤˜G + ˆP ⊤˜H¯D ⊤ u + ˆP ⊤˜W ¯Υ⊤ ˆΨc 0(2k−1−1)q×rs # , (57) b = − " X0 + tf ˆP ⊤˜FA + ˆP ⊤˜HB 0(2k−1−1)q×1 # . (58) tf ˆP ⊤˜E + ˆP ⊤˜FˆD ⊤ x + ˆP ⊤˜V ˆΥ⊤ −Iqs tf ˆP ⊤˜G + ˆP ⊤˜H¯D ⊤ u + ˆP ⊤˜W ¯Υ⊤ ˆΨc 0(2k−1−1)q×rs # , (57) (57) b = − " X0 + tf ˆP ⊤˜FA + ˆP ⊤˜HB 0(2k−1−1)q×1 # . (58) (58) Our task is to determine the matrices Ξ, Λ and b for the problem and then construct the QP solver. 3.1. Problem statement and Approximation process Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS 312 Clearly, Clearly, E = 0 1 −1 0 , F = 0 0 0 −1 , G = [ 0 1 ] , H = W = 0, V = Q = 0, T = [ 10 0 0 0 ] , R = 1. Using (1) and also (53) and (54) yield, in turn, Clearly, E = 0 1 −1 0 , F = 0 0 0 −1 , G = [ 0 1 ] , H = W = 0, V = Q = 0, T = [ 10 0 0 0 ] , R = 1. Using (1) and also (53) and (54) yield, in turn, Using (1) and also (53) and (54) yield, in turn, Ψ(1)Ψ⊤(1) = h 04×4 04×4 04×4 ψ i , ψ =   4 π 4 √ 2 π 4 √ 2 π 4 √ 2 π 4 √ 2 π 8 π 8 π 8 π 4 √ 2 π 8 π 8 π 8 π 4 √ 2 π 8 π 8 π 8 π  , t1 = 1/2, Ψc = h 2 √π , r 8 π , r 8 π , r 8 π ,−2 √π , r 8 π ,− r 8 π , r 8 π i . t1 = 1/2, Ψc = h 2 √π , r 8 π , r 8 π , r 8 π ,−2 √π , r 8 π ,− r 8 π , r 8 π i . By constructing the matrices P and L and putting all the ﬁndings on (59), (60) and (61), we By constructing the matrices P and L and putting all the ﬁndings on (59), (60) and (61), we solve the QP problem by calling quadprog in MATLAB. Also, we solve this example for different order of approximations and a comparison of the optimal costs is made in Table 1. The time histories of the optimal control and states for k = 2 and M = 5 are depicted in Fig.1 and it is clear that the optimal control compares very well with that was presented in [7]. However, as we see in Table 1, by changing k = 3, we get a better result and this means that by increasing the number of subintervals, we can obtain more accurate results. In [18, p. 3.5. Remark 3 For a system described by (26)–(28) with time-invariant weighting matrices in the PI, the entries in Q(t) and R(t) are constant and J = 1 2x⊤(tf)Tx(tf) + 1 2 R tf 0 x⊤(t)Qx(t) + u⊤(t)Ru(t) dt; thus the matrix Ξ in (56) becomes For a system described by (26)–(28) with time-invariant weighting matrices in the PI, the entries in Q(t) and R(t) are constant and J = 1 2x⊤(tf)Tx(tf) + 1 2 R tf 0 x⊤(t)Qx(t) + u⊤(t)Ru(t) dt; thus the matrix Ξ in (56) becomes Ξ = h tf L⊗Q+(Ψ(1)Ψ⊤(1)⊗T) 0qs×rs 0rs×qs tf L⊗R i . (61) (61) Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 IMAN MALMIR 313 313 IMAN MALMIR 4. Numerical examples 4.1. Example 1 4.1. Example 1 Consider a simple example to illustrate the method. A TD system ˙x1(t) = x2(t), x1(0) = 10 ˙x2(t) = −x1(t) −x2(t −1) + u(t), x2(0) = 0 , t ∈[0, 2] (62) x1(t) = x2(t) = 0, t ∈[−1, 0] (63) (62) (63) is to be controlled to minimize the cost functional is to be controlled to minimize the cost functional J = 5x2 1(2) + 1 2 Z 2 0 u2(t) dt. (64) J = 5x2 1(2) + 1 2 Z 2 0 u2(t) dt. (64) Find the optimal control, state and cost. Find the optimal control, state and cost. p tf = 2, so ﬁrst we deﬁne ϱ = t/2. We then select k = 2 and M = 4. Consequently, by (37) p tf = 2, so ﬁrst we deﬁne ϱ = t/2. We then select k = 2 and M = 4. Consequently, by (37) X0 = [x0, x0]⊤, x0 = √π 2 [10, 0, 0, 0, 0, 0, 0, 0]. X0 = [x0, x0]⊤, x0 = √π 2 [10, 0, 0, 0, 0, 0, 0, 0]. X0 = [x0, x0]⊤, x0 = √π 2 [10, 0, 0, 0, 0, 0, 0, 0]. X0 = [x0, x0]⊤, x0 = √π 2 [10, 0, 0, 0, 0, 0, 0, 0]. X [x , x ] , x 2 [10, 0, 0, 0, 0, 0, 0, 0]. Since ϱx = 1/2, it follows from (14) and (15) that ndx = 1 and Dx = 08×4 I4 04×4 . Since ϱx = 1/2, it follows from (14) and (15) that ndx = 1 and Dx = 08×4 I4 04×4 . Clearly, Stat., Optim. Inf. Comput. Vol. 5, December 2017 Clearly, 117], the author has used linear Legendre multiwavelets to solve this problem; the graph of optimal control which obtained by using k = 3 has a slight similarity with referenced graph which given by Banks and Burns in [7, p. 194] and the results do not provide a satisfactory approximation where the optimal control values are not compatible with the exact and approximated values (e.g., u∗(t = 0); see [18, p. 118] and the references therein). This example shows that this method is much better than the Khellat approach to solve such time-invariant TD problems outlined in [18]. For k = 2 and M = 5, we ﬁnd u∗(ϱ) = 1.6688ϱ4 −3.9117ϱ3 −6.44ϱ2 + 5.8148ϱ + 1.2475, ϱ ∈[0, 0.5] −24.384ϱ4 + 71.079ϱ3 −78.764ϱ2 + 35.048ϱ −3.0334, ϱ ∈[0.5, 1], x∗ 1(ϱ) = 1.5757ϱ4 + 4.8164ϱ3 −17.674ϱ2 + 0.010545ϱ + 9.9999, ϱ ∈[0, 0.5] −5.4685ϱ4 + 31.556ϱ3 −47.746ϱ2 + 13.564ϱ + 7.8387, ϱ ∈[0.5, 1], Stat., Optim. Inf. Comput. Vol. 5, December 2017 314 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS Table 1. Comparison of the accuracy of method (Example 1). Table 1. Comparison of the accuracy of method (Example 1). Source Estimated cost H.T. Banks, J.A. Burns [7] 3.3991 A.Y. Lee [11] 3.4827 F. Khellat [18] 3.43254 N. Haddadi, Y. Ordokhani, M. Razzaghi [19] 3.21663 This research k = 2 and M = 4 3.392588 This research k = 2 and M = 5 3.398603 This research k = 3 and M = 5 3.399132 This research k = 2 and M = 8 3.399112 x∗ 2(ϱ) = −4.3174ϱ4 + 7.4687ϱ3 + 5.8079ϱ2 −17.505ϱ + 0.00000, ϱ ∈[0, 0.5] −10.187ϱ4 + 19.625ϱ3 + 13.429ϱ2 −31.271ϱ + 3.8248, ϱ ∈[0.5, 1]. 0 0.2 0.4 0.6 0.8 1 −8 −6 −4 −2 0 2 4 6 8 10 Optimal States; k = 2 & M = 5 t x 2−1 xi (t) x1 (t) x2 (t) 0 0.2 0.4 0.6 0.8 1 −8 −6 −4 −2 0 2 4 6 8 10 Optimal States; k = 2 & M = 5 t x 2−1 xi (t) x1 (t) x2 (t) 0 0.2 0.4 0.6 0.8 1 −0.5 0 0.5 1 1.5 2 2.5 Optimal Control; k = 2 & M = 5 t x 2−1 u(t) u(t), this research; k = 2 & M = 5 u(t), by Banks and Burns [7] Figure 1. Clearly, Optimal states and controls for Example 1. 0 0.2 0.4 0.6 0.8 1 −0.5 0 0.5 1 1.5 2 2.5 Optimal Control; k = 2 & M = 5 t x 2−1 u(t) u(t), this research; k = 2 & M = 5 u(t), by Banks and Burns [7] Figure 1. Optimal states and controls for Example 1. 4.1.1. Solving Example 1 with terminal inequality constraints Consider the terminal inequality constraints which Lee in [11] imposed to this system, as ( ) ( ) (65) 4.1.1. Solving Example 1 with terminal inequality constraints Consider the terminal inequality constraints which Lee in [11] imposed to this system, as x1(tf) ≥2, x2(tf) ≤−5. (65) x1(tf) ≥2, x2(tf) ≤−5. (65) x1(tf) ≥2, x2(tf) ≤−5. (65) To solve this problem, we must rewrite (65) in the standard form: Λi.ez ≤bi.e. We write the inequality as −1 0 0 1 ˆΨ⊤(1)X ≤ −2 −5 ; hence we set Λi.e = Λ1 0 0 0 , bi.e = Xf 0 , where Λ1 = Φ ⊗ −1 0 0 1 , Φ = h 0 Ψ⊤(1) i and Xf = [0, 0, . . . , 0, −2, −5]⊤. With no additional effort, by adding Λi.e and bi.e to the algorithm which solved the unconstrained problem, for k = 2 and M = 4 we get J∗= 31.7842; also for the same k and M = 8, J∗= 31.7935. To solve this problem, we must rewrite (65) in the standard form: Λi.ez ≤bi.e. We write the inequality as −1 0 0 1 ˆΨ⊤(1)X ≤ −2 −5 ; hence we set Λi.e = Λ1 0 0 0 , bi.e = Xf 0 , where Λ1 = Φ ⊗ −1 0 0 1 , Φ = h 0 Ψ⊤(1) i and Xf = [0, 0, . . . , 0, −2, −5]⊤. With no additional effort, by adding Λi.e and bi.e to the algorithm which solved the unconstrained problem, for k = 2 and M = 4 we get J∗= 31.7842; also for the same k and M = 8, J∗= 31.7935. 4.2. Example 2 From the new proposed expansion in (42) for case II, we form We solve the problem for the two cases. From the new proposed expansion in (42) for case II, we form A = [∆1, ∆2, . . . , ∆11, 168 z }\| { 0, 0, . . . , 0]⊤, where ∆n′ = [ √π 8 , 7 z }\| { 0, 0, . . . , 0]. Constructing Ξ, Λ and b, we then obtain the solutions by using the interior-point-convex algorithm in MATLAB. The optimal performance indices computed by the present method for each of the cases and the optimal PI which obtained in [17] are given in Table 2. The time histories of u∗(t) and x∗(t) for the two approximations are depicted in Fig.2. We know from experience that the exact optimal state or control (if available) for a TD system is a piecewise-deﬁned function, in which the boundary point(s) (or some of these points) is (are) exactly equal to the delay(s). As we see in the optimal trajectories of Fig.2, for the ﬁrst approximation, irrespective of the unexpected jump near the point t = 0.3, one obvious boundary point is t = 0.6875, while for our approximation is t = 21/32 that is closer to the control delay t = 2/3; also in the optimal controls, this point for the ﬁrst and second cases is located at, in turn, 0.3125 and 11/32, which our result is closer to t = 1/3. These comparisons of two approximate solutions show that the second case is a better approximation and it provides more accurate results. Table 2. Comparison of the optimal PI (Example 2). Source Method J∗ H.R. Marzban, M. Razzaghi [17] hybrid of block-pulse and Legendre polynomials 0.37311241 This research k = 6 and M = 8 Chebyshev wavelets and case I 0.3686278302 This research k = 6 and M = 8 Chebyshev wavelets and case II 0.3747842350 0 0.2 0.4 0.6 0.8 1 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 Optimal State; k = 6 & M = 8 t x(t) Case I Case II 0 0.2 0.4 0.6 0.8 1 −1 −0.9 −0.8 −0.7 −0.6 −0.5 −0.4 −0.3 −0.2 −0.1 0 Optimal Control; k = 6 & M = 8 t u(t) Case I Case II Figure 2. Optimal states and controls for Example 2. Table 2. 4.2. Example 2 Let us illustrate the application of remark 2 with a simple ﬁrst order system containing delays in state and control variables. Given a TD system Let us illustrate the application of remark 2 with a simple ﬁrst order system containing delays in state and control variables. Given a TD system Let us illustrate the application of remark 2 with a simple ﬁrst order system containing delays in state and control variables. Given a TD system ˙x(t) = −x(t) + x(t −1 3) + u(t) −0.5u(t −2 3), 0 ≤t ≤1 (66) x(t) = 1, −1 3 ≤t ≤0 (67) u(t) = 0, −2 3 ≤t ≤0 (68) (66) (67) (68) Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 IMAN MALMIR 315 315 IMAN MALMIR and the PI and the PI J = 1 2 Z 1 0 x2(t) + 1 2u2(t) dt. (69) (69) Obtain the optimal control, state and PI. Obtain the optimal control, state and PI. We choose k = 6 and M = 8; according to remark 2, consider two app based on [17] as case I and the second we propose here as case II We choose k = 6 and M = 8; according to remark 2, consider two approximation cases: the ﬁrst case which based on [17] as case I and the second we propose here as case II tdu = 2 3 so ndx = [32/3] + 1, ndu = [64/3] + 1, I . tdx = 1 3, tdu = 2 3 so ndx = [32/3] + 1, ndu = [64/3] + 1, II . tdx ≈11 32, tdu ≈21 32 so ndx = 11, ndu = 21. I . tdx = 1 3, tdu = 2 3 so ndx = [32/3] + 1, ndu = [64/3] + 1, I . tdx = 1 3, tdu = 2 3 so ndx = [32/3] + 1, ndu = [64/3] + 1, II . tdx ≈11 32, tdu ≈21 32 so ndx = 11, ndu = 21. tdu ≈21 32 so ndx = 11, ndu = 21. II . tdx ≈11 32, tdu ≈21 32 so ndx = 11, ndu = 21. We solve the problem for the two cases. 4.3. Example 3 This example was ﬁrst studied in [8]. We are interested in ﬁnding the optimal control and state which when applied to a TD system expressed by ˙x(t) = t2 + 1 1 0 2 x(t −1 2) + 1 t + 1 u(t) + t + 1 t2 + 1 u(t −1 4) , 0 ≤t ≤1 (70) x(t) = [1, 1]⊤, −1 2 ≤t ≤0 (71) u(t) = 1, −1 4 ≤t ≤0 (72) d ib d b ˙x(t) = t2 + 1 1 0 2 x(t −1 2) + 1 t + 1 u(t) + t + 1 t2 + 1 u(t −1 4) , 0 ≤t ≤1 (70) ⊤ 1 ˙x(t) = t2 + 1 1 0 2 x(t −1 2) + 1 t + 1 u(t) + t + 1 t2 + 1 u(t −1 4) , 0 ≤t ≤1 (70 (70) x(t) = [1, 1]⊤, −1 2 ≤t ≤0 (71) u(t) = 1, −1 4 ≤t ≤0 (72) (71) (72) gives an optimal PI J∗described by J = 1 2 Z 1 0 x⊤(t) 1 t t t2 x(t) + (t2 + 1)u2(t) dt. (73) (73) We approximate x(t) and u(t) by dividing the time interval [0, 1] to four subintervals (k = 3) and by mean of 4th degree polynomials (M = 5). Formula (14) gives ndx = 2 and ndu = 1, hence according to Eqs. (42)–(43) We approximate x(t) and u(t) by dividing the time interval [0, 1] to four subintervals (k = 3) and by mean of 4th degree polynomials (M = 5). Formula (14) gives ndx = 2 and ndu = 1, hence according to Eqs. (42)–(43) A = [ p π 8 , p π 8 , 8 z }\| { 0, 0, . . . , 0, p π 8 , p π 8 , 8 z }\| { 0, 0, . . . , 0, 20 z }\| { 0, 0, . . . , 0]⊤, B = [ p π 8 , 4 z }\| { 0, 0, . . . , 0, 15 z }\| { 0, 0, . . . , 0]⊤. 4.2. Example 2 Comparison of the optimal PI (Example 2). 0 0.2 0.4 0.6 0.8 1 −1 −0.9 −0.8 −0.7 −0.6 −0.5 −0.4 −0.3 −0.2 −0.1 0 Optimal Control; k = 6 & M = 8 t u(t) Case I Case II 0 0.2 0.4 0.6 0.8 1 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 Optimal State; k = 6 & M = 8 t x(t) Case I Case II Figure 2. Optimal states and controls for Example 2. Stat., Optim. Inf. Comput. Vol. 5, December 2017 316 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS 4.3. Example 3 By using the expansion of t and t2, substituting the computed matrices in (56),(57) and (58) and calling quadratic programming in MATLAB, we thus obtain the solutions. In Table 3, a comparison is made between the numerical results of J∗obtained by this approach and those reported in [8], [20], [21] and [22]. The optimal curves are plotted in Fig.3 and it is obvious that the solutions of this approach represent smooth graphs. The simulation curves derived by this new algorithm which based on Chebyshev polynomials are much better than those which presented in [21] by using Bezier curves; the obtained graphs in [21] are hardly plausible solution curves. (58) d b Table 3. Comparison of the optimal PI (Example 3). Table 3. Comparison of the optimal PI (Example 3). Table 3. Comparison of the optimal PI (Example 3). Source Estimated cost functional G.P. Rao, L. Sivakumar [8] 1.56229959504408 X.T. Wang [20] 1.56224137355159 F. Ghomanjani, M.H. Farahi, M. Gachpazan [21] 1.536409753 H.R. Marzban [22] 1.562241373551585 This research k = 3 and M = 5 1.56224149098 This research k = 3 and M = 6 1.56224136508 This research k = 3 and M = 7 1.56224137354 For k = 3 and M = 7 we obtain For k = 3 and M = 7 we obtain u∗(t) =                      −0.703482968t6 + 4.104009t5 −1.2619225t4 −5.51571044t3 + 2.58918191t2 +3.86320787t −2.90681573, t ∈[0, 1 4] −1.90562493t6 + 1.12680481t5 + 12.9505209t4 −17.9693461t3 + 6.16088639t2 +3.58550249t −2.91835229, t ∈[ 1 4, 1 2] 4.95151613t6 −29.9265419t5 + 73.0830158t4 −87.3223313t3 + 54.8661848t2 −14.7597252t −0.147859839, t ∈[ 1 2, 3 4] −0.0621420831t6 −1.98062803t5 + 12.5883514t4 −28.079425t3 + 30.0233308t2 −14.6178289t + 2.12832355, t ∈[ 3 4, 1], Stat., Optim. Inf. Comput. Vol. 5, December 2017 317 IMAN MALMIR 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Optimal States; k = 3 & M = 7 t xi (t) x1 (t) x2 (t) 0 0.2 0.4 0.6 0.8 1 −3 −2.5 −2 −1.5 −1 −0.5 0 Optimal Control; k = 3 & M = 7 t u(t) u(t) Figure 3. Optimal states and control for Example 3. 4.3. Example 3 0 0.2 0.4 0.6 0.8 1 −3 −2.5 −2 −1.5 −1 −0.5 0 Optimal Control; k = 3 & M = 7 t u(t) u(t) 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Optimal States; k = 3 & M = 7 t xi (t) x1 (t) x2 (t) Figure 3. Optimal states and control for Example 3. x∗ 1(t) =                      0.596066128t6 −0.222156716t5 −1.38408007t4 + 1.19684481t3 + 2.43158515t2 +0.0931845651t + 0.999999999, t ∈[0, 1 4] 0.479277118t6 + 0.339521103t5 −3.17166627t4 + 4.71066779t3 + 0.844829548t2 −4.55436451t + 2.21261877, t ∈[ 1 4, 1 2] −1.20653996t6 + 10.0333344t5 −22.275601t4 + 24.4432887t3 −10.5364892t2 −1.13368726t + 1.79843736, t ∈[ 1 2, 3 4] −1.88346525t6 + 14.6590393t5 −39.6131801t4 + 55.2126686t3 −44.5329442t2 +20.8408825t −4.03182109, t ∈[ 3 4, 1], and x∗ 1(t) =                      0.596066128t6 −0.222156716t5 −1.38408007t4 + 1.19684481t3 + 2.43158515t2 +0.0931845651t + 0.999999999, t ∈[0, 1 4] 0.479277118t6 + 0.339521103t5 −3.17166627t4 + 4.71066779t3 + 0.844829548t2 −4.55436451t + 2.21261877, t ∈[ 1 4, 1 2] −1.20653996t6 + 10.0333344t5 −22.275601t4 + 24.4432887t3 −10.5364892t2 −1.13368726t + 1.79843736, t ∈[ 1 2, 3 4] −1.88346525t6 + 14.6590393t5 −39.6131801t4 + 55.2126686t3 −44.5329442t2 +20.8408825t −4.03182109, t ∈[ 3 4, 1], and and x∗ 2(t) =                      0.857069133t6 −1.48240708t5 −0.71044537t4 + 2.48230129t3 + 0.478271465t2 +0.0931831043t + 1.0, t ∈[0, 1 4] 1.0214618t6 + 0.459829797t5 −3.8630792t4 + 4.22455503t3 + 1.11394266t2 −4.54417613t + 2.10276577, t ∈[ 1 4, 1 2] −0.189539605t6 + 6.17628485t5 −15.7344435t4 + 17.852592t3 −7.4456427t2 −1.82914178t + 1.76388326, t ∈[ 1 2, 3 4] 0.323202163t6 + 3.2525986t5 −15.5957708t4 + 29.9041399t3 −31.1768748t2 +17.0923014t −3.60395733, t ∈[ 3 4, 1]. 4.3. Example 3 x∗ 2(t) =                      0.857069133t6 −1.48240708t5 −0.71044537t4 + 2.48230129t3 + 0.478271465t2 +0.0931831043t + 1.0, t ∈[0, 1 4] 1.0214618t6 + 0.459829797t5 −3.8630792t4 + 4.22455503t3 + 1.11394266t2 −4.54417613t + 2.10276577, t ∈[ 1 4, 1 2] −0.189539605t6 + 6.17628485t5 −15.7344435t4 + 17.852592t3 −7.4456427t2 −1.82914178t + 1.76388326, t ∈[ 1 2, 3 4] 0.323202163t6 + 3.2525986t5 −15.5957708t4 + 29.9041399t3 −31.1768748t2 +17.0923014t −3.60395733, t ∈[ 3 4, 1]. 4.3.1. Solving Example 3 with constraints Physical considerations imply that some constraints should be imposed on the optimal control systems and unconstrained systems are less happened. The proposed method is also applicable to such systems with state, state-control and interior point constraints and ﬁnal conditions. To illustrate this discussion, we impose a state constraint and a terminal condition on the present system as C 1 x1(t) ≤8(t −0.6)2 , 0 ≤t ≤1 (74) x1(t) ≤8(t −0.6)2 , 0 ≤t ≤1 (74) x1(1) = x2(1). (75) f ( ) 2 x1(t) ≤8(t −0.6)2 , 0 ≤t ≤1 (74 x1(t) ≤8(t −0.6)2 , 0 ≤t ≤1 (74) ) ≤( ) , ≤ ≤ ( ) x1(1) = x2(1). (75) Case 2. OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS 318 0 0.2 0.4 0.6 0.8 1 −0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Case 1; k = 3 & M = 7 t xi (t) 0 0.2 0.4 0.6 0.8 1 −4 −3.5 −3 −2.5 −2 −1.5 −1 −0.5 0 u(t) x1 (t) x2 (t) u(t) boundary of state constraint 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 xi (t) Case 2; k = 3 & M = 7 t 0 0.2 0.4 0.6 0.8 1 −5 −4 −3 −2 −1 0 1 2 u(t) x1 (t) x2 (t) u(t) Figure 4. Optimal states and controls for constrained problem. 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 xi (t) Case 2; k = 3 & M = 7 t 0 0.2 0.4 0.6 0.8 1 −5 −4 −3 −2 −1 0 1 2 u(t) x1 (t) x2 (t) u(t) 0 0.2 0.4 0.6 0.8 1 −0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Case 1; k = 3 & M = 7 t xi (t) 0 0.2 0.4 0.6 0.8 1 −4 −3.5 −3 −2.5 −2 −1.5 −1 −0.5 0 u(t) x1 (t) x2 (t) u(t) boundary of state constraint Case 1; k = 3 & M = 7 Figure 4. Optimal states and controls for constrained problem. Case 2. x1(1) = x2(1). x1(1) = x2(1). (75) (75) In case 1, we can write [ 1 0 0 0 ] ˆΨ⊤(t)X ≤ f1(t) 0 , where f1(t) = 8(t −0.6)2. To handle this constraint, we must satisfy it at discrete points tnm on every subinterval where we deﬁne: tnm = {(n −1)M + m}/s. For satisfying the obtained inequality constraints at these points, we have to add two matrices which computed from them to the quadprog function. If the obtained state touches the constraint, we set f1(t) −ϵ →f1(t), where ϵ ∈R>0 is an arbitrary constant (as small as possible) which enforced to prevent any violation. By adding the two matrices to the algorithm which solved the unconstrained problem, we ﬁnd J∗= 1.850110. But, by looking very closely at the obtained results, we see that there is a small violation. As already mentioned, by applying the latter condition, we get J∗= 1.850244, where we have no violation, and x∗ 1(t) does not touch the respective constraint. In case 2, we have the equality constraint; to handle this, we must add this constraint to (52). Hence we get J∗= 2.099227 and x(1) = [0.831905937, 0.831905937]⊤. The optimal control and state for the two cases are depicted in Fig.4. Stat., Optim. Inf. Comput. Vol. 5, December 2017 4.4. Example 4 This problem was ﬁrst studied in [20]. The problem is to ﬁnd the optimal control u∗(t) which minimizes the PI This problem was ﬁrst studied in [20]. The problem is to ﬁnd the optimal control u∗(t) which minimizes the PI This problem was ﬁrst studied in [20]. The problem is to ﬁnd the optimal control u∗(t) which minimizes the PI J = 1 2 Z 1 0   x⊤(t)   1 t 0 t t2 0 0 0 t2  x(t) + u⊤(t)I2u(t)   dt, (76) (76) subject to ˙x(t) =   t 1 t2 + 1 1 t 0 t2 + 1 0 t  x(t −1 3) +   1 t + 1 t + 1 1 1 1  u(t) +   1 t + 1 t 1 1 t2 + 1  u(t −2 3) +   t2 1 1 1 1 0 t 0 1  x(1 −t), 0 ≤t ≤1 (77) +   t2 1 1 1 1 0 t 0 1  x(1 −t), 0 ≤t ≤1 (77) 1 1 1 0 0 1  x(1 −t), 0 ≤t ≤1 (77) +   t 1 1 1 1 0 t 0 1  x(1 −t), 0 ≤t ≤1 (77) (77) x(t) = [1, 1, 1]⊤, −1 3 ≤t ≤0 (78) u(t) = [1, 1]⊤, −2 3 ≤t ≤0. (79) (78) (79) We choose k = 7 and M = 5; in a similar manner as explained in Example 3, we construct the required matrices and also we use the inverse time operational matrix of Chebyshev wavelets which described in (17). So, the obtained results are plotted in Fig.5. The optimal value of the PI by the procedure is given in Table 4 and for purposes of comparison we can ﬁnd the estimated values of the PI which reported in [20] and [23]. As can be seen, it should be obvious that the method is so accurate whereas the derived graphs appear smooth; this fact shows the effectiveness of the proposed method. Table 4. Computational results of J∗(Example 4). Source Method J∗ X.T. Wang [20] hybrid functions 3.3693347735 B.M. Mohan, S. Kumar Kar [23] shifted Legendre polynomials 3.1070469742 B.M. Mohan, S. Kumar Kar [23] block-pulse functions 3.1092085758 this research Ch. W. k = 7 and M = 5 3.1339565957 Table 4. Stat., Optim. Inf. Comput. Vol. 5, December 2017 4.5. Example 5 4.5. Example 5 Consider a linear time-varying system with different delays and terminal times described by (see [24]) r a linear time-varying system with different delays and terminal times described by (see [24]) ˙x(t) =   0 1 0 0 0 1 cos(t) 0 0  x(t) +   0 1 0 0 0 1 e−t 0 0  x(t −td) −   0 0 2 + sin(t)  u(t), 0 ≤t ≤tf (80) x(t) = [1, 0, sin(t)]⊤, −td ≤t ≤0. (81) (80) x(t) =   0 0 1 cos(t) 0 0  x(t) +   0 0 1 e−t 0 0  x(t −td) −   0 2 + sin(t)  u(t), 0 ≤t ≤tf (80) x(t) = [1, 0, sin(t)]⊤, −td ≤t ≤0. (81) x(t) = [1, 0, sin(t)]⊤, −td ≤t ≤0. (81) (81) ( ) [ This system is to be controlled to minimize the PI This system is to be controlled to minimize the PI J = x⊤(tf)I3x(tf) + Z tf 0 u2(t) dt, (82) (82) where the delay and terminal times are as the following three cases: where the delay and terminal times are as the following three cases: Case 1. td = 1, tf = 5, Case 2. td = 1, tf = 3, Case 3. td = 2, tf = 5. Case 1. td = 1, tf = 5, Case 2. td = 1, tf = 3, Case 3. td = 2, tf = 5. Case 1. td = 1, tf = 5, Case 2. td = 1, tf = 3, Case 3. td = 2, tf = 5. Case 1. td = 1, tf = 5, Case 2. td = 1, tf = 3, Case 3. td = 2, tf = 5. Since the delay and terminal time are changed, ndx and consequently A and Dx will be changed; this affects on the optimal PI J∗. In case 1, when we solve the QP problem, then by just changing A and Dx according to the new value of ϱx, we are able to solve case 3 of the problem and this is a distinct advantage of the method over some conventional methods. Selecting k = 5 and M = 8, then the values of J∗by the method are reported in Table 5 and the optimal states and controls shown in Fig.6. 4.4. Example 4 Computational results of J∗(Example 4). Source Method J∗ X.T. Wang [20] hybrid functions 3.3693347735 B.M. Mohan, S. Kumar Kar [23] shifted Legendre polynomials 3.1070469742 B.M. Mohan, S. Kumar Kar [23] block-pulse functions 3.1092085758 this research Ch. W. k = 7 and M = 5 3.1339565957 Table 4. Computational results of J∗(Example 4). Table 4. Computational results of J∗(Example 4). Stat., Optim. Inf. Comput. Vol. 5, December 2017 319 IMAN MALMIR 0 0.2 0.4 0.6 0.8 1 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 t xi (t) Optimal States; k = 7 & M = 5 x1 (t) x2 (t) x3 (t) 0 0.2 0.4 0.6 0.8 1 −4 −3.5 −3 −2.5 −2 −1.5 −1 −0.5 0 0.5 t ui (t) Optimal Controls; k = 7 & M = 5 u1 (t) u2 (t) Figure 5. Optimal states and controls for Example 4. 0 0.2 0.4 0.6 0.8 1 −4 −3.5 −3 −2.5 −2 −1.5 −1 −0.5 0 0.5 t ui (t) Optimal Controls; k = 7 & M = 5 u1 (t) u2 (t) 0 0.2 0.4 0.6 0.8 1 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 t xi (t) Optimal States; k = 7 & M = 5 x1 (t) x2 (t) x3 (t) Figure 5. Optimal states and controls for Example 4. 4.5. Example 5 C id li ti i t ith diff t d l d t i l ti d ib d b ( [24]) †The numerical results generated by HP ENVY 15-j013cl Notebook PC in MATLAB R2012b. Stat., Optim. Inf. Comput. Vol. 5, December 2017 4.5. Example 5 0 0.2 0.4 0.6 0.8 1 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 Optimal States for case 2: td = 1, tf = 3; k = 5 & M = 8 x1 (t) x2 (t) x3 (t) 0 0.2 0.4 0.6 0.8 1 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 Optimal States for case 1: td = 1, tf = 5; k = 5 & M = 8 t x 5−1 xi (t) x1 (t) x2 (t) x3 (t) O ti l St t f 3 t 2 t 5 k 5 & M 8 Optimal States for case 2: td = 1, tf = 3; k = 5 & M = 8 Optimal States for case 1: td = 1, tf = 5; k = 5 & M = 8 0 0.2 0.4 0.6 0.8 1 −0.5 0 0.5 1 Optimal States for case 3: td = 2, tf = 5; k = 5 & M = 8 t x 5−1 xi (t) x1 (t) x2 (t) x3 (t) 0 0.2 0.4 0.6 0.8 1 −0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Optimal Control; k = 5 & M = 8 t x 5−1 u(t) Case 1 Case 2 Case 3 Figure 6. Optimal states and controls for Example 5. 4.6. Example 6 This example is adopted from [25]. Consider a TD system which contains multiple delays as follows: This example is adopted from [25]. Consider a TD system which contains multiple delays as follows: ˙x(t) = 0 1 −0.5e−t 0.5 x(t) + −0.5e−t 0 0.1 −0.5 x(t −τ1) + 0 0.1 0 −0.6 x(t −τ2) + 0 1 u(t −h1) + 0.5e−t 1 u(t −h2), ˙x(t) = 0 1 −0.5e−t 0.5 x(t) + −0.5e−t 0 0.1 −0.5 x(t −τ1) + 0 0.1 0 −0.6 x(t −τ2) + 0 1 u(t −h1) + 0.5e−t 1 u(t −h2), (83) (83) x(t) = [0, cos(πt/3)]⊤, −τ2 ≤t ≤0. (84) x(t) = [0, cos(πt/3)]⊤, −τ2 ≤t ≤0. (84) x(t) = [0, cos(πt/3)]⊤, −τ2 ≤t ≤0. (84) (84) We want to ﬁnd the optimal control u∗(t) which minimizes We want to ﬁnd the optimal control u∗(t) which minimizes J = x⊤(tf)I2x(tf) + Z tf 0 u2(t) dt, (85) (85) subject to Eqs. (83)–(84) with tf = 16 and different delays as subject to Eqs. (83)–(84) with tf = 16 and different delays as subject to Eqs. (83)–(84) with tf = 16 and different delays as f Case 1. τ1 = 1, τ2 = 2, h1 = 0, and h2 = 2, f Case 1. τ1 = 1, τ2 = 2, h1 = 0, and h2 = 2, Case 2. τ1 = 1, τ2 = 2, h1 = 1, and h2 = 2. se 2. τ1 = 1, τ2 = 2, h1 = 1, and h2 = 2. Here, the state equation is ˙x(ϱ) = tf(E(ϱ)x(ϱ) + F1(ϱ)x(ϱ −ϱ1x) + F2x(ϱ −ϱ2x) + H1u(ϱ −ϱ1u) + H2(ϱ)u(ϱ −ϱ2u)). Since we have two state delay, we need to deﬁne two column vectors as A1 and A2 in order to expand (84) by (42). Therefore in (57)–(58), we set Λ11 = tf(ˆP ⊤˜E + ˆP ⊤˜F1 ˆD ⊤ 1x + (P⊤⊗F2)ˆD ⊤ 2x) −Iqs, Λ12 = tf((P⊤⊗H1)D⊤ 1u + ˆP ⊤˜H2D⊤ 2u), and b11 = X0 + tf(ˆP ⊤˜F1A1 + (P⊤⊗F2)A2). By choosing k = 6 and M = 8 and using these results, Fig.7 shows the results of our approximations. Here, the state equation is ˙x(ϱ) = tf(E(ϱ)x(ϱ) + F1(ϱ)x(ϱ −ϱ1x) + F2x(ϱ −ϱ2x) + H1u(ϱ −ϱ1u) + H2(ϱ)u(ϱ −ϱ2u)). 4.5. Example 5 Because of the nature of the control cost expression u2(t), we have to pay higher cost for larger energy consumption. As one can see, the values of delay and ﬁnal time play a signiﬁcant role in reducing J∗; these facts lead us immediately to the conclusion that a TD system with a longer terminal time probably have more opportunities to be controlled with lower energy cost by using little energy. Table 5. Computational results (Example 5). assumptions J∗ CPU time† td = 1, tf = 5 0.327972 1.280 seconds td = 1, tf = 3 0.333568 1.354 seconds td = 2, tf = 5 0.203085 1.385 seconds Table 5. Computational results (Example 5). assumptions J∗ CPU time† td = 1, tf = 5 0.327972 1.280 seconds td = 1, tf = 3 0.333568 1.354 seconds td = 2, tf = 5 0.203085 1.385 seconds Table 5. Computational results (Example 5). †The numerical results generated by HP ENVY 15-j013cl Notebook PC in MATLAB R2012b. Stat., Optim. Inf. Comput. Vol. 5, December 2017 320 OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS 0 0.2 0.4 0.6 0.8 1 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 Optimal States for case 1: td = 1, tf = 5; k = 5 & M = 8 t x 5−1 xi (t) x1 (t) x2 (t) x3 (t) 0 0.2 0.4 0.6 0.8 1 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1 Optimal States for case 2: td = 1, tf = 3; k = 5 & M = 8 x1 (t) x2 (t) x3 (t) 0 0.2 0.4 0.6 0.8 1 −0.5 0 0.5 1 Optimal States for case 3: td = 2, tf = 5; k = 5 & M = 8 t x 5−1 xi (t) x1 (t) x2 (t) x3 (t) 0 0.2 0.4 0.6 0.8 1 −0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Optimal Control; k = 5 & M = 8 t x 5−1 u(t) Case 1 Case 2 Case 3 Figure 6. Optimal states and controls for Example 5. 4.6. Example 6 Since we have two state delay, we need to deﬁne two column vectors as A1 and A2 in order to expand (84) by (42). Therefore in (57)–(58), we set Λ11 = tf(ˆP ⊤˜E + ˆP ⊤˜F1 ˆD ⊤ 1x + (P⊤⊗F2)ˆD ⊤ 2x) −Iqs, Λ12 = tf((P⊤⊗H1)D⊤ 1u + ˆP ⊤˜H2D⊤ 2u), and b11 = X0 + tf(ˆP ⊤˜F1A1 + (P⊤⊗F2)A2). By choosing k = 6 and M = 8 and using these results, Fig.7 shows the results of our approximations. Stat., Optim. Inf. Comput. Vol. 5, December 2017 321 IMAN MALMIR As mentioned previously, by changing the values of the delays, the optimal value of the PI J∗varies considerably regarding the optimal ﬁnal state x∗(tf) (this directly affects on the optimal value of the terminal PI) and control u∗(t). In case 1 we ﬁnd J∗= 0.135229 and in case 2, J∗= 0.081048. 0 2 4 6 8 10 12 14 16 −1.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Optimal States; k = 6 & M = 8 t xi (t) x1 (t); case 1 x2 (t); case 1 x1 (t); case 2 x2 (t); case 2 0 2 4 6 8 10 12 14 16 −0.2 −0.15 −0.1 −0.05 0 0.05 0.1 0.15 Optimal Control; k = 6 & M = 8 t u(t) u(t); case 1 u(t); case 2 Figure 7. Optimal states and controls for Example 6. 0 2 4 6 8 10 12 14 16 −1.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Optimal States; k = 6 & M = 8 t xi (t) x1 (t); case 1 x2 (t); case 1 x1 (t); case 2 x2 (t); case 2 0 2 4 6 8 10 12 14 16 −0.2 −0.15 −0.1 −0.05 0 0.05 0.1 0.15 Optimal Control; k = 6 & M = 8 t u(t) u(t); case 1 u(t); case 2 Figure 7. Optimal states and controls for Example 6. 4.7. Example 7 p This problem was studied in [1]. A second order linear time-varying multi-delay system expressed by This problem was studied in [1]. A second order linear time-varying multi-delay system expressed by ˙x1(t) = x2(t) + x1(t −1) ˙x2(t) = tx1(t) + 2x1(t −1) + x2(t −1) + x2(t −0.8) + u(t) −u(t −0.5) , t ≥0 ˙x1(t) = x2(t) + x1(t −1) ˙x2(t) = tx1(t) + 2x1(t −1) + x2(t −1) + x2(t −0.8) + u(t) −u(t −0.5) , t ≥0 (86 (86)    x1(t) = 1, −1.0 ≤t ≤0 x2(t) = 1, −0.8 ≤t ≤0 u(t) = 5(t + 1), −0.5 ≤t ≤0 (87) (87) is to be controlled to minimize the cost functional J = 1 2x2 1(3) + x2 2(3) + 1 2 Z 3 0 2x2 1(t) + 2x1(t)x2(t) + x2 2(t) + 1 t + 2u2(t) dt. (88) (88) (88) Calculate the minimum value of J. Calculate the minimum value of J. the minimum value of J. escaling the time interval by setting ϱ = t/3, the problem converted to ﬁnding u∗(ϱ) and x∗(ϱ) which 1 0 Z 1 2 1 1 Calculate the minimum value of J. After rescaling the time interval by setting ϱ = t/3, the problem converted to ﬁnding u∗(ϱ) and x∗(ϱ) which minimize After rescaling the time interval by setting ϱ = t/3, the problem converted to ﬁnding u∗ minimize After rescaling the time interval by setting ϱ = t/3, the problem converted to ﬁnding u∗(ϱ) and x∗(ϱ) which minimize 1 J = 1 2x⊤(1) 1 0 0 2 x(1) + 3 2 Z 1 0 x⊤(ϱ) 2 1 1 1 x(ϱ) + 1 3ϱ + 2u2(ϱ) dϱ, subject to ˙x(ϱ) = 3 0 1 3ϱ 0 x(ϱ) + 1 0 2 1 x(ϱ −1 3) + 0 0 0 1 x(ϱ −4 15) + 0 1 u(ϱ) + 0 −1 u(ϱ −1 6) , ( x(ϱ) = [1, 1]⊤, −1 3 ≤ϱ ≤0 u(ϱ) = 15ϱ + 5, −1 6 ≤ϱ ≤0. Using the procedure in case II of Example 2, we set ϱx1 ≈21/64, ϱx2 ≈17/64, and ϱu ≈11/64 to obtain more accurate results; hence we choose k = 7. OPTIMAL CONTROL OF LINEAR TD SYSTEMS BY CHEBYSHEV WAVELETS the piecewise-deﬁned function containing eleven sub-functions. Although increasing the number of subintervals is undesirable, accurate information about the location of boundary points and the values of sub-functions is important so that by using a smooth curve ﬁtting technique, we can replace the piecewise-deﬁned function by an alternative function; for example, x∗ 2(t) ∼= g(t), t ∈[0, 0.5]. In Table 6 a comparison is made, so it is found that the results of applying this method are in good agreement. Table 6. Comparison of estimated optimal cost with different methods (Example 7). Source Method J∗ M. Malek-Zavarei, M. Jamshidi [1] sensitivity approach 24.0200500 M. Malek-Zavarei, M. Jamshidi [1] nondelay conversion approach 22.0472483 This research Ch. W. k = 7 and M = 5 22.467638686 This research Ch. W. k = 7 and M = 8 22.467638704 0 0.5 1 1.5 2 2.5 3 −5 −4 −3 −2 −1 0 1 2 Optimal states; k = 7 & M = 8 t xi (t) x1 (t) x2 (t) 0 0.5 1 1.5 2 2.5 3 −15 −10 −5 0 Optimal control; k = 7 & M = 8 t u(t) u(t) Figure 8. Optimal states and control for Example 7. Table 6. Comparison of estimated optimal cost with different methods (Example 7). Source Method J∗ M. Malek-Zavarei, M. Jamshidi [1] sensitivity approach 24.0200500 M. Malek-Zavarei, M. Jamshidi [1] nondelay conversion approach 22.0472483 This research Ch. W. k = 7 and M = 5 22.467638686 This research Ch. W. k = 7 and M = 8 22.467638704 0 0.5 1 1.5 2 2.5 3 −5 −4 −3 −2 −1 0 1 2 Optimal states; k = 7 & M = 8 t xi (t) x1 (t) x2 (t) 0 0.5 1 1.5 2 2.5 3 −15 −10 −5 0 Optimal control; k = 7 & M = 8 t u(t) u(t) Figure 8. Optimal states and control for Example 7. 4.7. Example 7 By selecting M = 5, 8 and using the proposed approach, the QP is solved in MATLAB within, in turn, 2.311 and 4.324 seconds. x∗(t) and u∗(t) are plotted in Fig.8. As mentioned in section 4.2, for the optimal state x∗ 2, one boundary point seems to be t ≈0.5, while we ﬁnd x∗ 2 on the interval [0, 0.5] as Using the procedure in case II of Example 2, we set ϱx1 ≈21/64, ϱx2 ≈17/64, and ϱu ≈11/64 to obtain more accurate results; hence we choose k = 7. By selecting M = 5, 8 and using the proposed approach, the QP is solved in MATLAB within, in turn, 2.311 and 4.324 seconds. x∗(t) and u∗(t) are plotted in Fig.8. As mentioned in section 4.2, for the optimal state x∗ 2, one boundary point seems to be t ≈0.5, while we ﬁnd x∗ 2 on the interval [0, 0.5] as Stat., Optim. Inf. Comput. Vol. 5, December 2017 322 4.8. Example 8 Consider an example of a typical control problem occurring in the chemical and petroleum industries with transport lag introduced by D.W. Ross [26]. This problem also investigated in [27] by using Lyapunov redesign technique for linear TD systems. The system described by ˙x(t) =   −4.93 −1.01 0 0 −3.20 −5.30 −12.8 0 6.40 0.347 −32.5 −1.04 0 0.833 11.0 −3.96  x(t) +   1.92 0 0 0 0 1.92 0 0 0 0 1.87 0 0 0 0 0.724  x(t −1) +   1 0 0 1 0 0 0 0  u(t), (89) ˙x(t) =   −4.93 −1.01 0 0 −3.20 −5.30 −12.8 0 6.40 0.347 −32.5 −1.04 0 0.833 11.0 −3.96  x(t) +   1.92 0 0 0 0 1.92 0 0 0 0 1.87 0 0 0 0 0.724  x(t −1) +   1 0 0 1 0 0 0 0  u(t), (89) where x(t) = [x1(t), x2(t), x3(t), x4(t)]⊤and u(t) = [u1(t), u2(t)]⊤; the performance criterion is J = Z ∞ 0        x⊤(t)   1 0 0 0 0 10 0 0 0 0 1 0 0 0 0 100  x(t) + u⊤(t)u(t)        dt, (90) (90) where we have where we have α(t) = [0.042, 0.040, 0.035, 0.030]⊤, −1 ≤t ≤0. (91) α(t) = [0.042, 0.040, 0.035, 0.030]⊤, −1 ≤t ≤0. (91) where we have α(t) = [0.042, 0.040, 0.035, 0.030]⊤, −1 ≤t ≤0. (91) (91) Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 Stat., Optim. Inf. Comput. Vol. 5, December 2017 323 IMAN MALMIR We ﬁrst determine the value of k. Since the proposed method does not deal with inﬁnite time problems, we consider the ﬁnite horizon version of this problem; so, assume tf = 8. We select k = 5 and M = 8. By using Remarks 1 and 3 and implementation of the algorithm in MATLAB, we solve the problem easily. The optimal states and inputs as Fig.9 are obtained. We can see in the obtained curves that the convergence rate of the presented method is higher than the convergence rate of the method developed in [27]. 4.8. Example 8 0 1 2 3 4 5 6 7 8 −0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 t x(t) Optimal states; k = 5 & M = 8 x1 (t) x2 (t) x3 (t) x4 (t) 0 1 2 3 4 5 6 7 8 −0.18 −0.16 −0.14 −0.12 −0.1 −0.08 −0.06 −0.04 −0.02 0 0.02 t u(t) Optimal inputs; k = 5 & M = 8 u1 (t) u2 (t) Figure 9. Optimal states and controls for Example 8. 0 1 2 3 4 5 6 7 8 −0.18 −0.16 −0.14 −0.12 −0.1 −0.08 −0.06 −0.04 −0.02 0 0.02 t u(t) Optimal inputs; k = 5 & M = 8 u1 (t) u2 *(t) Figure 9. Optimal states and controls for Example 8. 5. Conclusion In this paper a state and control parameterization method is introduced to solve optimal control of linear time- varying multiple delays system which containing inverse time. We have transformed the original TD problem into a new one and obtained the optimal trajectory, control and PI by solving a simple QP problem instead of using of such techniques can lead to major difﬁculties, such as the generalized Riccati method. The problems with combined state and control constraints can be handled by this method. Furthermore, since the method can incorporate the initial and ﬁnal conditions and interior point constraints directly into the algorithm, there is no need for the separate operations of applying these conditions to the obtained solutions. In a minimum-energy system, it is interesting to see how changing the parameters like terminal time, lags and control weighted matrix affects on the PI. An efﬁcient method for solving a TD optimal control problem should easily be able to resolve the problem for various cases, such as changing the values of delays, initial vector functions and weighting matrices; the proposed method has fairly this capability. Also, illustrative examples demonstrate that this wavelet-based method compares very well with other methods in the accuracy and order of approximation. It is worth mentioning that the convergence of the method is clearly evident in the obtained curves. The presented QP model of a TD optimal control problem has a very simple structure and implementation of it is easy and convenient. As we have seen, it is important to ﬁnd accurate information about the solutions in terms of the location of boundary points and polynomial equations of sub-functions to achieve high accuracy. Since the product of the delays and the number of subintervals are not often integers, the accuracy of the method may vary with the choice of k. We have seen that the accuracy may be improved by increasing k. Of course, this enhanced accuracy is usually obtained at a cost-namely, increased computation time; hence future work includes improving the choice of k to match systems with different delays. Acknowledgement The author highly appreciates the editors and two anonymous referees for their detailed comments and constructive criticisms that greatly improved this paper. Stat., Optim. Inf. Comput. Vol. 5, December 2017 324 REFERENCES alek-Zavarei, M. Jamshidi, Time-Delay Systems: Analysis, Optimization and Applications, North-Holland, 1978. 2. K.B. Datta, B.M. Mohan, Orthogonal functions in systems and control, Advanced Series in Electrical and Computer Engineering, World Scientiﬁc Publishing Co., Vol. 9, 1995. g 3. G.L. Kharatishvili. 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Kumar Kar, Orthogonal functions approach to optimal control of delay systems with reverse ti the Franklin Institute, 347, no. 9, pp. 1723–1739, 2010. , , , pp , 24. H.L. Liu, G.Y. Tang, S.Y. Han, Optimal Control for Linear Time-Varying Systems with Multiple Time-Delays, Proceedings of 2011 International Conference on Modelling, Identiﬁcation and Control, Shanghai, China, pp. 387–393, 2011. 25. H.L. Liu, G.Y. Tang, X. Yang, Minimum-energy control for time-varying systems with multiple state and input delays, International Journal of Systems Science, 47, no. 12, pp. 3036–3043, 2016. y pp 26. D.W. Ross, Controller design for time lag systems via a quadratic criterion, IEEE Transactions on Automat pp. 664–672, 1971. 27. L.R. Guerrero, S. Mondi´e, O.J. S. S´anchez, Guaranteed Cost Control Using Lyapunov Redesign for Uncertain Linear Time Delay Systems, IFAC Workshop on Time Delay Systems, June 28-30, Ann Arbor, MI, USA, 48, no. 12, pp. 392–397, 2015. Stat., Optim. Inf. Comput. 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https://openalex.org/W4237243743	https://www.qeios.com/read/15TNMV/pdf	English	null	Giant Cell Fibroblastoma	Definitions	2,020	cc-by	84	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Giant Cell Fibroblastoma National Cancer Institute National Cancer Institute Qeios ID: 15TNMV · https://doi.org/10.32388/15TNMV Source National Cancer Institute. Giant Cell Fibroblastoma. NCI Thesaurus. Code C4700. A rare, locally aggressive fibroblastic neoplasm typically affecting young patients (predominantly boys). It manifests with painless nodules in the dermis or subcutaneous tissues. Morphologically, it contains giant cells and wide vessel-like spaces. This tumor can recur but metastases have not been reported. Qeios ID: 15TNMV · https://doi.org/10.32388/15TNMV 1/1
https://openalex.org/W4310987533	https://link.springer.com/content/pdf/10.1134/S0021364022602512.pdf	English	null	Repulsion of a Néel-Type Skyrmion from a Pearl Vortex in Thin Ferromagnet–Superconductor Heterostructures	JETP letters	2,022	cc-by	6,346	INTRODUCTION a chiral ferromagnetic film due to stray fields (see Fig. 1). Contrary to [28], we take into account the change of the skyrmion profile due to magnetic field induced by the Pearl vortex. Performing non-pertur- bative treatment of the effect of vortex magnetic field on the one hand by analytical approach and on the other hand by micromagnetic modeling, we find that the free energy of the system as a function of the dis- tance a between the skyrmion and the vortex experi- ences drastic changes with increase in the dimension- less strength γ of the vortex magnetic field, cf. Eq. (6). In particular, has (i) the single minimum at for , (ii) two minima at and at with for , (iii) two minima at and at with for , and (iv) the single minimum at for (cf. Fig. 2). With increase in γ the distance between the centers of a Néel-type skyrmion and a Pearl vortex is reduced and jumps to zero abruptly at (cf. Fig. 3). All three critical values and depend on the dimensionless DMI strength (cf. Fig. 4). In gen- eral, we can make somewhat counter-intuitive state- ment: the repulsion between the skyrmion and the vortex is suppressed with increase in the dimensionless vortex magnetic field. F F min = > 0 a a − γ γcr, < = 0 a min = a a F F min (0) > ( ) a − γ γ γ cr, cr < < = 0 a min = a a F F min (0) < ( ) a + γ γ γ cr cr, < < = 0 a + γ γ cr, < min a + γ γcr, = ± γcr, γcr Mutual influence of magnetism and superconduc- tivity in heterostructures has long history of research [1–5]. Recently, superconductor–ferromagnet (SF) bilayers hosting topologically nontrivial magnetic configurations have attracted much attention [6–8]. Such topologically stable configurations can be stabi- lized by Dzyaloshinskii–Moriya interaction (DMI) in ferromagnetic films [9]. Skyrmions in SF heterostruc- tures induce Yu–Shiba–Rusinov-type bound states [10, 11], host Majorana modes [12–20], affect the Josephson effect [21], and change the superconduct- ing critical temperature [22]. Skyrmions and superconducting vortices can form bound pairs in SF heterostructures due to interplay of spin–orbit coupling and proximity effect [23, 24]. In addition, vortices and skyrmions interact via stray fields [25–28]. Repulsion of a Néel-Type Skyrmion from a Pearl Vortex in Thin Ferromagnet–Superconductor Heterostructures E. S. Andriyakhinaa, b, S. Apostoloffa, and I. S. Burmistrova, c, * a Landau Institute for Theoretical Physics, Russian Academy of Sciences, Chernogolovka, Moscow region, 142432 Russia b Moscow Institute for Physics and Technology (National Research University), Moscow, 141700 Russia c Laboratory for Condensed Matter Physics, National Research University Higher School of Economics, Moscow, 101000 Russia e-mail: burmi@itp.ac.ru Received October 16, 2022; revised October 22, 2022; accepted October 25, 2022 Repulsion of a Néel-Type Skyrmion from a Pearl Vortex in Thin Ferromagnet–Superconductor Heterostructures E. S. Andriyakhinaa, b, S. Apostoloffa, and I. S. Burmistrova, c, a Landau Institute for Theoretical Physics, Russian Academy of Sciences, Chernogolovka, Moscow region, 142432 Russia b Moscow Institute for Physics and Technology (National Research University), Moscow, 141700 Russia c Laboratory for Condensed Matter Physics, National Research University Higher School of Economics, Moscow, 101000 Russia *e-mail: burmi@itp.ac.ru Received October 16, 2022; revised October 22, 2022; accepted October 25, 2022 Received October 16, 2022; revised October 22, 2022; accepted October 25, 2022 In this paper we study repulsion of a Néel-type skyrmion in a chiral ferromagnetic film from a superconduct- ing Pearl vortex due to the stray fields. Taking into account an effect of the vortex magnetic field on the sky- rmion non-perturbatively, we find that the repulsion between them is suppressed with increase in the dimen- sionless strength of the vortex magnetic field. This manifests itself in complicated evolution of the free energy with increase in the vortex magnetic field and reduction of the equilibrium distance between the centers of Néel-type skyrmion and Pearl vortex. ISSN 0021-3640, JETP Letters, 2022, Vol. 116, No. 11, pp. 825–830. © The Author(s), 2022. This article is an open access publication. ISSN 0021-3640, JETP Letters, 2022. © The Author(s), 2022. This article is an open access publication. Russian Text © The Author(s), 2022, published in Pis’ma v Zhurnal Eksperimental’noi i Teoreticheskoi Fiziki, 2022, Vol. 116, No. 11, pp. 801–807. ISSN 0021-3640, JETP Letters, 2022, Vol. 116, No. 11, pp. 825–830. © The Author(s), 2022. This article is an open access publication. ISSN 0021-3640, JETP Letters, 2022. © The Author(s), 2022. This article is an open access publication. Russian Text © The Author(s), 2022, published in Pis’ma v Zhurnal Eksperimental’noi i Teoreticheskoi Fiziki, 2022, Vol. 116, No. 11, pp. 801–807. SKYRMION–VORTEX INTERACTION Minimizing the free energy with with respect to a skyrmion angle , one can derive the Euler–Lagrange equation, m F[ ] BV = 0 θ( )r Following [28], our setup consists of ferromagnetic and superconducting films of thicknesses dF and dS, respectively. We assume that both films are thin, dS ≪λL and dF ≪ R, where λL is the London penetra- tion length and denotes the skyrmion radius. In addi- tion, we assume the presence of a thin insulating layer between superconducting and ferromagnetic films in order to suppress the proximity effect. The supercon- ducting film hosts a pair of Pearl vortex and antivortex separated by a distance much larger than the Pearl pen- etration length [30] (see Fig. 1). R λ λ2 = / L S d (4) + θ Δ θ − θ + ℓ ℓ ℓ ε 2 2 2 2 2 ( ) sin ( ) ( ) sin 2 ( ) 2 = 0. / 2 w w r w r r r r r r (4) Here a dimensionless parameter con- trols the strength of DMI, the domain wall width sets a natural length scale in the problem and is the radial part of Laplacian. ε = /2 D AK ℓ = / w A K Δ θ ∂ ∂θ = ( )/ r r r r r In order to solve Eq. (4) one needs to specify the boundary conditions. Assuming naturally that the film is ferromagnetically magnetized away from the skyr- mion, ( ). At the center of the skyrmion magnetization has the opposite direction, . Since at only skyrmions of positive chi- rality are stabilized in the ferromagnet, corre- sponds to the condition . θ →∞ ( ) = 0 r = 1 z m − = 1 z m ε > 0 − = 1 z m θ → π ( 0) = r The free energy of a thin chiral ferromagnetic film subjected to the magnetic field produced by a Pearl vortex is given by BV (1) + = ∇ + ∇⋅ − ⋅∇ + − − ⋅  m r m m m m B F 2 2 2 V = 0 [ ] { ( ) [ ( ) ] (1 ) \| }. SHIFTED SKYRMION AT γ →0 In the absence of the vortex the magnetization cor- responding to a Néel-type skyrmion can be sought in the cylindrical coordinate system as [32] (3) θ + θ m e e = sin ( ) cos ( ). r z r r (3) INTRODUCTION Recently, stable skyrmion-vortex coexistence has been experimentally observed in [Ir1Fe0.5Co0.5Pt1]10/MgO/Nb sandwich structure [29]. In [28] two of the authors predicted that a Néel-type skyrmion and a Pearl vortex interacting via stray fields are repelled from each other to be located at a finite distance. However, the analysis of [28] has been limited to the lowest order perturbation theory in the magnetic field induced by the vortex. In this work, we study interaction between a super- conducting Pearl vortex and a Néel-type skyrmion in 825 ANDRIYAKHINA et al. 826 Fig. 1. (Color online) Sketch of a ferromagnet (blue)– superconductor (green) heterostructure. A thin insulating layer is not shown. The ferromagnetic layer hosts a Néel- type skyrmion (Sk). The superconducting film hosts a vor- tex (V). The distance between their centers is a. where denotes the flux quantum. The free energy is normalized in such a way that for the ferromagnetic state, , and without the Pearl vortex, . We note that we neglect interac- tion between the skyrmion and antivortex situated at a large distance away. φ0 = /2 hc e m F[ ] F = 0 = 1 z m BV = 0 Fig. 1. (Color online) Sketch of a ferromagnet (blue)– superconductor (green) heterostructure. A thin insulating layer is not shown. The ferromagnetic layer hosts a Néel- type skyrmion (Sk). The superconducting film hosts a vor- tex (V). The distance between their centers is a. Fig. 1. (Color online) Sketch of a ferromagnet (blue)– superconductor (green) heterostructure. A thin insulating layer is not shown. The ferromagnetic layer hosts a Néel- type skyrmion (Sk). The superconducting film hosts a vor- tex (V). The distance between their centers is a. SKYRMION–VORTEX INTERACTION F z z z s z d d A D m m K m M Here, is the unit vector along direction of the magnetization M, stands for saturation magnetiza- tion of the film. The exchange, DMI, and perpendic- ular anisotropy energy constants are denoted as A, D, and K, respectively. We assume that these parameters are positive, . The magnetic field of the Pearl vortex centered at the position with coordinate a is given as [31]: m( )r s M , , > 0 A K D Knowing the solution of Eq. (4) (in the absence of the vortex magnetic field), we can cal- culate [28] the interaction energy as a function of dis- tance between the centers of the skyrmion and the vortex, θ θ0 ( ) = ( ) r r a (5) ∞ δ π γ θ + θ −  F 0 0 0 ( ) 4 = { ( )sin ( ) ( )[cos ( ) 1]}, F a a r z a Ad drr b r r b r r (5) δ π F( ) 4 F a Ad (5) g [ ] (2) − + − φ ∇ + λ π  q r a q B 2 \| \| ( ) V 0 2 = sgn( ) , (1 2 ) (2 ) q z i d e z q q (5 ∞ π γ θ + θ −  0 0 0 4 = { ( )sin ( ) ( )[cos ( ) 1]}, F a a r z Ad drr b r r b r r Fig. 2. (Color online) Sketch of dependence for different ranges of γ. Left panel, : There is the single minimum at nonzero distance amin. Middle panel with : There is the global minimum at nonzero distance amin and the local one at . Middle panel with : There is the global minimum at zero distance and the local one at . Right panel, : There is the single minimum at . NEARLY CENTERED SKYRMION, →0 a Now we study at what parameters the coaxial con- figuration of the skyrmion and the vortex ( ) is unstable. The magnetization of such skyrmion also can be sought as given by Eq. (3), due to the radial symmetry of the problem. Then the minimization of the free energy with respect to the skyrmion angle yields the Euler–Lagrange equation similar to Eq. (4), but with instead of zero in the right hand side. Boundary con- ditions remain the same: and . = 0 a m F[ ] θ( )r γ θ − θ 0 0 [ ( )cos ( ) ( )sin ( )] r z b r r b r r θ →∞ ( ) = 0 r θ →∞ π ( ) = r Now we study at what parameters the coaxial con- figuration of the skyrmion and the vortex ( ) is unstable. The magnetization of such skyrmion also can be sought as given by Eq. (3), due to the radial symmetry of the problem. Then the minimization of the free energy with respect to the skyrmion angle yields the Euler–Lagrange equation similar = 0 a m F[ ] θ( )r (7) ∞ − λ +  ℓ 0 1/0 / 1 0 ( ) ( ) ( ) = 2 . ( 2 ) a r z w dqqJ qa J qr b r q (7) Assuming the skyrmion radius to be much smaller than the Pearl length, , that requires , we can treat the functions in the limit as λ ! R λ ! 2 / S L d R / ( ) a r z b r λ ∼ ! r R (8)   ≈   π + +   ≈ Θ − ℓ ℓ 2 2 4 ( ) , ( ) ( ) ( ) ( / ) ( ). a w z a r w ar b r K a r a r b r r r a When the solution for a finite magni- tude of γ is obtained, we can calculate the interaction energy for small . The idea is the same as beyond Eq. (5). For small spacing , one can neglect the reshaping of the skyrmion in the leading approxi- mation and use Eq. (5), but with , γ θ θ ( ) = ( ) r r δF( ) a ℓ ! SKYRMION–VORTEX INTERACTION F( ) a − γ γcr, < − γ γ γ cr, cr < < = 0 a + γ γ γ cr cr, < < = 0 a min a + γ γ cr, < = 0 a (2) − + − φ ∇ + λ π  q r a q B 2 \| \| ( ) V 0 2 = sgn( ) , (1 2 ) (2 ) q z i d e z q q γ θ + θ −  0 0 0 = { ( )sin ( ) ( )[cos ( ) 1]}, a a r z drr b r r b r r (2) Fig. 2. (Color online) Sketch of dependence for different ranges of γ. Left panel, : There is the single minimum at nonzero distance amin. Middle panel with : There is the global minimum at nonzero distance amin and the local one at . Middle panel with : There is the global minimum at zero distance and the local one at . Right panel, : There is the single minimum at . F( ) a − γ γcr, < − γ γ γ cr, cr < < = 0 a + γ γ γ cr cr, < < = 0 a min a + γ γ cr, < = 0 a JETP LETTERS Vol. 116 No. 11 2022 REPULSION OF A NÉEL-TYPE SKYRMION 827 Fig. 4. (Color online) Phase diagram. The solid curve shows dependence extracted from micromagnetic simulations. The black dashed curve corresponds to . In the blue shaded region, the distance between the skyrmion and the vortex is nonzero, amin > 0, cf. Fig. 2. The points A–F correspond to the respective panels (a–f) in Fig. 5. γ ε cr( ) − γ ε cr, ( ) Fig. 3. (Color online) Distance amin between the skyrmion and the vortex versus for several values of γ ranging from 0.01 to 0.3. The dashed curve illustrates analytical predic- tion for , see text below Eq. (8). For each value of the distance amin varies from to . Once γ becomes larger than the system reaches coaxial phase. The shaded regions guide the con- stant value of the vortex strength γ, see the color bar inset. ε ε min( ) a γ →0 ε γ → min( 0) a γ min cr ( ) a γcr Fig. 4. (6) Equation (5) is valid in the leading approximation on small vortex strength . Notation means that we subtract the energy of the lonely skyrmion itself and the energy of the homogeneous ferromagnet in the vortex field from the total free energy . The functions and means the dimensionless r- and z-projections of the vortex field averaged by rotation of the system around the center of the skyr- mion, i.e., over all possible directions of the vector a. They are given as γ ! 1 δF m F[ ] ( ) a rb r ( ) a zb r BV Equation (5) is valid in the leading approximation on small vortex strength . Notation means that we subtract the energy of the lonely skyrmion itself and the energy of the homogeneous ferromagnet in the vortex field from the total free energy . The γ ! 1 δF m F[ ] SKYRMION–VORTEX INTERACTION (Color online) Phase diagram. The solid curve shows dependence extracted from micromagnetic simulations. The black dashed curve corresponds to . In the blue shaded region, the distance between the skyrmion and the vortex is nonzero, amin > 0, cf. Fig. 2. The points A–F correspond to the respective panels (a–f) in Fig. 5. γ ε cr( ) − γ ε cr, ( ) Fig. 3. (Color online) Distance amin between the skyrmion Fig. 3. (Color online) Distance amin between the skyrmion and the vortex versus for several values of γ ranging from 0.01 to 0.3. The dashed curve illustrates analytical predic- tion for , see text below Eq. (8). For each value of the distance amin varies from to . Once γ becomes larger than the system reaches coaxial phase. The shaded regions guide the con- stant value of the vortex strength γ, see the color bar inset. ε ε min( ) a γ →0 ε γ → min( 0) a γ min cr ( ) a γcr Fig. 3. (Color online) Distance amin between the skyrmion and the vortex versus for several values of γ ranging from 0.01 to 0.3. The dashed curve illustrates analytical predic- tion for , see text below Eq. (8). For each value of the distance amin varies from to . Once γ becomes larger than the system reaches coaxial phase. The shaded regions guide the con- stant value of the vortex strength γ, see the color bar inset. ε ε min( ) a γ →0 ε γ → min( 0) a γ min cr ( ) a γcr It should be emphasized that in the analysis above we neglect a change of the skyrmion profile due to the vortex field when calculating Eq. (5). Indeed, such a reshaping should be taken into account in the next approximation on small vortex strength γ. For small γ an account of the next order correction leads only to a small correction of the magnitude of amin, but does not change the result qualitatively. where the dimensionless strength γ of the vortex mag- netic field is given as (6) γ λ φ π ℓ 0 = ( / )( /8 ). w s M A MICROMAGNETIC MODELING To explore the skyrmion stable state in the field of the Pearl vortex with change of and γ, we perform micromagnetic simulations. We utilize Object Ori- ented MicroMagnetic Framework (OOMMF) [33] by dint of Ubermag [34] Python packages. ε The skyrmion profiles obtained by means of micro- magnetic simulations for and γ corresponding to the points A, B, and C in Fig. 4 are presented in Fig. 5 (upper row). As expected, in all three cases skyrmions are situated right on the top of the vortex, i.e., . In lower row of Fig. 5 we demonstrate skyr- mions for parameters and γ corresponding to the points D, E, and F in Fig. 4. In this case the nonzero distance of the skyrmion from the vortex is clearly seen. ε min = 0 a ε The system is modeled as a set of classical magnetic vectors placed in the center of mesh cells. The distance is measured in units of the domain wall width . We note that for the micromagnetic modeling we set the magnetic anisotropy parameter and DMI . Periodical boundary conditions (in -plane) are imposed to simulate an isolated region of a ferro- magnet–insulator-superconductor structure. We cre- ate a skyrmion by initiating a closed region with a flipped magnetization and letting it relax in the pres- ence of the Heisenberg exchange, DMI and magnetic anisotropy interactions as well as in the vortex- induced magnetic field. ℓw = 1 K ε = 2 D xy We draw a reader’s attention to the spatial structure of the magnetization of the shifted skyrmions corre- sponding to the points D, E, and F. Naturally, as the effective strength γ of the vortex magnetic field increases, the magnetization profile deviates from the radially symmetric one. This leads to the following. Firstly, the center of the skyrmion is getting closer the center of the vortex, and secondly, the radius of the skyrmion increases. In addition to that, the projection of the magnetization on the plane (shown as black arrows in Fig. 5) partially aligns along the direction of the magnetic field. These changes in the profile lead to a decrease in the total free energy of the dis- placed skyrmion as a function of γ. However, the free energy of a skyrmion placed exactly above the vortex decreases faster with increasing γ. NEARLY CENTERED SKYRMION, →0 a In our simulations we have found that by making the mesh finer, one observes the declining tendency for towards the theoretical value marked by the dashed curve in Fig. 3. However, for small values of one needs to account both for smaller mesh cells and for larger sample size, which is computationally expensive. γ →0 min a ε min a ε ε min a ε Here we introduce . At r ~ and these functions can be estimated as δ − 0 / / / ( ) = ( ) ( ) a a r z r z r z b r b r b r λ R ! →0 a and . Note that though the result (9) has been obtained in the limit of small , it allows to predict the existence of a minimum of at for an arbitrary value . δ ≈ℓ 2 3 ( ) /(4 ) a z w b r a r δ ≈ λ ℓ 2 2 ( ) /(8 ) a r w b r a r a F( ) a = 0 a γ > 0 At and for the free energy has a maximum at , because . Hence, the skyrmion is repelled from the vortex in agreement with [28]. With increase in γ the difference changes sign at and the mini- mum at appears in the free energy. Therefore, for the skyrmion can be stable right at the vor- tex. The dependence of on extracted from Eq. (9) is shown in Fig. 4 by the dashed curve. As one can see decreases with increase in and vanishes at . We expect similar dependence of on . Our theoretical analysis demonstrates that vanishes at which is only slightly larger than . We note that vanishes at the value of which lies between and . We stress that with relevant precision . NEARLY CENTERED SKYRMION, →0 a w a a γθ ( )r (8) Here, denotes the Heaviside step function and is the complete elliptic integral of the first kind. Θ( )z ( ) K z The minimum of determines the stable posi- tion amin of the skyrmion. The resulting dependence of amin on at is shown in Fig. 3 by the dashed line. As one can see, amin is of the order of and decreases with increase in . δF( ) a ε γ →0 ℓ 2 w ε (9) ∞ γ γ δ −δ γ δ θ π + δ θ −  F F 0 ( ) (0) = { ( )sin ( ) 4 ( )[cos ( ) 1]}. a r F a z a drr b r r Ad b r r (9) ∞ γ δ −δ γ δ θ π  F F 0 ( ) (0) = { ( )sin ( ) 4 a r F a drr b r r Ad (9) γ + δ θ − ( )[cos ( ) 1]}. a zb r r γ + δ θ − ( )[cos ( ) 1]}. a zb r r JETP LETTERS Vol. 116 No. 11 2022 ANDRIYAKHINA et al. 828 Here we introduce . At r ~ and these functions can be estimated as and . Note that though the result (9) has been obtained in the limit of small , it allows to predict the existence of a minimum of at for an arbitrary value . δ − 0 / / / ( ) = ( ) ( ) a a r z r z r z b r b r b r λ R ! →0 a δ ≈ℓ 2 3 ( ) /(4 ) a z w b r a r δ ≈ λ ℓ 2 2 ( ) /(8 ) a r w b r a r a F( ) a = 0 a γ > 0 in the case of , decreases with increase in at constant magnitude of γ. Similarly, decreases with the increase in γ at a constant value of . We note a discrepancy between the theory and the micromag- netic simulations that becomes more prominent for smaller , see Fig. 3. We believe that this happens due to discretization effects which are inevitable in the numerical approach. NEARLY CENTERED SKYRMION, →0 a γ →0 −≈ ε εcr, < 0.488 = 0 a δ δ F F ( ) < (0) a δ −δ F F ( ) (0) a − γ γcr, = = 0 a − γ γcr, > − γcr, ε − γcr, ε −≈ εcr, 0.488 + γcr, ε + γcr, + ≈ εcr, 0.493 − εcr, γcr ≈ εcr 0.491 − εcr, + εcr, − + ≈ ε ε ε cr, cr cr, 0.49 The results of micromagnetic simulations are con- sistent with evolution of the free energy with γ illustrated in Fig. 2 schematically. The phase diagram in and γ plane extracted from the obtained results is shown in Fig. 4. Two phases of stable position of the skyrmion, right on the top of the vortex (white clear upper region) and at the finite distance (blue shaded lower region), are separated by the solid line . One can see that drops to zero when reaches approxi- mately 0.49 in accordance with theoretical predictions. As we have mentioned above there is the lower and upper critical values at which minimum at disappears and minimum at appears, respectively. However, we cannot resolve these values within our micromagnetic modeling. ε min a γ γ ε cr = ( ) γcr ε γ ∓ε cr, ( ) min = > 0 a a = 0 a SUMMARY skyrmion becomes greater than the energy of the sky- rmion located exactly above the vortex. In this work, we have extended the study of interac- tion between a superconducting Pearl vortex and a Néel-type skyrmion in a chiral ferromagnetic film to non-perturbative regime with respect to the stray fields induced by the vortex. Contrary to the previous work [28] of two of us limited to the regime of a weak dimensionless vortex magnetic field, , we have found that the increase in γ suppresses repulsion of skyrmion and vortex and leads to reduction of the dis- tance between the centers of a Néel-type skyr- mion and a Pearl vortex as shown in Fig. 3. Most sur- prisingly, we have discovered the existence of interest- ing evolution of the free energy of the system with γ. In particular, at the free energy has the only minimum at whereas at it has the only minimum at , see Figs. 2 and 4. γ →0 min a − γ γcr, < F( ) a min = a a + γ γcr, > = 0 a To clarify significance of critical values of dimen- sionless vortex strength and let us consider the case of small but finite concentration of skyrmions and vortices. Then at one can expect the phase of (dipole-like) pairs consisting of skyrmion and vortex separated by a distance . At one can observe the phase of (point-like) pairs of skyrmion and vortex sitting on the top of each other. In the inter- mediate range there is the phase in which there are finite concentrations of dipole-like and point-like skyrmion-vortex pairs. Under assump- tion that the system can reach the global minimum of the free energy, there will be a true thermodynamic transition at between the phases with dipole-like and point-like pairs, respectively. Since the dimen- sionless vortex strength γ depends on material param- eters, see Eq. (6), and is proportional to the thickness of superconducting film , it could be possible to see the transitions described above with thickness change. ± γcr, γcr − γ γcr, < min a + γ γcr, > − + γ γ γ cr, cr, < < γcr S d Finally, we mention that it would be interesting to generalize our results to the case of skyrmions and vor- tices in confined geometries, e.g., nanodots etc. MICROMAGNETIC MODELING As a con- sequence, when γ exceeds , the energy of the shifted xy F min ( ) a F( = 0) a γcr We point out that the system is not subject to any other external fields, so that the only source of Zee- man energy comes from the interaction with the vor- tex. In our simulations we consider a Pearl vortex with zero-sized core to be pinned in the origin of the grid, see Eq. (2). The micromagnetic simulations allow us to find the dependence of the distance between the skyrmion and the vortex on for a finite value of γ, see Fig. 3. As min a ε JETP LETTERS Vol. 116 No. 11 2022 REPULSION OF A NÉEL-TYPE SKYRMION 829 Fig. 5. (Color online) Distributions of magnetization for different values of and γ. The Pearl vortex is located at the center of each picture, i.e., at . The white contours illustrate a level set of the projection. The most extreme contour in each panel separates the skyrmion magnetization from purely ferromagnetic spin ordering. The black arrows reflect the magnitude and direction of the projection of the magnetization m on the plane. The upper row corresponds to vortex strength γ slightly above the critical , when skyrmion abruptly changes its position and becomes coaxial with the vortex. The lower row illustrates dis- tribution of for γ which is slightly less than , i.e., for the case of nonzero amin. One can clearly see that the skyrmions in the lower row are shifted from the sample’s center where the Pearl vortex is settled. The panels (a–f) match with the respective points A–F marked in Fig. 4. (a) (b) (c) (d) (e) (f) ε = = 0 x y z m xy γcr z m γcr (b) (a) (c) (d) (d) (f) (e) (f) (e) olor online) Distributions of magnetization for different values of and γ. The Pearl vortex is located at the ε s of and γ. The Pearl vortex is located at the center o ε Fig. 5. (Color online) Distributions of magnetization for different values of and γ. The Pearl vortex is located at the center of each picture, i.e., at . The white contours illustrate a level set of the projection. The most extreme contour in each panel separates the skyrmion magnetization from purely ferromagnetic spin ordering. MICROMAGNETIC MODELING The black arrows reflect the magnitude and direction of the projection of the magnetization m on the plane. The upper row corresponds to vortex strength γ slightly above the critical , when skyrmion abruptly changes its position and becomes coaxial with the vortex. The lower row illustrates dis- tribution of for γ which is slightly less than , i.e., for the case of nonzero amin. One can clearly see that the skyrmions in the lower row are shifted from the sample’s center where the Pearl vortex is settled. The panels (a–f) match with the respective points A–F marked in Fig. 4. ε = = 0 x y z m xy γcr z m γcr ACKNOWLEDGMENTS We are grateful to A. Fraerman, M. Kuznetsov, and M. Shustin for useful discussions and to O. Tretiakov and We are grateful to A. Fraerman, M. Kuznetsov, and M. Shustin for useful discussions and to O. Tretiakov and P. Vorobyev for collaboration on a related project. We acknowledge the computing time provided to us at com- puter facilities at Landau Institute. 12. W. Chen and A. P. Schnyder, Phys. Rev. B 92, 214502 (2015). P. Vorobyev for collaboration on a related project. We acknowledge the computing time provided to us at com- puter facilities at Landau Institute. 13. G. Yang, P. Stano, J. Klinovaja, and D. Loss, Phys. Rev. B 93, 224505 (2016). 14. U. Güngördü, S. Sandhoefner, and A. A. Kovalev, Phys. Rev. B 97, 115136 (2018). 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https://openalex.org/W2143916184	https://pure.mpg.de/pubman/item/item_3162785_1/component/file_3162786/Cooper_2014.PDF	English	null	You Turn Me Cold: Evidence for Temperature Contagion	PloS one	2,014	cc-by	8,361	OPEN ACCESS Introduction: During social interactions, our own physiological responses influence those of others. Synchronization of physiological (and behavioural) responses can facilitate emotional understanding and group coherence through inter-subjectivity. Here we investigate if observing cues indicating a change in another’s body temperature results in a corresponding temperature change in the observer. Methods: Thirty-six healthy participants (age; 22.9¡3.1 yrs) each observed, then rated, eight purpose-made videos (3 min duration) that depicted actors with either their right or left hand in visibly warm (warm videos) or cold water (cold videos). Citation: Cooper EA, Garlick J, Featherstone E, Voon V, Singer T, et al. (2014) You Turn Me Cold: Evidence for Temperature Contagion. PLoS ONE 9(12): e116126. doi:10.1371/journal.pone. 0116126 Editor: Marcus Gray, The University of Queensland, Australia Editor: Marcus Gray, The University of Queensland, Australia Four control videos with the actors’ hand in front of the water were also shown. Temperature of participant observers’ right and left hands was concurrently measured using a thermistor within a Wheatstone bridge with a theoretical temperature sensitivity of ,0.0001˚C. Temperature data were analysed in a repeated measures ANOVA (temperature 6 actor’s hand 6 observer’s hand). Results: Participants rated the videos showing hands immersed in cold water as being significantly cooler than hands immersed in warm water, F(1,34)5256.67, p,0.001. Participants’ own hands also showed a significant temperature- dependent effect: hands were significantly colder when observing cold vs. warm videos F(1,34)513.83, p50.001 with post-hoc t-test demonstrating a significant reduction in participants’ own left (t(35)523.54, p50.001) and right (t(35)522.33, p50.026) hand temperature during observation of cold videos but no change to warm videos (p.0.1). There was however no evidence of left-right mirroring of these temperature effects p.0.1). Sensitivity to temperature contagion was also predicted by inter-individual differences in self-report empathy. Copyright: 2014 Cooper et al. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and repro- duction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data are accessible at Figshare (http:// dx.doi.org/10.6084/m9.figshare.1248937). Funding: This work was supported by Wellcome Trust Intermediate Clinical Fellowships awarded to NAH; Wellcome Trust Intermediate Clinical Fellowships awarded to VV; and European Research Council support to HDC. Abstract Introduction: During social interactions, our own physiological responses influence those of others. Synchronization of physiological (and behavioural) responses can facilitate emotional understanding and group coherence through inter-subjectivity. Here we investigate if observing cues indicating a change in another’s body temperature results in a corresponding temperature change in the observer. Methods: Thirty-six healthy participants (age; 22.9¡3.1 yrs) each observed, then rated, eight purpose-made videos (3 min duration) that depicted actors with either their right or left hand in visibly warm (warm videos) or cold water (cold videos). Four control videos with the actors’ hand in front of the water were also shown. Temperature of participant observers’ right and left hands was concurrently measured using a thermistor within a Wheatstone bridge with a theoretical temperature sensitivity of ,0.0001˚C. Temperature data were analysed in a repeated measures ANOVA (temperature 6 actor’s hand 6 observer’s hand). Results: Participants rated the videos showing hands immersed in cold water as being significantly cooler than hands immersed in warm water, F(1,34)5256.67, p,0.001. Participants’ own hands also showed a significant temperature- dependent effect: hands were significantly colder when observing cold vs. warm videos F(1,34)513.83, p50.001 with post-hoc t-test demonstrating a significant reduction in participants’ own left (t(35)523.54, p50.001) and right (t(35)522.33, p50.026) hand temperature during observation of cold videos but no change to warm videos (p.0.1). There was however no evidence of left-right mirroring of these temperature effects p.0.1). Sensitivity to temperature contagion was also predicted by inter-individual differences in self-report empathy. n.harrison@bsms.ac.uk . These authors contributed equally to this work. RESEARCH ARTICLE . These authors contributed equally to this work. Ella A. Cooper1., John Garlick2., Eric Featherstone3, Valerie Voon4, Tania Singer2,5, Hugo D. Critchley1,2,6,7, Neil A. Harrison1,2,6,7 Ella A. Cooper1., John Garlick2., Eric Featherstone3, Valerie Voon4, Tania Singer2,5, Hugo D. Critchley1,2,6,7, Neil A. Harrison1,2,6,7* 1. Brighton and Sussex Medical School, University of Sussex, Brighton, United Kingdom, 2. Institute of Cognitive Neuroscience, UCL, London, United Kingdom, 3. Wellcome Trust Centre for Neuroimaging, UCL, London, United Kingdom, 4. Behavioural and Clinical Neurosciences Institute, University of Cambridge, Cambridge, United Kingdom, 5. Department of Social Neuroscience, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany, 6. Sackler Centre for Consciousness Science, University of Sussex, Brighton, United Kingdom, 7. Sussex Partnership NHS Trust, Brighton, United Kingdom *n.harrison@bsms.ac.uk Introduction Adaptive social behavior is dependent on the efficient communication of affective and motivational signals between individuals that together facilitate under- standing of others’ mental and emotional states. In humans, perception of these signals is associated with a marked tendency to mimic, which is well described for emotional facial expressions [1], body postures [2], gesticulations [3] and elements of speech [4]. This tendency, which typically occurs without conscious intent, has been proposed to facilitate emotional understanding across individuals. Empirical data demonstrating correlations between tendency to mimic emotional facial expressions and self-report measures of empathy [5, 6] support the encapsulation of mimicry within the broader concept of empathy [7]. Following de Vignemont and Singer we define empathy as occurring if: ‘(i) one is in an affective state; (ii) this state is isomorphic to another person’s affective state; (iii) this state is elicited by the observation or imagination of another person’s affective state; (iv) one knows that the other person is the source of one’s own affective state’ [8]. In contrast we define the narrower concept of emotional contagion as sharing of affect (points i, ii and iii above) in the absence of an awareness that the other is the source of one’s own affective state (point iv above). Though somatically mediated motor signals have traditionally dominated research in this field, recent evidence has demonstrated that effects of emotion contagion can be observed for facial temperature [52] and have even been observed at the level of hormones [9]. Thus, observing a familiar person or a stranger undergoing a Trier Stress test increases cortisol levels not only in the stressed person but also passive observers watching the scene through a one-way mirror or TV screen. Similarly, evidence for contagion effects have also been observed at the level of the autonomic nervous system in the domain of facial flushing, pupil size and skin temperature. For example, pupillary signals have demonstrated a role in signalling the intensity of sadness [6, 10] and skin temperature the experience of anger [11, 12]. Perceptual sensitivity to another’s pupil size during sadness has also been shown to predict inter-individual differences in empathy [6]. Similar to somatic motor responses, autonomic contagion has also been described during social exchange. For example, during psychotherapy heart rates of therapists and their clients tend to speed up and slow down together [13]. Temperature Contagion Conclusions: We illustrate physiological contagion of temperature in healthy individuals, suggesting that empathetic understanding for primary low-level physiological challenges (as well as more complex emotions) are grounded in somatic simulation. PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 OPEN ACCESS The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Temperature Contagion whether such contagious effects generalize across different axes of the autonomic nervous system is currently unknown. whether such contagious effects generalize across different axes of the autonomic nervous system is currently unknown. Until a decade ago, neuroscientific approaches to the study of empathy were lacking. However, with the discovery of mirror neurons within the premotor cortex, which respond during both performance and observation of a conspecific performing the same action, a potential neural mechanism mediating how we understand other people’s actions and intentions was proposed [15, 16]. Shortly after the formulation of such action–perception models of motor behavior to imitation [17], they were extended to the domain of emotions with the first empathy models [18] suggested that perception of another’s emotional state should automatically activate a similar representation within the viewer together with associated autonomic and somatic responses. Subsequently, a huge number of human functional imaging studies have provided empirical support for such shared networks in the domain of feeling and emotional states. Most have been performed in the domain of pain and show an overlapping anterior insula and anterior midcingulate cortex (aMCC) network underlying both the first-hand experience of pain as well as its observation in others [19, 20] for meta-analyses see [21, 22]. Such shared neuronal networks of empathy have also been observed in the domain of neutral touch [23–25] as well as pleasant and unpleasant touch [26, 27] disgust and taste [28, 29] as well as positive affect such as joy or reward [30, 31]. In sum, multiple studies have found evidence for our human capacity to share affective states with each other, be it at the level of motor mimicry, autonomic or the neuronal activity. To date however, no study has asked whether sharing of autonomic To date however, no study has asked whether sharing of autonomic physiological responses also extends to peripheral skin temperature. All home- othermic animals including humans rigidly regulate their core body temperature through a variety of involuntary thermoregulatory responses, such as shivering and non-shivering thermogenesis, cutaneous vasomotor responses, sweating, piloerection and panting [32]. Of these, sympathetically mediated changes in peripheral skin blood-flow (manifest as a change in peripheral temperature), is the most acutely sensitive to environmental temperature change [33]. However, changes in peripheral body temperature are additionally linked to changes in emotional state [11, 12], e.g. PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Introduction Pupil size has also been demonstrated to decrease during both the experience [14] and observation of sadness in others [10]. However, 2 / 18 Participants Thirty-eight healthy participants with normal or corrected to normal vision were recruited via advertisement on the UCL psychology online research website. Two participants were subsequently excluded from the analysis of temperature responses due to technical failure and 14 from the heart rate analysis due to battery failure. Thus a total of 36 participants (13 males, mean 22.9¡3.1 years) were included in analysis of temperature response and 22 (8 males, mean 22.9¡3.5 years) in the combined analyses of heart rate and temperature responses. Written informed consent was obtained in accordance with the declaration of Helsinki (Helsinki 1991) and the procedures were approved by the joint Ethics Committee of the National Hospital and Institute of Neurology, London. Temperature Contagion hand (reflectional symmetry). 3) That these temperature changes would occur in the absence of more general measures of arousal e.g. change in heart rate. 4) That contagion of another’s peripheral temperature change would be greater in participants with high emotional empathy as measured through psychological trait questionnaires. PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 hot, clammy hands in anxiety or facial flushing in embarrassment, and can be modulated by mental imagery, hypnotic suggestion [34] and disruption of the sense of body ownership using the rubber hand illusion [35] and illusory self-identification with an avatar [64]. Together these findings suggest sensitivity of peripheral body temperature to top-down cognitive processes and a complimentary role in social communication. To investigate emotional contagion in the domain of body temperature we measured the left and right hand temperature of thirty-six healthy volunteers while viewing videos of two actors placing their right or left hand in warm or cold water. We predicted: 1) that viewing another’s hand in warm/cold water in the absence of any emotional cues would be associated with congruent temperature changes in the viewer’s hand. 2) That viewed changes in the right hand would be associated with congruent (contagious) temperature changes in the viewer’s left PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 3 / 18 Temperature Contagion Fig. 1. Experimental Timeline. The experimental timeline along the bottom shows video playback, red bars represent playback of warm, blue cold and beige control videos. The graph on the top left shows an example of single participants left (black) and right (green) hand temperature responses to a single illustrative cold video. The orange line illustrates changes in ambient temperature. The pictures on the top right show snap shots from the warm, cold and neutral videos. Fig. 1. Experimental Timeline. The experimental timeline along the bottom shows video playback, red bars represent playback of warm, blue cold and beige control videos. The graph on the top left shows an example of single participants left (black) and right (green) hand temperature responses to a single illustrative cold video. The orange line illustrates changes in ambient temperature. The pictures on the top right show snap shots from the warm, cold and neutral videos. doi:10.1371/journal.pone.0116126.g001 Study design The study adopted a randomised within-subject design, with each participant viewing all ten of the videos in counter-balanced order. After each video participants gave subjective ratings for both the observed water temperature (‘‘How Hot or Cold is the Water?’’) and the temperature of the actor’s hand (‘‘How Hot or Cold is the Actor’s Hand?’’) using a keyboard controlled visual analogue scale ranging from ‘Very Cold’ (far left) through ‘Neutral’ (centre) to ‘Very Hot’ (far right). Each video was separated by a 60 second inter-trial interval. The task was written and presented, and behavioural responses logged via a desktop computer running Cogent software on a Matlab platform (Mathwork, Nantick MA). See Fig. 1 for study timeline. Video temperature stimuli Ten custom temperature stimuli videos were produced. Each video began with one of two actors (one male, one female) sitting in front of a transparent container partially filled with water. In four of the videos the actor then gradually added hot water from a steaming kettle into the container, checking the temperature of the water every few seconds with one hand. One video showed the male actor placing his right hand in the water and one his left. The other two videos showed the same procedure with the female actor. Four additional videos showed each actor filling the container with a bag of ice then testing the cold water with his/her left or right hand. The first 40 s of each video showed the actor cautiously filling the semi-filled transparent container with water from a steaming kettle or ice from a bag and intermittently testing the water temperature with his or her hand. The remainder of the videos used in subsequent analyses focused exclusively on the actor’s hand placed in the water. These sections of the videos showed the water and the actor’s hand only with no facial or other body movement cues that may communicate emotional state. Two additional control videos (each of 120 s duration) showed the same combination of factors i.e. bowl of water and actors hand, however in these videos no hot water/ice was added to the water and the actor’s hand was held in front of the water container. One control video showed the female actor’s left hand and the other the male actors’ right hand (screen-shots illustrated in Fig. 1). PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 4 / 18 Temperature Contagion as well as ambient room temperature, were recorded throughout the study. Minute (362 mm) thermistors were attached to the palmar distal phalanges on the fourth finger of each hand to measure temperature change. Convection and conductive heat transfer to the environment was minimised by attaching the thermistors with micropore tape and placing participant’s hands palm upwards on cushions throughout video playback. Ambient temperature was recorded throughout using a third thermistor suspended 20–30 cm in front of the hands. To minimise muscle movement related temperature change participants were asked to keep their hands as still as possible during video payback and use the index finger of both hands to input responses once the video had finished playing. Heart rate was simultaneously monitored using a pulse oxymeter (Nonin 8600; Nonin Medical) attached to each participant’s left small finger. as well as ambient room temperature, were recorded throughout the study. Minute (362 mm) thermistors were attached to the palmar distal phalanges on the fourth finger of each hand to measure temperature change. Convection and conductive heat transfer to the environment was minimised by attaching the thermistors with micropore tape and placing participant’s hands palm upwards on cushions throughout video playback. Ambient temperature was recorded throughout using a third thermistor suspended 20–30 cm in front of the hands. To minimise muscle movement related temperature change participants were asked to keep their hands as still as possible during video payback and use the index finger of both hands to input responses once the video had finished playing. Heart rate was simultaneously monitored using a pulse oxymeter (Nonin 8600; Nonin Medical) attached to each participant’s left small finger. Each thermistor was connected to a separate Wheatstone bridge (detailed below) with outputs, pulse oximetry signals and stimulus timing pulses all passed to a Cambridge Electrical Designs (CED) Power 1401 data acquisition interface then recorded at a rate of 100 Hz on a second PC running the program Spike2 (see Fig. 2). Physiological data recording All testing was performed in a dedicated testing room kept at a constant temperature of 21˚C. Participant’s right and left hand temperature and heart rate, PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 5 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Custom temperature Gauge Each 10 kV thermistor (EPCOS NTC B57861S103F40) was connected to a custom Wheatstone bridge built using three additional 10 kV (+/2 1%) resisters balanced with a rheostat (Fig. 3A). Each of the three Wheatstone bridges were then connected to a CED 1902 low noise, high-gain isolated pre-amplifier via 8-pin DIN plugs. Amplified potential differences were then passed to the CED 1401 data acquisition interface as described above. The thermistors used (Resistance (R0) 510 kV, B25/100 (beta) 53988K at a rated temperature (T0) 525˚C) had a near linear resistance change (Equation 1, Fig. 3B) and rate of change of resistance (dR/ dT) (Equation 2, Fig. 3C) over the physiological temperature range of interest (20–40˚C). The 10 Volt range and 16 bit resolution of the CED 1401 data acquisition devise coupled with the 30-fold gain of the CED 1902 amplifier enabled us to achieve a theoretical temperature sensitivity in the order of 0.00002˚C that was near linear over the physiological temperature range of interest (20–40˚C). (Equations 3–5, Fig. 3D). Finally, we calibrated the high-sensitivity temperature gauge against a digital thermometer (Kane-May 8004 digital thermometer) recording four points over the physiological temperature range 22–35˚C. This demonstrated a linear relationship between voltage and temperature change (R250.98, p50.001, Temperature ˚C521.0876 Voltage (V) +20.059 (Fig. 3E). To ensure that results were not influenced by potential differences in the sensitivity of the two finger thermistors half the participants had thermistor 1 attached to the left hand and thermistor 2 to the right hand and vice versa. Equation 1: R ~ R0 x e(beta=K - beta=K0) Equation 2: dR=dT ~ -beta x R=K2 6 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Temperature Contagion Fig. 2. Experimental Setup. Graphic illustrating the experimental setup. Thermistors were attached to the participants’ left and right hands with a third thermistor used to record ambient temperature. All three thermistors were connected to Wheatstone bridges (labelled Thermistor transducer) with the output passed to a CED 1902 signal amplifier. Output from the CED 1902 and the Pulse oxymeter (attached to the left hand) were fed into a CED Power 1401 data acquisition interface and the digitized output recorded in a PC running Spike2. A second PC running cogent in Matlab presented all of the task stimuli and passed a timing pulse to the CED Power 1401 to ensure accurate temporal alignment of the data. Fig. 2. Experimental Setup. Graphic illustrating the experimental setup. Thermistors were attached to the participants’ left and right hands with a third thermistor used to record ambient temperature. All three thermistors were connected to Wheatstone bridges (labelled Thermistor transducer) with the output passed to a CED 1902 signal amplifier. Output from the CED 1902 and the Pulse oxymeter (attached to the left hand) were fed into a CED Power 1401 data acquisition interface and the digitized output recorded in a PC running Spike2. A second PC running cogent in Matlab presented all of the task stimuli and passed a timing pulse to the CED Power 1401 to ensure accurate temporal alignment of the data. doi:10.1371/journal.pone.0116126.g002 Equation 3: dV=dR~ Vpd x 1= RzRpd ð Þ -R= RzRpd ð Þ2 Equation 4: dV=dT ~ dV=dR x dR=dT q = = = Equation 5: Temp Resolution in K ~ Range1401=Gain1902=216 ð Þ= dV=dT ð Þ T5 temperature in ˚C, T0525˚C, R5 resistance, R05 10 kV, K (temperature in K) 5T+273.15, beta 53988 K, K05T0+273.15, Range1401 510 V, Gain1902 530, Rpd (potential divider resistance) 510,000, Vpd (potential divider supply voltage) 524 V. PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 7 / 18 Temperature Contagion Fig. 3. Temperature Gauge Characteristics. A) Graphical illustration of the structure of each of the three Wheatstone bridges each built using a 10 kV thermistor and three additional 10 kV thermistors. B) Resistance properties of the thermistors illustrating a near linear response over physiological temperature range of interest (20–40˚C). C) Rate of change in resistance for the thermistors across the physiological temperature range. D) Theoretical temperature resolution of the temperature gauge across the physiological temperature range. E) Calibration of the temperature gauge. Fig. 3. Temperature Gauge Characteristics. A) Graphical illustration of the structure of each of the three Wheatstone bridges each built using a 10 kV thermistor and three additional 10 kV thermistors. B) Resistance properties of the thermistors illustrating a near linear response over physiological temperature range of interest (20–40˚C). C) Rate of change in resistance for the thermistors across the physiological temperature range. D) Theoretical temperature resolution of the temperature gauge across the physiological temperature range. E) Calibration of the temperature gauge. doi:10.1371/journal.pone.0116126.g003 Temperature Contagion [37, 38]. The BEES contains 30 items e.g. ‘‘It upsets me to see someone being mistreated’’ rated on a 9-point agree/disagree scale and provides a well-validated measure of emotional empathy. The IRI contains 28 items rated on a 5-point does/does not describe me well scale. It provides a composite measure of dispositional empathy as well as sub-scales of Perspective Taking (PT) "I sometimes try to understand my friends better by imagining how things look from their perspective", Empathic Concern (EC) ‘‘I often have tender, concerned feelings for people less fortunate than me’’, Personal Distress (PD) "Being in a tense emotional situation scares me’’ and Fantasy Scales (FS) "When I am reading an interesting story or novel, I imagine how I would feel if the events in the story were happening to me’’. BEES and Davis IRI total empathy score as well as the Davis sub-scores were then used in a step-wise multiple regression analysis in SPSS21 to investigate whether inter-individual differences in empathy predicted contagion of another’s temperature change. Temperature contagion was defined as an individual’s mean increase in temperature to all warm videos minus their mean decrease in temperature to all cool videos (averaged across both left and right hands). Ratings of observed temperature stimuli Ratings of observed temperature stimuli Repeated measures ANOVAs with factors temperature (warm, cool) and observed hand (left, right) confirmed that our experimental manipulation significantly modulated participants’ ratings of both the water and actors hand temperature, with both rated as appearing significantly warmer in the warm compared to cool conditions F(1,34)5449.25, p,0.001 and F(1,34)5256.67, p,0.001 respectively. There was no significant main effect of observed hand (left, right) or observed hand by temperature interaction for either rating demonstrating that the perceived temperature was equivalent for left and right hand video stimuli in both warm and cool conditions. We therefore collapsed ratings for observed (left, right) hand and repeated the ANOVAs including the neutral condition (warm, neutral, cool). This again confirmed that stimulus type (warm, neutral, cool) significantly affected ratings of the observed temperature for both the water and hand; F(1,34)5305.79, p,0.001 and F(1,34)5201.16, p,0.001 respectively. Post-hoc paired-sample t-tests confirmed significant differences (all p,0.001) between each pair of stimuli (Fig. 4A). Physiological data analysis Left and right hand temperature recordings were first corrected for minor fluctuations in room temperature by subtracting ambient temperature recordings at the equivalent time-point then de-trended to remove linear drifts across the experimental session. The container-filling phase (40 s), plus the subsequent 10 s (and equivalent pre-video playback period in the control condition) was used to measure video specific baseline temperature recordings. Video induced changes in participants’ own hand temperature over the remaining 130 s were then calculated in 10 s epochs by subtracting baseline temperature from ambient corrected left and right hand temperatures. Data were then averaged within subjects to obtain a mean response to each video type e.g. warm/neutral/cool and left/right observed hand then analysed in second level repeated measures ANOVAs using SPSS 21. Video induced changes in heart rate were analysed in an equivalent manner. Empathy Questionnaires Subjects completed two questionnaires: the Mehrabian Balanced Emotional Empathy Score (BEES) [36] and the Davis Interpersonal Reactivity Index (IRI) 8 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Temperature Contagion Fig. 4. Behavioural and Temperature responses to Warm and Cool stimuli. A) Participants mean subjective ratings for the observed water temperature (‘‘How Hot or Cold is the Water?’’) and temperature of the actor’s hand (‘‘How Hot or Cold is the Actor’s Hand?’’) reported using a keyboard controlled visual analogue scale ranging from ‘Very Cold’ (far left) through ‘Neutral’ (centre) to ‘Very Hot’ (far right). B) Participants mean temperature change for their right (right leaning diagonals) and left (left leaning diagonals) when viewing warm (red) cold (blue) and neutral (grey) stimuli. Bold diagonals denote congruent responses (e.g. participants’ left hand response when viewing left hand stimuli) and non-bold diagonals incongruent responses (e.g. e.g. participants’ left hand response when viewing right hand stimuli). C) Mean time course response to viewing all warm videos for the left (red) and right (orange) hand displayed in 10 s epochs. D) Mean time course response to viewing all cold videos for the left (dark blue) and right (light blue) hand displayed in 10 s epochs. Fig. 4. Behavioural and Temperature responses to Warm and Cool stimuli. A) Participants mean subjective ratings for the observed water temperature (‘‘How Hot or Cold is the Water?’’) and temperature of the actor’s hand (‘‘How Hot or Cold is the Actor’s Hand?’’) reported using a keyboard controlled visual analogue scale ranging from ‘Very Cold’ (far left) through ‘Neutral’ (centre) to ‘Very Hot’ (far right). B) Participants mean temperature change for their right (right leaning diagonals) and left (left leaning diagonals) when viewing warm (red) cold (blue) and neutral (grey) stimuli. Bold diagonals denote congruent responses (e.g. participants’ left hand response when viewing left hand stimuli) and non-bold diagonals incongruent responses (e.g. e.g. participants’ left hand response when viewing right hand stimuli). C) Mean time course response to viewing all warm videos for the left (red) and right (orange) hand displayed in 10 s epochs. D) Mean time course response to viewing all cold videos for the left (dark blue) and right (light blue) hand displayed in 10 s epochs. Fig. 4. Behavioural and Temperature responses to Warm and Cool stimuli. A) Participants mean subjective ra Fig. 4. Behavioural and Temperature responses to Warm and Cool stimuli. Temperature analysis Repeated measures ANOVA with factors observed temperature (warm, cool), observed hand (left, right) and participants own hand (left, right) demonstrated a significant main effect of observed temperature on participants’ own hand temperature F(1,35)513.83, p50.001, with post-hoc t-test demonstrating a significant reduction in participants’ own left (t(35)523.54, p50.001) and right PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 9 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Heart rate analysis Heart rate analysis Repeated measures ANOVA with factors observed temperature (warm, cool), observed hand (left, right) and participants own hand (left, right) demonstrated no significant effect of observed temperature on participants own heart rate (all main effects and interactions p.0.1). Relationship between temperature contagion and empathy Finally we investigated whether self-reported empathy scores predicted an individual’s contagion of another’s observed temperature. Multiple regression analysis demonstrated that both the BEES and the Empathic Concern (EC) sub- scale of the Davis IRI (but not the other Davis sub-scales) significantly predicted contagion of observed temperature changes (F(2,35)56.82, p,0.003) with an adjusted R250.25. Exploration of the factors within this model demonstrated a nuanced relationship between empathy and contagion of another’s temperature change; specifically in this model the BEES (which provides a single composite measures of empathy) negatively predicted temperature contagion (t(35)523.68, p50.001, b520.92) while the EC sub-scale of the Davis (which selectively measures empathic concern) positively predicted temperature contagion (t(35)52.79, p50.009, b50.69) (Fig. 5). PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 A) Participants mean subjective ratings for the observed water temperature (‘‘How Hot or Cold is the Water?’’) and temperature of the actor’s hand (‘‘How Hot or Cold is the Actor’s Hand?’’) reported using a keyboard controlled visual analogue scale ranging from ‘Very Cold’ (far left) through ‘Neutral’ (centre) to ‘Very Hot’ (far right). B) Participants mean temperature change for their right (right leaning diagonals) and left (left leaning diagonals) when viewing warm (red) cold (blue) and neutral (grey) stimuli. Bold diagonals denote congruent responses (e.g. participants’ left hand response when viewing left hand stimuli) and non-bold diagonals incongruent responses (e.g. e.g. participants’ left hand response when viewing right hand stimuli). C) Mean time course response to viewing all warm videos for the left (red) and right (orange) hand displayed in 10 s epochs. D) Mean time course response to viewing all cold videos for the left (dark blue) and right (light blue) hand displayed in 10 s epochs. doi:10.1371/journal.pone.0116126.g004 (t(35)522.33, p50.026) hand temperature during observation of cool videos but no change to warm videos (p.0.1) (Fig. 4B). We also observed a significant main effect of participant hand (F(1,35)54.78, p50.036) and a significant participant hand 6 observed temperature interaction (F(1,35)513.49, p50.001). Post-hoc t- tests showed that this was driven by a significantly greater reduction in left versus right hand temperature when viewing cool videos (paired t(35)523.80, p50.001) demonstrating greater sensitivity of participants left hand to observed changes in temperature. Importantly, we did not observe significant observed 6 own hand or temperature 6 observed 6 own hand interactions (p.0.1) suggesting that induced changes in temperature were not influenced by laterality of the observed hand. Finally, there was no change in participants’ own left or right hand temperature when they observed the neutral videos (p.0.1) (data illustrated in Fig. 4B). 10 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Temperature Contagion The time scale of induced changes in participants’ own hand temperature when viewing warm and videos are illustrated in Fig. 4C and 4D respectively and demonstrate a maximum 0.2˚C temperature drop 2 minutes into the cool videos and a maximum 0.033˚C temperature rise occurring 10–50 s after onset of the warm videos. PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Discussion Here we aimed to investigate emotional contagion in the domain of body temperature by showing healthy volunteers video clips of actors with their hands in warm or cold water while simultaneously recording their own right and left hand temperature. We show that healthy participants exhibit contagion of another’s hand temperature even in the absence of visible emotional or facial cues. This temperature contagion was particularly prominent for cool stimuli and was more marked for the observers’ left hand. However, it should be noted that ice cubes were clearly visible throughout the cool condition but steam visible only at the beginning of the warm videos which may have contributed to this finding. We found no interaction between the laterality of the hand observed and experiencing the change in temperature arguing against common rotational or mirror symmetry effects described in naturalistic social interactions [7]; though note that viewed hands were oriented perpendicular to their own. Interestingly, self-report measures of empathy additionally predicted inter-individual differences in sensitivity to temperature contagion. This demonstration of contagion of 11 / 18 Temperature Contagion preoptic area (POA) resulting in an inhibition of its tonic discharge. This ultimately results in disinhibition of sympathetic premotor neurons within the rostral medullary raphe (rMR) orchestrating cutaneous vasoconstriction, tachycardia, skeletal muscle shivering [32, 40] and non-shivering thermogenesis in brown adipose tissue [12]. p Interestingly, different effectors mechanisms are associated with partially separable central control systems [41], expressed physiologically as a greater sensitivity of vasoconstrictive responses to temperature change [33]. This difference in central control mechanism may also underpin why, in our current study, we saw isolated changes in hand temperature (likely mediated by a direct POA-rMR pathway) but not heart rate (mediated by an intermediate projection to the dorsomedial hypothalamus (DMH)) [32]. WARM sensitive neurons projecting via the dorsal parabrachial nucleus (LPBd) play a similar role in orchestrating cutaneous vasodilation and tachycardia in response to environ- mental warming [42]. In addition to bottom-up feed-forward pathways triggered by changes in skin temperature, thermoregulatory responses are also sensitive to top-down influences for example though visual imagery, temperature biofeedback and hypnotic suggestion [43]. This was first highlighted by Hadfield in 1920 in a case report of a patient who was able to selectively increase and decrease their right and left hand temperature by almost 3˚C through suggestions of heat or cold [44]. Subsequently, similar selective increases and decreases in left and right hand temperature have been demonstrated in response to hypnotic suggestion in adults [45] and children [46] as well as biofeedback [47, 48], instructed imagery [49] or combinations of these techniques [50, 51]. More recently, synchronous changes in facial temperature have also been reported in mothers observing their child at play [52]. A review of these studies [43] has highlighted that temperature decreases are typically easier to elicit and of greater magnitude than temperature increases, and occur in the absence of heart rate change, as we observed. These top-down influences on thermoregulatory responses have also been exploited clinically in the treatment of Raynauld’s syndrome [53, 54], though large inter-individual variability in ability to regulate finger temperature has limited its more widespread clinical adoption [55]. Direct cooling of the hand has been shown to increase blood flow (an indirect measure of neuronal activity) within the posterior insula [56], a region proposed to provide a cortical representation of all visceral afferent input [57, 58]. PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Temperature Contagion Fig. 5. Relationship between sensitivity to temperature contagion, BEES and the empathic concern subscale of the Davis IRI. A) Relationship between sensitivity to temperature contagion and BEES. B) Relationship between BEES and empathic concern (EC) subscale of the Davis IRI. C) Relationship between EC residuals (after regressing out relationship with BEES) and sensitivity to temperature contagion. D) 3D scatter plot illustrating the relationship between sensitivity to temperature contagion (TC), BEES and empathic concern subscale of ARI (EC). Fig. 5. Relationship between sensitivity to temperature contagion, BEES and the empathic concern subscale of the Davis IRI. A) Relationship between sensitivity to temperature contagion and BEES. B) Relationship between BEES and empathic concern (EC) subscale of the Davis IRI. C) Relationship between EC residuals (after regressing out relationship with BEES) and sensitivity to temperature contagion. D) 3D scatter plot illustrating the relationship between sensitivity to temperature contagion (TC), BEES and empathic concern subscale of ARI (EC). doi:10.1371/journal.pone.0116126.g005 observed body temperature extends the range of human mimetic responses to another axis of the autonomic nervous system. It also lends empirical support to extension of perception-action mechanisms to non-volitional, non-emotional responses exclusively mediated by the autonomic nervous system [9, 10]. Maintaining a stable internal thermal environment is critical to the life- preserving actions of bioactive proteins, such as enzymes and ion channels. As a consequence, core body temperature is rigidly regulated by the brains of all homeothermic animals including humans through a variety of involuntary thermoregulatory responses, such as shivering and non-shivering thermogenesis, cutaneous vasomotor responses, sweating, piloerection and panting [32]. In response to a reduction in environmental temperature skin temperature falls rapidly and triggers firing of strategically located COOL-sensitive neurons [39]. Feed-forward thermal afferent information is then relayed via the external lateral parabrachial nucleus (LPBel) to the hypothalamic thermoregulatory centre in the 12 / 18 Temperature Contagion experimentally using the rubber hand illusion [35] or in the clinical disorder cold- type complex regional pain syndrome (CRPS) [60]. In both of these conditions unilateral disruption of body ownership is associated with a localised reduction in body temperature suggesting that the conscious sense of our physical self and its physiological regulation are linked. During experimental induction of the rubber hand illusion activity changes are observed within insula cortex as well as premotor and intraparietal cortex [61] suggesting a potential role for the insula in reported temperature changes. In CRPS patients (a neurological disorder associated with pain, abnormal temperature regulation and often dystonia in a single limb) changes in limb temperature were reported dependent upon its location in space [60]. For example, when the affected (cool) limb was moved across the midline its temperature spontaneously increased with a converse effect described for the healthy limb. On the basis of these findings the authors argued for a space-based rather than somatotopic frame of reference with descending projections from parietal cortex onto brainstem autonomic centres hypothesised as the mechanism through which changes in the spatial location of the limb result in associated temperature change. It is thus possible that inter-personal comparator processes within the intraparietal junction play a similar role in temperature contagion. Finally, in contrast to our prediction of a simple relationship between sensitivity to temperature contagion and empathy score we found a more nuanced relationship. Specifically, an inverse relationship between BEES emotional empathy score and sensitivity to temperature contagion. i.e. those individual who scored highest on the BEES showed the least sensitivity to temperature contagion. However, when we included both the BEES and the Davis IRI (including subscores) into a stepwise linear regression analysis we showed that though BEES continued to show a negative relationship to temperature contagion the empathic concern subscale of the IRI showed a positive relationship. The basis for these findings is currently unclear, though may relate to subtle differences in the concepts captured by the BEES and EC scales. For example, though we showed a tight positive correlation between BEES and EC scores (R250.65, p,0.001) across participants it was participants with relatively high EC compared to BEES scores that showed the greatest propensity to temperature contagion. Alternately this finding may relate to the nature of our experimental stimuli in which we were careful not to show discernable emotional cues. PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 Whether similar increases in insula activity are also associated with temperature changes observed in a conspecific is currently unknown, though the wealth of fMRI studies showing shared empathic in other domains would predict that they would. In this regard, it is also instructive to note that in monkeys many POA thermosensitive neurons are additionally affected by non-thermal emotional stimuli such as rewards or aversive stimuli [59] suggesting that hypothalamic POA-rMR effector pathways may be recruited by top-down cognitive processes. y y y g Insight into the mechanism underlying temperature contagion may also be usefully informed by studies of disrupted body ownership induced either 13 / 18 Acknowledgments We are grateful to Tina Gonsalvez for help in producing the videos used in this study. Author Contributions Conceived and designed the experiments: NAH TS HDC EF. Performed the experiments: JG NAH. Analyzed the data: NAH JG EF VV EAC. Contributed reagents/materials/analysis tools: EF. Wrote the paper: EAC NAH TS JG VV HDC EF. It would be important for future studies to clarify the precise nature of the relationship between individual differences in emotion contagion, empathic distress and concern particularly to such low-level contagion phenomena. To conclude, here we show that healthy individuals are sensitive to observable signals of another’s peripheral body temperature and further show contagion of their temperature, particularly in the context of cold. Inter-individual differences in temperature contagion are marked and show a complex relationship to inter- individual difference in empathy. Interestingly, abnormal temperature regulation is also observed in disorders of social cognition such as autism [62] and PLOS ONE \| DOI:10.1371/journal.pone.0116126 December 31, 2014 14 / 18 Temperature Contagion Schizophenia [63] suggesting interest in measuring temperature contagion in these populations. Schizophenia [63] suggesting interest in measuring temperature contagion in these populations. 15. 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https://openalex.org/W2145746529	https://europepmc.org/articles/pmc4116186?pdf=render	English	null	Perinatal Exposure of Mice to the Pesticide DDT Impairs Energy Expenditure and Metabolism in Adult Female Offspring	PloS one	2,014	cc-by	11,197	Abstract Dichlorodiphenyltrichloroethane (DDT) has been used extensively to control malaria, typhus, body lice and bubonic plague worldwide, until countries began restricting its use in the 1970s. Its use in malaria control continues in some countries according to recommendation by the World Health Organization. Individuals exposed to elevated levels of DDT and its metabolite dichlorodiphenyldichloroethylene (DDE) have an increased prevalence of diabetes and insulin resistance. Here we hypothesize that perinatal exposure to DDT disrupts metabolic programming leading to impaired metabolism in adult offspring. To test this, we administered DDT to C57BL/6J mice from gestational day 11.5 to postnatal day 5 and studied their metabolic phenotype at several ages up to nine months. Perinatal DDT exposure reduced core body temperature, impaired cold tolerance, decreased energy expenditure, and produced a transient early-life increase in body fat in female offspring. When challenged with a high fat diet for 12 weeks in adulthood, female offspring perinatally exposed to DDT developed glucose intolerance, hyperinsulinemia, dyslipidemia, and altered bile acid metabolism. Perinatal DDT exposure combined with high fat feeding in adulthood further impaired thermogenesis as evidenced by reductions in core temperature and in the expression of numerous RNA that promote thermogenesis and substrate utilization in the brown adipose tissue of adult female mice. These observations suggest that perinatal DDT exposure impairs thermogenesis and the metabolism of carbohydrates and lipids which may increase susceptibility to the metabolic syndrome in adult female offspring. Citation: La Merrill M, Karey E, Moshier E, Lindtner C, La Frano MR, et al. (2014) Perinatal Exposure of Mice to the Pesticide DDT Impairs Energy Expenditure and Metabolism in Adult Female Offspring. PLoS ONE 9(7): e103337. doi:10.1371/journal.pone.0103337 Editor: Barbara T. Alexander, Univ Mississippi Medical Center, United States of America Editor: Barbara T. Alexander, Univ Mississippi Medical Center, United States of America Received May 10, 2014; Accepted June 25, 2014; Published July 30, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data are available in Figshare at: http://dx.doi. org/10.6084/m9.figshare.1084396. Abstract Funding: This research was supported through funding by the National Institute of Health (R00 ES019919, R03 DK082724, U24 DK092993, U24 DK097154, T32 ES007059, and P60 DK020541), the American Diabetes Association, and United States Department of Agriculture - Agricultural Research Service Intramural Project 5306-51530-019-00D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. * Email: mlamerrill@ucdavis.edu odds of T2D [7]. Further, exposure to DDE, the primary metabolite of DDT, has also been associated with increased odds of excess body weight, insulin resistance, and dyslipidemia in people [8]. Because the World Health Organization still recom- mends the use of DDT for malaria control, DDT’s potential contribution to the pathogenesis of T2D continues to be relevant to public health. Furthermore, obesity and T2D have increased at alarming rates in countries where DDT is still in use. For instance, the prevalence of diabetes among rural Indian adults increased 3.8 fold from 1989 to 2006 [9] and although a confluence of factors contribute to this rise, the contribution of endocrine disrupting chemicals cannot be ruled out without further study. Since epidemiological associations cannot establish causality, we tested Michele La Merrill1,2,3, Emma Karey1, Erin Moshier2, Claudia Lindtner3,4, Michael R. La Frano5,6, John W. Newman5,6,7, Christoph Buettner3,4 1 Department of Environmental Toxicology, University of California Davis, Davis, California, United States of America, 2 Department of Preventive Medicine, Mount Sinai School of Medicine, New York, New York, United States of America, 3 Metabolism Institute, Mount Sinai School of Medicine, New York, New York, United States of America, 4 Division of Endocrinology, Diabetes and Bone Disease, Mount Sinai School of Medicine, New York, New York, United States of America, 5 West Coast Metabolomic Center, University of California Davis, Davis, California, United States of America, 6 Department of Nutrition, University of California Davis, Davis, California, United States of America, 7 Obesity and Metabolism Research Unit, USDA-ARS-Western Human Nutrition Research Center, Davis, California, United States of America Perinatal Exposure of Mice to the Pesticide DDT Impairs Energy Expenditure and Metabolism in Adult Female Offspring Michele La Merrill1,2,3, Emma Karey1, Erin Moshier2, Claudia Lindtner3,4, Michael R. La Frano5,6, John W. Newman5,6,7, Christoph Buettner3,4 July 2014 \| Volume 9 \| Issue 7 \| e103337 Effects of Perinatal DDT on Metabolism in Mice whether exposure to DDT and DDE predisposes to T2D in mice at doses comparable to human exposures [10–13]. whether exposure to DDT and DDE predisposes to T2D in mice at doses comparable to human exposures [10–13]. by indirect calorimetry in 1 female mouse/litter (4 mice/perinatal treatment) for 7 days after 2 days of acclimation. Cold tolerance was assessed 3 days after indirect calorimetry in 5 female mice/ perinatal treatment, as described below. Both obesity and T2D are characterized by impaired energy expenditure and reduced thermogenesis, which are often the result of reduced brown adipose tissue (BAT) activity [5]. Mouse studies also suggest that impaired thermogenesis may precede insulin resistance [14]. Therefore, increasing energy expenditure by activating BAT thermogenesis may hold promise as a therapeutic strategy for managing both obesity and T2D [15–18]. For the high fat feeding study, littermates were randomized to high fat diet (HFD, 4.73 kcal/g, 20% protein, 35% carbohydrate, and 45% fat per kcal; D12451, Research Diets, New Brunswick, NJ) or a low fat diet (LFD, 3.85 kcal/g, 20% protein, 70% carbohydrate, and 10% fat per kcal; D12450B, Research Diets) feeding following their glucose tolerance test (GTT) at 6 months of age for a total of 12 weeks (n = 8 mice/sex/litter per perinatal treatment and diet combination). Perturbations in the perinatal environment can impair meta- bolic programming that can consequently increase susceptibility to obesity and T2D in adulthood. A classic example of the importance of the perinatal environment on the future risk of T2D has been historically provided by the Dutch famine. Fetuses carried to term that were exposed to fetal malnutrition suffered from increased rates of the metabolic syndrome and glucose intolerance as adult offspring [19,20]. The consequences of impaired fetal development on metabolic programming can be modeled in rodents, where fetal under-nutrition results in the development of the metabolic syndrome and glucose intolerance in adult offspring [21,22]. Here we confirm the hypothesis that perinatal exposure to DDT impairs energy expenditure and metabolism leading to insulin resistance and the metabolic syndrome in adult offspring. We demonstrate for the first time that developmental exposure to DDT impairs BAT thermogenesis. All mice had access to food and water ad libitum (other than during stated fasting periods) in sterile ventilated cages in an American Association for the Accreditation of Laboratory Animal Care-approved facility. At the end of all the studies, mice were sacrificed by exsanguination under isoflurane anesthesia. Energy homeostasis We measured food intake in mice on a monthly basis from 2–6 months (n = 15 DDT- and n = 14 vehicle- exposed litters). In 2 month old mice, each mouse/sex/litter/treatment was singly housed and food intake was assessed daily over a 5 day period. In group- housed mice aged 3–6 months, we measured food intake per cage over a 2 day period. In the HF feeding study, we measured weekly food intake of singly housed mice fed LFD or HFD. Food intake (g or kcal) was calculated as the difference in food weight divided by the number of mice that had access to it and the number of days they had access, and transformed to kcal when comparing HFD and LFD (SAS 9.2, SAS Institute, Cary, NC). We measured core temperature monthly in all mice aged 2–5 and 8 months old by inserting a thermocouple into the rectum (BAT-10, Physitemp, Clifton, NJ). DDT exposure To mimic the commercial formulation of DDT used as a pesticide in the US prior to its ban, 77.2% p,p9-DDT (98.5% purity neat, AccuStandard, New Haven, CT) and 22.8% o,p9- DDT (100% purity neat, AccuStandard) were dissolved at a final concentration of 0.17 g DDT mixture/L organic olive oil [23], hereafter referred to as DDT. We selected the dosing period to span liver and white- and brown- adipose tissue ontogenesis and programming [24–28]. From 11.5 days post coitus (determined by vaginal plug) to postnatal day (PND) 5, we administered 1.7 mg DDT/kg body weight (per os, n = 15 dams, 10 mL solution/g mouse), or equivalent volume of olive oil vehicle (hereafter referred to as vehicle or VEH, per os, n = 14 dams) daily to primigravid C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, Fig. S1). To determine the internal dose after transplacental and lactational transfer to pups [29], 4 additional dams were treated identically with DDT, and 24 hours after the final dose their sera was analyzed for o,p9-DDT, p,p9-DDT, and p,p9-DDE by gas chromatography using electron capture detection with detection limits of 0.36, 0.27 and 0.12 pg/mL serum respectively [30]. Body composition We measured body weight and percent fat mass by MRI (3in1- EchoMRI, Echo Medical Systems, Houston, TX) of each offspring monthly in 1–6 month old mice (n = 15 DDT- and n = 14 vehicle- exposed litters). The body mass of all mice was also measured at PND 5. During HF feeding, mice were weighed weekly and we used MRI to determine body composition at 8 months of age (n = 8 mice/sex/litter per perinatal treatment and diet combina- tion). Effects of Perinatal DDT on Metabolism in Mice All animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by the US National Institutes of Health. The procedures of this study were approved by the Mount Sinai School of Medicine Institutional Animal Care and Use Committee protocol #LA11-00116. Introduction The prevalence of type 2 diabetes (T2D) is rising worldwide [1], creating a tremendous burden for health care systems in developed and developing nations. The estimated cost associated with T2D was $245 billion last year in the United States alone [2]. This rise in the incidence of T2D is largely attributed to increasing obesity rates [3]. Obesity and T2D are characterized by insulin resistance and are often associated with perturbations in energy expenditure [4,5]. Numerous epidemiological studies have suggested that exposure to persistent organic pollutants (POPs) contribute to the rising incidence of T2D [6]. For example, exposure to the organochlo- rine pesticide DDT has been found to be associated with increased July 2014 \| Volume 9 \| Issue 7 \| e103337 1 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Effects of Perinatal DDT on Metabolism in Mice July 2014 \| Volume 9 \| Issue 7 \| e103337 Study Design For the energy balance studies, we housed 4 females/perinatal treatment (1 mouse/litter) individually in metabolic chambers, which continuously measured oxygen consumption, carbon dioxide production, food intake, and 3 plane-locomotor activity through open-circuit, indirect calorimetry (CLAMS, Columbus Instruments, Columbus, OH). During this 9 day period, we provided mouse powdered diet (NIH 07) and water ad libitum while they were maintained at 21–22uC for a 12-hour light/dark cycle. The respiratory exchange ratio (RER) was calculated as the quotient of vCO2/vO2. We culled litters to 6 pups after the final DDT dose on PND 5, to minimize infanticide common among C57BL/6J dams while normalizing lactational transfer of DDT and maternal behavior effects (Fig. S1). We evaluated timing of puberty daily by monitoring vaginal opening (VO) and preputial separation (PPS), in all female and male mice respectively, beginning at PND 18. On PND 21 we weaned pups (1 cage/sex/litter/ treatment). We assessed body composition, energy homeostasis, metabolic parameters, and glucose tolerance up to 6 months of age when we randomized mice into 2 study arms to assess: 1) energy balance or 2) the metabolic consequences of high fat feeding (Fig. S1). Three days after the indirect calorimetry was completed, we individually housed 5 female mice/perinatal treatment (1 mouse/ litter) at 4uC with bedding enrichment. Core body temperature was measured immediately upon exposure to 4uC, and every 30 minutes thereafter until 90 minutes, when 50% of the mice For the energy balance study, 10 females from 10 litters and 8 metabolic chambers were available. Energy balance was assessed PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e103337 2 Effects of Perinatal DDT on Metabolism in Mice reached core temperatures of ,30uC after being kept for 90- minutes at 4uC. Two days later, we housed each of the 10 female mice for 1 hour at 4uC prior to sacrifice. quantified by LC-MS/MS using modifications of a previously published method [35]. Separation of bile acids was achieved using reversed phase UPLC with a 1.7 mm, 2.16100 mm Acquity BEH C18 column (Waters, Milford, MA) using a 16 min gradient (Solvent A = 0.1% formic acid; Solvent B = 0.1% formic acid in acetonitrile) on a Waters Acquity UPLC (Waters) and detected on an API 4000 QTrap (AB Sciex, Framingham, MA, USA) by multiple reaction monitoring (MRM) after negative mode electrospray ionization. Western blot analysis Protein expression was assessed as previously described [36]. Briefly, liver was homogenized and 10–20 mg of protein was subjected to Western blot analysis with primary antibodies against fatty acid synthase (FAS, BD Biosciences, San Jose, CA), phospho- acetyl-CoA carboxylase (ACC), ACC, phospho- adenosine triphosphate citrate lyase (ATPCL), ATPCL, adipose triglyceride lipase (ATGL), AKT Thr308 and Ser473, AKT, phospho- glycogen synthase kinase 3 (GSK3), GSK3, phospho-ERK, and ERK (all antibodies from Cell Signaling Technology, Danvers, MA unless stated otherwise) and insulin receptor (IR) beta (Santa Cruz Biotechnology, Dallas, TX). The secondary antibody was DyLight conjugated goat anti-rabbit or anti-mouse IgG (ThermoScientific). Signal intensity of proteins of interest was divided by the intensity of the housekeeping protein HSC 70 (Santa Cruz Biotechnology) to determine fold change (Odyssey, LI-COR, Lincoln, NE). We have confirmed the specificity of ATGL in adipose tissue specific ATGL knockout vs. wild type mice, and all other antibodies have been characterized and validated in prior peer review publications [(Table S1), [37–41]]. GTT Glucose tolerance was assessed in 1 mouse/sex/litter/perinatal treatment aged 2, 3 and 6 months (randomized selection from n = 15 DDT- and n = 14 vehicle- exposed litters) and 1 mouse/ sex/litter for each perinatal treatment and adult diet combination at eight months (randomized selection from n = 8 litters/ treatment). Two-month-old mice received 2.0 mg D-glucose/g mouse body weight (i.p.), since at young age mice (as well as humans) are more glucose tolerant, and all other mice received 1.0 mg D-glucose/g mouse body weight (i.p.). For all GTT, we fasted mice for 5 hours, and collected sub-mandibular blood to determine fasting blood glucose (AlphaTrak, Abbott Laboratories, Abbott Park, IL) and fasting plasma metabolic parameters. We measured blood glucose 15, 30, 45, 60, 90, and 120 minutes after a D-glucose bolus for GTT. We determined total AUC using the trapezoidal approach (PROC SQL, SAS 9.4, SAS Institute, Cary, NC). Pathology mRNA was extracted with the RNeasy kit (Qiagen, Hilden, Germany) to synthesize cDNA using reverse transcription PCR (Applied Biosystems, Foster City, CA). Semi-quantitative PCR was performed using proprietary TaqMan primers for transcripts: peroxisome proliferative activated receptor gamma coactivator 1 alpha (Ppargc1a), iodothyronine type II deiodinase (Dio2), twist- related protein 1 (Twist1) and uncoupling protein 1 (Ucp1) in BAT using TATA box binding protein (Tbp) as an endogenous control; 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (Hmgcr), low density lipoprotein receptor (Ldlr), and cytochrome P450 7a1 (Cyp7a1) in livers with glucuronidase beta (Gusb) as an endoge- nous control (Applied Biosystems). Additional semi-quantitative PCR was performed in BAT using SYBR Green with published primers: glucose transporter type 4 (Glut4, F: CATCCCA- CAAGGCACCCCTC, R: CATGCCACCCACAGAGAAGA [42]), lipoprotein lipase (Lpl, F:TGGAGAAGCCATCCGTGTG, R: TCATGCGAGCACTTCACCAG [42]), adipose triglyceride lipase (Pnpla, F: TGTTTCAGACGGAGAGAACGTC, R: TGAGAATGGGGACACTGTGATG [43]), and carnitine pal- mitoyltransferase 2 (Cpt2, F: CCAGCGGATAAACCACAACA, R: AAGCCACCGAGGCTCACTG [42]) using Tbp (F: GAAGCTGCGGTACAATTCCAG, R: CCCCTTGTACCCT- TCACCAAT [44]) as endogenous control. Endogenous controls did not vary significantly across treatment groups. The 2-ddCT method was used to approximate relative transcript fold change between treatment groups (coefficient of replicate variation ,10%) [45]. Livers were harvested from 1 female fed HFD per perinatal treatment group (randomized selection from n = 1 female/litter in 7 VEH and 8 DDT litters) into OCT medium, frozen 5 mM thick sections were stained with oil red O, and fields with represen- tative staining of the sample and the least clear space (vessels, ducts, etc.) were selected. Histopathological evaluations were assessed by a veterinary pathologist who was blinded to the treatments. Hepatic lipids Hepatic triglycerides and cholesterol esters were measured after a modified Folch extraction (randomized selection from n = 1 female/litter in 7 VEH and 8 DDT litters fed HFD) [32]. Total cholesterol [33] and triglycerides [34] were measured enzymati- cally from these livers (Fisher Diagnostics, Middletown, VA). Metabolic parameters Trunk blood glucose was measured in all mice culled on PND 5. Sub-mandibular blood was collected from mice fasted for 5 hours (1 mouse/sex/litter/treatment) at ages 1–6 months old (random- ized selection from n = 15 DDT- and n = 14 vehicle- exposed litters) and 8 months old (randomized selection from n = 8 litters/ treatment) to determine blood glucose (AlphaTrak) and plasma insulin by ELISA (Mercodia, Uppsala, Sweden). Fasting plasma triglycerides, total cholesterol, and non-esterified fatty acids (NEFA) were evaluated enzymatically (Wako Chemicals, Rich- mond, VA). Homeostasis model assessment- insulin resistance (HOMA-IR) was calculated as the product of fasting glucose and insulin divided by 22.5 [31]. Study Design Residues were quantified against 7 point curves of authentic standards purchased from Steraloid Inc. (Newport, RI), Sigma-Aldrich (St. Louis, MO), and Medical Isotopes, Inc. (Pelham, NH) using internal standard methodolo- gies. Statistical analyses y Phenotypes were assessed at specific ages by modeling the fixed effect of perinatal DDT and the random effect of litter while stratifying by gender (PROC MIXED, SAS). Longitudinal analyses of body mass, fat mass, and temperature were conducted using the age-specific model as described above, with the addition of age and an agetreatment term as fixed effects, and individual mouse identifiers as random effects. To assess circadian effects during indirect calorimetry, photoperiods and a photoperiodper- inatal treatment term were included as fixed effects in the age- specific model. Body weight and fat mass were considered as potential covariates in indirect calorimetry analyses [46] but because these body composition variables were not significantly different between perinatal treatments and caused no reduction in data significance or difference in interpretation, these covariates were not included in the final indirect calorimetry models. When examining phenotypes from the HF feeding study, diet and a dietperinatal treatment term were included as fixed effects in the age-specific and longitudinal models described above. GTTs, fasting metabolic parameters, protein expression, mRNA, lipids, and bile acids were evaluated without random effects because only one mouse per litter was included (PROC GLM, SAS). To evaluate the potential covariate behavior of hepatic bile acids and other metabolic factors which may influence or be influenced by cholesterol metabolism, imDEV v 1.4.2 [47] was employed to normalize data, transform variables to mean centers with unit variance, and conduct partial least squares discriminate analysis (PLS DA; oscores pls). The dependent variable was the perinatal treatment group while the independent variables were hepatic bile acids, Cyp7a1 fold-change, hepatic cholesterol and triglycerides, plasma cholesterol, % body fat, and age. Measures with variable importance in projection (VIP) scores .1 were considered important discriminators of treatment [48]. Timing of puberty was evaluated using survival analysis with treatment as a fixed effect and litter as a repeated event (PROC PHREG, SAS). In all models, we used a significance threshold of p,0.05 for main effects and p,0.1 for interaction effects. Figure 1. Effect of perinatal DDT on the development of female mice. (A) Female body mass at PND5 (n = 15 DDT- and n = 14 VEH- exposed litters). (B) Trunk blood glucose in PND5 females culled from litters .6 pups (n = 7 DDT- and n = 7 VEH- exposed litters). Hepatic bile acids A 15 mg liver sample (randomized selection from n = 1 female/ litter in 7 VEH and 8 DDT litters fed HFD) was pulverized on dry ice, enriched with deuterated bile acid analogs, butylated hydroxytoluene and ethylinediaminetetraacetic acid, and extract- ed twice with 500 mL methanol. The combined extract was dried, reconstituted in 100 mL methanol:acetonitrile (50:50, v/v) with internal standards 1-phenyl-3-hexanoic acid urea and 1-cyclo- hexyl-3-dodecanoic acid urea (Sigma-Aldrich, St. Louis, MO) and filtered through 0.1 mm filters. Tissue bile acids were isolated and July 2014 \| Volume 9 \| Issue 7 \| e103337 PLOS ONE \| www.plosone.org 3 Effects of Perinatal DDT on Metabolism in Mice Figure 1. Effect of perinatal DDT on the development of female mice. (A) Female body mass at PND5 (n = 15 DDT- and n = 14 VEH- exposed litters). (B) Trunk blood glucose in PND5 females culled from litters .6 pups (n = 7 DDT- and n = 7 VEH- exposed litters). (C) Proportion of mice with vaginal opening as a function of age (n = 15 DDT- and n = 14 VEH- exposed litters). p,0.05 DDT vs. vehicle controls. Data are represented as least squares (LS) means + SEM. doi:10.1371/journal.pone.0103337.g001 Statistical analyses (C) Proportion of mice with vaginal opening as a function of age (n = 15 DDT- and n = 14 VEH- exposed litters). p,0.05 DDT vs. vehicle controls. Data are represented as least squares (LS) means + SEM. doi:10.1371/journal.pone.0103337.g001 Perinatal DDT exposure increases adiposity in young adults Perinatal DDT exposure increases adiposity in young adults To examine whether perinatal DDT exposure causes the metabolic syndrome in adult mouse offspring, dams were exposed to DDT from mid-gestation to PND 5. The resulting internal doses were 2.2 (60.1) ng p,p9-DDE/mL, 51.1 (610.2) ng p,p- DDT/mL, while o,p9-DDT was undetected [mean (6 SEM)]. These exposures were sufficiently low as to have no impact on length of gestation, weight gain during gestation, or litter size (Fig. S2) and fall within the range of contemporary human exposures [10,49]. Perinatal DDT exposure decreased body weight and blood glucose in male mice (Fig. S3A–B) and decreased glucose in female mice (Fig. 1B) on PND 5. Although DDT and DDE are ligands of the estrogen and androgen receptors [50], the timing of puberty in either sex was not significantly affected by the perinatal DDT exposure (Fig. 1C, Fig S3C). Perinatal DDT exposure caused a transient increase in body weight and adiposity of young adult female mice (Fig. 2A–C) but no differences in female lean mass (Fig. 2D), or male body composition were observed (Fig. S3D–E). Further, glucose tolerance, fasting glucose, insulin, and lipid levels were not altered by perinatal DDT exposure in either sex during their first 6 months of post-weaning life (Fig. S4, Table S2). These data suggest that mice exposed to perinatal DDT manifested only subtle features of the metabolic syndrome until adulthood. Since perinatal DDT exposure had no effect on food intake of offspring (Fig. 2E, Fig. S3F), the increased adiposity transiently observed among young female mice exposed to DDT could have resulted from impairments in thermogenesis and energy expenditure. Perinatal exposure to DDT increases susceptibility to HFD- induced insulin resistance Female mice maintained on a LFD and exposed perinatally to DDT had equivocal glucose tolerance compared to vehicle treated controls (Fig. 6A). However, after HF feeding for 12 weeks, female mice exposed perinatally to DDT developed glucose intolerance and insulin resistance as evidenced by higher AUC during GTT, fasting hyperinsulinemia, and increased HOMA-IR (Fig. 6A–C). Of note, this constellation is often observed in prediabetic patients that commonly have the metabolic syndrome. Further, the expression and activation states of AKT and ERK, proteins involved in insulin signaling, were moderately decreased in female mice perinatally exposed to DDT and fed a HFD (Fig. 6D–E), although it remains unclear how much these moderate decreases in hepatic insulin signaling contribute to the observed glucose intolerance and insulin resistance. However, male mice perinatally exposed to DDT developed glucose intolerance when fed LFD, but not HFD (Fig. S8A–B). Thus, perinatal exposure to DDT increases the susceptibility to HFD- induced glucose intolerance in female but not male mice (Fig. 6, Fig. S6). The sex differences will require further studies in the future to test if this is estrogen dependent, but of note, female mice (and humans) are somewhat protected from HF feeding- induced glucose intolerance [58]. Figure 2. Effect of perinatal DDT on body composition and energy balance in female mice. Monthly (A) body mass (p = 0.05 at 2 months, p = 0.08 at 3 months), (B) percent adiposity (p = 0.05 at 3 months), (C) fat mass, (D) lean mass, and (E) food intake (A–E: n = 15 DDT- and n = 14 VEH- exposed litters). (F) Monthly core temperature (1 female/litter at 2 month time point, all females/litter thereafter, p = 0.07 at 2 months, ageDDT pi = 0.07). p,0.05, p,0.001, *p,0.0001 DDT vs. vehicle controls. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g002 thermogenesis. To further establish that female mice perinatally exposed to DDT had compromised thermogenesis, we evaluated cold tolerance in female mice exposed to 4uC. Female mice with perinatal DDT exposure exhibited marked cold intolerance, necessitating the termination of the cold tolerance test after 90 minutes (Fig. 3F). To determine if high fat feeding, a commonly used metabolic stressor to model human obesity, could unveil a metabolic phenotype caused by perinatal DDT exposure, mice were fed either LFD or HFD for 12 weeks beginning at 6 months of age. Perinatal exposure to DDT increases susceptibility to HFD- induced insulin resistance HF feeding increased caloric intake, adiposity, and body mass equally in all groups (Fig. 4A–C, Fig. S5), indicating that any metabolic alterations observed in HF fed mice that were perinatally exposed to DDT occurred independently of caloric intake and/or body composition. Perinatal DDT exposure reduces energy expenditure y To assess thermogenesis, core body temperature was measured longitudinally. Perinatal DDT exposure did not affect the core body temperature of male mice (Fig. S3G), while it decreased the core temperature of female mice, an effect that became more pronounced with age (Fig. 2F). At 6 months, female mice perinatally exposed to DDT had decreased oxygen consumption (Fig. 3A) and a more pronounced RER reduction in the light period than in the dark period, indicative of greater fasting lipid utilization (Fig. 3B), although food intake appeared equivocal across treatment groups (Fig. 3C). Female mice perinatally exposed to DDT also had decreased energy expenditure, while physical activity was not altered (Fig. 3D–E), suggesting that decreased energy expenditure was the result of decreased July 2014 \| Volume 9 \| Issue 7 \| e103337 PLOS ONE \| www.plosone.org 4 Effects of Perinatal DDT on Metabolism in Mice Figure 2. Effect of perinatal DDT on body composition and energy balance in female mice. Monthly (A) body mass (p = 0.05 at 2 months, p = 0.08 at 3 months), (B) percent adiposity (p = 0.05 at 3 months), (C) fat mass, (D) lean mass, and (E) food intake (A–E: n = 15 DDT- and n = 14 VEH- exposed litters). (F) Monthly core temperature (1 female/litter at 2 month time point, all females/litter thereafter, p = 0.07 at 2 months, ageDDT pi = 0.07). p,0.05, p,0.001, *p,0.0001 DDT vs. vehicle controls. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g002 thermogenesis. Indeed HFD and DDT exposures reduced the expression of Dio2 (Fig. 5B), which encodes an enzyme that locally activates thyroid hormone by deiodinating T4 to T3 to stimulate thermogenesis in BAT. However there was no significant change in Ucp1 RNA expression (Fig. 5C), a protein that uncouples respiration to increase thermogenesis [51,52]. Consistent with the regulatory role of PGC1A in substrate utilization [14,53–56], perinatal DDT exposure and adult HF feeding decreased the expression of glucose transporter Glut4, lipoprotein transporter Lpl (no diet effect), lipolysis rate-limiting Pnpla, and the fatty acid oxidation shuttle Cpt2 (Fig. 5D–G). Because transcription of Ppargc1a and its downstream targets was suppressed, we investigated negative regulation of Ppargc1a across DDT and HFD treatments. Indeed, expression of Twist1, a negative regulator of Ppargc1a [57], was increased in BAT of 9 month old mice exposed perinatally to DDT and fed a HFD during adulthood (Fig. 5H). July 2014 \| Volume 9 \| Issue 7 \| e103337 Perinatal DDT exposure and high fat feeding cause mild dyslipidemia in female mice To evaluate whether perinatal DDT exposure impaired lipid metabolism in adult HFD fed- mice, we measured fasting triglycerides, cholesterol, and NEFAs (Fig. 7, Fig. S6). Perinatal DDT exposure significantly increased fasting cholesterol in female mice fed HFD, although it had a negligible effect on cholesterol in female mice fed LFD (Fig. 7A). A similar, but non-significant trend was observed in fasting triglycerides levels in female mice (Fig. 7B), whereas NEFAs were not altered by perinatal DDT or HFD in female mice (Fig. 7C). Thus perinatal DDT exposure combined with HFD caused mild dyslipidemia in female (Fig. 7A) but not male mice (Fig. S6E-G). We also examined whether perinatal DDT exposure altered the expression of mRNA and proteins associated with the regulation of lipid homeostasis in livers from female mice fed HFD. mRNA expression of Ldlr, a cholesterol transporter, was significantly decreased, whereas that of Cyp7a1, the rate limiting enzyme in the synthesis of bile acids from cholesterol, was significantly increased by perinatal DDT exposure (Fig. 7D). Changes in hepatic bile acid metabolism among females fed HFD were consistent with these changes in Cyp7a1 expression Perinatal DDT exposure reduced core temperature in 9 month old female mice (Fig. 4D). HFD feeding led to higher body core temperatures in non-exposed mice, however this thermogenic adaptation to HFD was absent in mice perinatally exposed to DDT (Fig. 4D). Perinatal DDT exposure paradoxically reduced body core temperature by more than 1uC in mice fed HFD (p, 0.0001). Thus, perinatal DDT impairs thermogenesis which is further worsened by HFD, indicating that developmental DDT exposure increases susceptibility to HFD induced- impairment in energy balance. We next examined if perinatal DDT exposure altered molecular mechanisms controlling thermogenesis in BAT of 9 month old female mice (Fig. 5). Compared to LFD-fed control mice, adult HF feeding and perinatal DDT exposure caused a reduction in mRNA expression of Ppargc1a (Fig. 5A), a master regulator of PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e103337 5 Effects of Perinatal DDT on Metabolism in Mice Figure 3. Effect of perinatal DDT on energy expenditure and cold tolerance in six month old female mice. (A) Oxygen consumption, (B) RER (periodDDT pinteraction,0.0001), (C) cumulative food intake, (D) energy expenditure, and (E) movement (over 5 day period and 4 female/ treatment, 1 mouse/litter). (F) Core temperature (5 females/treatment, 1 female/litter). p,0.05, p,0.01, *p,0.001 DDT vs. vehicle controls. Perinatal DDT exposure and high fat feeding cause mild dyslipidemia in female mice Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g003 Figure 3. Effect of perinatal DDT on energy expenditure and cold tolerance in six month old female mice. (A) Oxygen consumption, (B) RER (periodDDT pinteraction,0.0001), (C) cumulative food intake, (D) energy expenditure, and (E) movement (over 5 day period and 4 female/ treatment, 1 mouse/litter). (F) Core temperature (5 females/treatment, 1 female/litter). p,0.05, p,0.01, *p,0.001 DDT vs. vehicle controls. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g003 control groups and were closely associated with hepatic lipids (VIP.1, Fig. 7E, Fig. S7, Table S3). Hepatic protein expression of two lipogenic enzymes ACC and ATPCL was also increased, consistent with increased hepatic lipogenesis as a consequence of perinatal DDT exposure (Fig. 7F). This did not appear to contribute to steatosis, as hepatic lipid content was not increased due to perinatal DDT exposure among female mice fed HFD (Fig. S8). due to perinatal DDT exposure. Specifically, conjugated bile acids, including GLCA, TLCA, and TDCA, and the C27 precursor TriHCA, increased with DDT treatment and these conjugated bile acid changes were closely correlated with hepatic Cyp7a1 expression and plasma cholesterol (VIP.1, Fig. 7E, Fig. S7, Table S3). Conversely, unconjugated bile acids, including HCA, HDCA, and b-MCA, were relatively lower in the DDT exposed versus due to perinatal DDT exposure. Specifically, conjugated bile acids, including GLCA, TLCA, and TDCA, and the C27 precursor TriHCA, increased with DDT treatment and these conjugated bile acid changes were closely correlated with hepatic Cyp7a1 expression and plasma cholesterol (VIP.1, Fig. 7E, Fig. S7, Table S3). Conversely, unconjugated bile acids, including HCA, HDCA, and b-MCA, were relatively lower in the DDT exposed versus Figure 4. Effects of perinatal DDT and adult HFD feeding on body composition and energy balance in female mice (n = 1 female/litter in 7 VEH and 8 DDT litters). (A) Weekly body mass (HFDage pi,0.0001). (B) Percent adiposity in 8 month old mice. (C) Cumulative caloric intake over 12 weeks of HFD or LFD feeding. (D) Core temperature in 8 month old mice (DDTHFD pi = 0.01). p,0.05, p, 0.01, p,0.0001 A–B) LFD vs. HFD only; D) significance between groups indicated by bars. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g004 Discussion Here we investigated the hypothesis that perinatal DDT exposure impairs energy expenditure and metabolism in adult mice. Our results suggest that perinatal DDT exposure decreases energy expenditure, and impairs the regulation of thermogenesis, glucose-, and lipid- homeostasis, which contribute to the development of insulin resistance and the metabolic syndrome in adult female mice. Obesity and thermogenesis Perinatal DDT exposure increased adiposity in young adult female mice. This transient increase in adiposity may have resulted from the profoundly impaired thermogenesis we observed after DDT exposure, which was greater than what was observed in UCP1 knock-out mice [59]. Impaired thermogenesis may have been a consequence of reduced energy supply and utilization in BAT as perinatal DDT exposure significantly decreased expres- sion of lipolytic Pnpla and substrate transporters (Glut4, Lpl, Cpt2) [14,53–56]. Because food intake and physical activity were equal among groups, the reduced energy expenditure would be predicted to increase fat mass more profoundly than what we observed. Whether increased caloric content in feces accounts for this discrepancy will require further study [60]. Figure 4. Effects of perinatal DDT and adult HFD feeding on body composition and energy balance in female mice (n = 1 female/litter in 7 VEH and 8 DDT litters). (A) Weekly body mass (HFDage pi,0.0001). (B) Percent adiposity in 8 month old mice. (C) Cumulative caloric intake over 12 weeks of HFD or LFD feeding. (D) Core temperature in 8 month old mice (DDTHFD pi = 0.01). p,0.05, p, 0.01, p,0.0001 A–B) LFD vs. HFD only; D) significance between groups indicated by bars. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g004 July 2014 \| Volume 9 \| Issue 7 \| e103337 PLOS ONE \| www.plosone.org 6 Effects of Perinatal DDT on Metabolism in Mice Figure 5. Effects of perinatal DDT and adult HFD feeding on RNA expression in BAT from 9 month old female mice (n = 1 female/ litter in 8 VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HFD litters). (A) Ppargc1a- (DDTHFD pi,0.01), (B) Dio2- (DDTHFD pi,0.01), (C) Ucp1-, (D) Glut4- (DDTHFD pi,0.05), (E) Lpl- (DDTHFD pi,0.05), (F) Pnpla- (DDTHFD pi,0.05), (G) Cpt2- (DDTHFD pi,0.05), and (H) Twist1- (DDTHFD pi,0.1) fold change (relative to LFD + VEH and adjusted by Tbp). p,0.05, p,0.01 significance between groups indicated by bars. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g005 Figure 5. Effects of perinatal DDT and adult HFD feeding on RNA expression in BAT from 9 month old female mice (n = 1 female/ litter in 8 VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HFD litters). Insulin resistance and thermogenesis Epidemiological studies have demonstrated associations be- tween adult DDT and DDE exposures and diabetes in humans [7,8,61,62]. Similarly, adult rodents exposed to high doses of DDT had impaired insulin secretion, glucose intolerance, and elevated gluconeogenesis [63,64]. Here we find that female mice perinatally exposed to low doses of DDT and subsequently fed a HFD in adulthood developed impaired glucose tolerance, increased fasting insulin, and HOMA-IR, indicative of insulin resistance and increased risk for T2D. We have observed that Ppargc1a expression in BAT of adult female offspring was reduced after perinatal DDT exposure. Ppargc1a plays an important role linking thermogenesis to risk of T2D as illustrated by genetic ablation studies of Ppargc1a in mice, after which thermogenesis was impaired and susceptibility to HFD-induced insulin resistance, but not obesity, was increased [14]. Whether the reduced Ppargc1a expression in BAT of DDT-exposed mice accounts for their reduced thermogenesis and insulin resistance needs further study. It is plausible that the effects of perinatal DDT on thermogenesis and insulin resistance in mice may be relevant to humans who suffered elevated DDT exposure perinatally. Lipid homeostasis and adaptive energy metabolism We also observed that perinatal DDT exposure caused a modest increase in circulating lipids after HFD feeding, which may in part be due to increased hepatic de novo lipogenesis as suggested by the increased protein expression of key lipogenic enzymes in the liver. Previous studies suggest that acute DDT administration increases hepatic cholesterol and triglyceride synthesis, resulting in their increased circulation in both rats and monkeys [65,66]. It is plausible that DDT and HFD exposures caused higher hepatic de novo lipogenesis, which was compensated for by increased lipid Obesity and thermogenesis (A) Ppargc1a- (DDTHFD pi,0.01), (B) Dio2- (DDTHFD pi,0.01), (C) Ucp1-, (D) Glut4- (DDTHFD pi,0.05), (E) Lpl- (DDTHFD pi,0.05), (F) Pnpla- (DDTHFD pi,0.05), (G) Cpt2- (DDTHFD pi,0.05), and (H) Twist1- (DDTHFD pi,0.1) fold change (relative to LFD + VEH and adjusted by Tbp). p,0.05, p,0.01 significance between groups indicated by bars. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g005 utilization, as evidenced by reduced RER, and increased hepatic bile acid conjugation, to prevent the development of hepatic steatosis in the female mice here [67,68]. Developmental origins of metabolic disruption Several lines of evidence implicate developmental origins in DDT-induced metabolic syndrome. First, when the developmental stage of newborn mice was equivalent to that of a human fetus at term, pups exposed to DDT exhibited reduced body weights and glucose levels. This may contribute to their increased susceptibility to the metabolic syndrome since human studies have demonstrat- ed that low birth weight and low neonatal glucose can be associated with the metabolic syndrome in adulthood [20,69,70]. Second, perinatal DDT exposure was associated with early-adult obesity in female offspring here and across generations in other rodent studies [71,72]. While sex differences are common in mouse models of metabolic disease [73], and both DDT and DDE have estrogenic and anti-androgenic properties [50] the basis for the sex differences observed in our study will have to be delineated in future studies. Further, these data corroborate a prospective study of women where higher cord blood levels of DDE were associated with significantly increased BMI of the adult daughters in a dose dependent manner [74]. Conclusions F) Hepatic FAS- (p = 0.14), A phosphorylation - (p = 0.13), ACC total-, ATPCL phosphorylation (p = 0.07), ATPCL total-, and ATGL- (p = 0.22) fold change (relative LFD + VEH and adjusted by HSC 70) in 9 month old female mice f Figure 6. Effects of perinatal DDT and adult HFD feeding o glucose homeostasis in adult female mice (n = 1 female/litter 8 VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HF litters). (A) Glucose tolerance test and resulting AUC (DDTHF pi = 0.08), (B) fasting insulin (DDTHFD pi = 0.08) and (C) HOMA- (DDTHFD pi = 0.07) in 8 month old mice. (D) Hepatic IRb total, AKT 4 phosphorylation, AKT 308 phosphorylation, AKT total, GSK 3 phospho Figure 7. Effects of perinatal DDT and adult HFD feeding on lipid homeostasis in adult female mice (n = 1 female/litter in 8 VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HFD litters). Fasting- (A) total cholesterol (HFDDDT pi = 0.02), (B) triglycerides (HFDDDT pi = 0.16), and (C) NEFA in 8 month old mice. (D) Hepatic Hmgcr-, Ldlr-, and Cyp7a1- fold change (relative to LFD + VEH and adjusted by Gusb) in 9 month old female mice fed HFD. (E) Bile acid metabolites discriminated by DDT or VEH exposure (VIP.1). Inset: The PLS-DA Scores Plot demonstrates clear discriminatory separation of mice with or without perinatal DDT exposure along latent variable 1 (LV1). Symbols represent individual animals. Metabolite abbreviations followed by ‘‘(s)’’ indicate ‘‘screening’’ data without the benefit of authentic calibration standards. F) Hepatic FAS- (p = 0.14), ACC phosphorylation - (p = 0.13), ACC total-, ATPCL phosphorylation - (p = 0.07), ATPCL total-, and ATGL- (p = 0.22) fold change (relative to LFD + VEH and adjusted by HSC 70) in 9 month old female mice fed HFD. p,0.05, p,0.01, *p,0.001 significance between groups Figure 7. Effects of perinatal DDT lipid homeostasis in adult female m VEH + LFD 8 DDT + LFD 7 VEH + HF Figure 7. Effects of perinatal DDT and adult HFD feeding on lipid homeostasis in adult female mice (n = 1 female/litter in 8 VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HFD litters). Conclusions Our study is the first to establish a link between developmental exposure to DDT and a moderately increased risk for the metabolic syndrome in adult offspring. Further, we know of no other studies implicating impaired thermogenesis in toxicant- induced insulin resistance and the metabolic syndrome. Given that circulating DDT and DDE levels in our mice fell within the range of DDT and DDE found in serum of contemporary people living July 2014 \| Volume 9 \| Issue 7 \| e103337 PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e103337 7 Effects of Perinatal DDT on Metabolism in Mice Figure 6. Effects of perinatal DDT and adult HFD feeding on glucose homeostasis in adult female mice (n = 1 female/litter in 8 VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HFD litters). (A) Glucose tolerance test and resulting AUC (DDTHFD pi = 0.08), (B) fasting insulin (DDTHFD pi = 0.08) and (C) HOMA-IR (DDTHFD pi = 0.07) in 8 month old mice. (D) Hepatic IRb total, AKT 473 phosphorylation, AKT 308 phosphorylation, AKT total, GSK 3 phosphor- ylation, GSK 3 total, ERK phosphorylation, and ERK total fold change (relative to LFD + VEH and adjusted by HSC 70) as assessed by Western blot analysis (n = 1 female/litter in 7 VEH and 8 DDT litters) in 9 month old mice fed HFD. p,0.05, p,0.01, *p,0.001 significance between groups indicated by bars. Data are represented as LS means + SEM. d i 0 3 /j l 0 0333 006 Figure 7. Effects of perinatal DDT and adult HFD feeding o lipid homeostasis in adult female mice (n = 1 female/litter in VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HFD litter Fasting- (A) total cholesterol (HFDDDT pi = 0.02), (B) triglycerid (HFDDDT pi = 0.16), and (C) NEFA in 8 month old mice. (D) Hepa Hmgcr-, Ldlr-, and Cyp7a1- fold change (relative to LFD + VEH a adjusted by Gusb) in 9 month old female mice fed HFD. (E) Bile ac metabolites discriminated by DDT or VEH exposure (VIP.1). Inset: T PLS-DA Scores Plot demonstrates clear discriminatory separation mice with or without perinatal DDT exposure along latent variable (LV1). Symbols represent individual animals. Metabolite abbreviatio followed by ‘‘(s)’’ indicate ‘‘screening’’ data without the benefit authentic calibration standards. Supporting Information Figure S1 Schematic of study design. 1.7 mg DDT/kg body weight was administered to dams daily on gestational day 11 through postnatal day 5 and litters were culled to 6 mice at postnatal day 6. Mice were metabolically screened from 2–6 months when they were randomized into 2 independent study arms: energy balance and HFD or LFD feeding. Indirect calorimetry and cold tolerance were assessed in 6-month-old mice. Metabolic parameters (e.g. body composition, fasting lipids, glucose, and insulin, GTT) were assessed during 12 weeks of HFD or LFD feeding until mice were sacrifice at 9 months of age. (DOCX) Figure S1 Schematic of study design. 1.7 mg DDT/kg body weight was administered to dams daily on gestational day 11 through postnatal day 5 and litters were culled to 6 mice at postnatal day 6. Mice were metabolically screened from 2–6 months when they were randomized into 2 independent study arms: energy balance and HFD or LFD feeding. Indirect calorimetry and cold tolerance were assessed in 6-month-old mice. Metabolic parameters (e.g. body composition, fasting lipids, glucose, and insulin, GTT) were assessed during 12 weeks of HFD or LFD feeding until mice were sacrifice at 9 months of age. (DOCX) Figure S2 Effect of prenatal DDT on gestation. (A) Gestation length, (B) gestation weight gain, and (C) litter size (n = 15 DDT litters and n = 14 VEH litters). Data are represented as LS means 6 SEM. (DOCX) Figure S8 Lipid content of adult female mice fed HFD. (A) Oil red O stained liver from 9 month old female mice fed HFD for 12 weeks with exposure to perinatal vehicle (top) or DDT (bottom, bar = 50 microns). (B) Hepatic triglycerides and cholesterol in 9 month old female mice fed HFD. Data are represented as LS means + SEM. (DOCX) Figure S3 Effect of perinatal DDT on body composition and energy balance in male mice. (A) Body mass at PND5 (n = 15 DDT- and n = 14 vehicle- exposed litters). (B) Trunk glucose in PND5 males culled from litters .6 pups (n = 7 DDT and n = 7 VEH litters). (C) Proportion of males with preputial separation as a function of age (n = 15 DDT and n = 14 VEH litters). Conclusions Fasting- (A) total cholesterol (HFDDDT pi = 0.02), (B) triglycerides (HFDDDT pi = 0.16), and (C) NEFA in 8 month old mice. (D) Hepatic Hmgcr-, Ldlr-, and Cyp7a1- fold change (relative to LFD + VEH and adjusted by Gusb) in 9 month old female mice fed HFD. (E) Bile acid metabolites discriminated by DDT or VEH exposure (VIP.1). Inset: The PLS-DA Scores Plot demonstrates clear discriminatory separation of mice with or without perinatal DDT exposure along latent variable 1 (LV1). Symbols represent individual animals. Metabolite abbreviations followed by ‘‘(s)’’ indicate ‘‘screening’’ data without the benefit of authentic calibration standards. F) Hepatic FAS- (p = 0.14), ACC phosphorylation - (p = 0.13), ACC total-, ATPCL phosphorylation - (p = 0.07), ATPCL total-, and ATGL- (p = 0.22) fold change (relative to LFD + VEH and adjusted by HSC 70) in 9 month old female mice fed HFD. p,0.05, p,0.01, p,0.001 significance between groups indicated by bars. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g007 Figure 6. Effects of perinatal DDT and adult HFD feeding on glucose homeostasis in adult female mice (n = 1 female/litter in 8 VEH + LFD, 8 DDT + LFD, 7 VEH + HFD and 8 DDT + HFD litters). (A) Glucose tolerance test and resulting AUC (DDTHFD pi = 0.08), (B) fasting insulin (DDTHFD pi = 0.08) and (C) HOMA-IR (DDTHFD pi = 0.07) in 8 month old mice. (D) Hepatic IRb total, AKT 473 phosphorylation, AKT 308 phosphorylation, AKT total, GSK 3 phosphor- ylation, GSK 3 total, ERK phosphorylation, and ERK total fold change (relative to LFD + VEH and adjusted by HSC 70) as assessed by Western blot analysis (n = 1 female/litter in 7 VEH and 8 DDT litters) in 9 month old mice fed HFD. p,0.05, p,0.01, *p,0.001 significance between groups indicated by bars. Data are represented as LS means + SEM. doi:10.1371/journal.pone.0103337.g006 PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e103337 8 Effects of Perinatal DDT on Metabolism in Mice intake over 12 week HFD and LFD feeding period (n=8 litters/ treatment, 1 mouse/litter). (D) Rectal temperature in 8 month old mice (n=8 litters/treatment, 1 mouse/litter). p,0.05, p,0.01, p, 0.0001 LFD vs. HFD. Data are represented as LS means 6 SEM. Acknowledgments We wish to thank Leslie Roteta for help with parts of the animals studies and Drs. Gary Schwartz and Derek LeRoith for sharing their BAT- 10 and Echo-MRI respectively; Drs. Myrto Petreas and June-Soo Park for quantitating sera DDTs; Drs. James Godbold, Philip Landrigan, and Michael Gallo for their comments. The USDA is an equal opportunity provider and employer. Table S1 Table of Information about Western Blot Antibodies. (DOCX) Table S2 Effects of perinatal DDT on metabolic param- eters in mice fed regular chow. LS Means (6 SEM) shown. (DOCX) Table S3 Liver Bile Acid concentrations (nmol/g). Values are reported as mean 6 SEM). (DOCX) Figure S4 Effects of perinatal DDT on glucose tolerance. GTT in (A) female and (B) male 2 month old mice (2.0 mg glucose/ kg body mass, 1.5 mg glucose/kg body mass, n =15 DDT-exposed litters and n =14 oil-exposed litters, 1 mouse/litter), in (C) female and (D) male 3 month old mice (1.0 mg glucose/kg body mass, 1.5 mg glucose/kg body mass, n = 12 litters/treatment, 1 mouse/litter), and in (E) female and (F) male 6 month old mice (1.0 mg glucose/kg body mass, n = 15 DDT-exposed litters and n =14 oil-exposed litters, 1 mouse/litter). Data are represented as LS means 6 SEM. (DOCX) Conclusions (DOCX) in northern Europe and in malaria-infested regions, and of the pregnant mothers of US adults who are currently in their mid- fifties [7,49,75,76] this is an important observation. Thus, it is likely that perinatal DDT exposure represents a risk factor for reduced energy expenditure in adult humans, even decades after DDT usage had ceased. The exact mechanism of this metabolic toxicity is unknown and how much it contributes to insulin resistance and dyslipidemia in the current obesity and diabetes epidemic will require further studies. But even small dysmetabolic effects of DDT and DDE are likely to be important from a public health perspective due to the ubiquity of these exposures among aging US and European populations as well as current populations of some developing and circumpolar countries. Given that the ban of DDT has reduced DDT exposure among the US population by about 100 fold [77], replacing DDT in malaria control should decrease DDT exposures over time and may reduce the susceptibility to the metabolic syndrome in future generations. Figure S6 Joint effects of perinatal DDT and HFD on glucose and lipid homeostasis in male mice. (A) Glucose tolerance test at 1 mg glucose/kg body mass and (B) correspond- ing AUC, in 8 month old mice (n = 8 litters/treatment, 1 mouse/ litter). (C) Insulin, (D) HOMA-IR, (E) total cholesterol, (F) triglycerides, and (G) NEFA (mg/l plasma) in 8 month old mice fasted for 5 hrs (n = 8 litters/treatment, 1 mouse/litter). LFD vs. HFD. p,0.05 significance between groups indicated by bars. Data are represented as LS means 6 SEM. (DOCX) Figure S7 Partial Least Squares Discrimination Analy- sis (PLS-DA) of hepatic bile acids, triglycerides, and other metabolic factors which may influence or be influenced by cholesterol metabolism segregate treat- ment groups. (B) Relative weight of independent variable to this segregation are shown in the PLS Loadings Plot. Hierarchical cluster analyses were used to segregate variables and 4 unique clusters are displayed with unique colors. Variables with the variable importance in projection VIP scores .0.8 or ,20.8 are labeled, .1 or ,21 shown as triangles. (C) All LV1 VIP scores. Metabolite abbreviations followed by ‘‘(s)’’ indicate ‘‘screening’’ data relative areas generated from the expected precursor . product ion mass transition in the LC-MS/MS system, without the benefit of authentic calibration standards. (DOCX) Supporting Information Monthly (D) body mass and (E) percent adiposity, (F) food intake, and (G) rectal temperature ((n = 15 DDT and n = 14 VEH litters, 1 mouse/litter at 2 month time point, all males/litter thereafter). Data are represented as LS means 6 SEM. p,0.05 DDT vs. vehicle controls. (DOCX) References 1. Shaw JE, Sicree RA, Zimmet PZ (2010) Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 87: 4–14. 28. Wang QA, Tao C, Gupta RK, Scherer PE (2013) Tracking adipogenesis during white adipose tissue development, expansion and regeneration. Nat Med 19: 1338–1344. 2. American Diabetes A (2013) Economic costs of diabetes in the U.S. in 2012. Diabetes Care 36: 1033–1046. 29. 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(2013) Prenatal Exposure to the Pesticide DDT and Hypertension Diagnosed in Women before Age 50: A Longitudinal Birth Cohort Study. Environ Health Perspect 121: 594–599. 65. Nagaoka S, Masaki H, Aoyama Y, Yoshida A (1986) Effects of excess dietary tyrosine or certain xenobiotics on the cholesterogenesis in rats. Journal of Nutrition 116: 726–732. 66. Sanyal S, Agarwal N, Dudeja PK, Mahmood A, Subrahmanyam D (1982) Effect of a single oral dose of DDT on lipid metabolism in protein-calorie malnourished monkeys. Indian Journal of Biochemistry and Biophysics 19: 111–114. 77. Eskenazi B, Chevrier J, Rosas LG, Anderson HA, Bornman MS, et al. (2009) The Pine River statement: human health consequences of DDT use. Environ Health Perspect 117: 1359–1367. PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e103337 11
https://openalex.org/W2568179435	https://europepmc.org/articles/pmc5248579?pdf=render	English	null	The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation	PeerJ	2,017	cc-by	12,178	ABSTRACT Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C- terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested. Submitted 18 October 2016 Accepted 8 December 2016 Published 18 January 2017 Corresponding author Rüdiger H. Ettrich, ettrich@nh.cas.cz Academic editor Joanna Moraczewska Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.2887 Copyright 2017 Bialevich et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Submitted 18 October 2016 Accepted 8 December 2016 Published 18 January 2017 Corresponding author Rüdiger H. Ettrich, ettrich@nh.cas.cz Academic editor Joanna Moraczewska Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.2887 Copyright Subjects Biochemistry, Biophysics, Computational Biology, Molecular Biology Keywords E. coli, Multisubunit enzyme complex, Molecular modeling, Domain interactions, DNA restriction enzymes Subjects Biochemistry, Biophysics, Computational Biology, Molecular Biology Keywords E. coli, Multisubunit enzyme complex, Molecular modeling, Domain interactions, DNA restriction enzymes How to cite this article Bialevich et al. (2017), The helical domain of the EcoR124I motor subunit participates in ATPase activity and ds- DNA translocation. PeerJ 5:e2887; DOI 10.7717/peerj.2887 Vitali Bialevich1,2,, Dhiraj Sinha1,2,, Katsiaryna Shamayeva1, Alena Guzanova3, David Řeha1,2, Eva Csefalvay1, Jannette Carey1,4, Marie Weiserova3 and Rüdiger H. Ettrich1,2,5 1 Center for Nanobiology and Structural Biology, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Nove Hrady, Czech Republic 1 Center for Nanobiology and Structural Biology, Institute of Microbiology of the Academy of Sciences of t Czech Republic, Nove Hrady, Czech Republic 2 Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Nove Hrady, Czech Republic 3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic 4 Chemistry Department, Princeton University, Princeton, NJ, United States 5 College of Medical Sciences, Nova Southeastern University, Fort Lauderdale, FL, United States * These authors contributed equally to this work. 2 Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Nove Hrady, Czech Republic 3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic 4 Chemistry Department, Princeton University, Princeton, NJ, United States 5 College of Medical Sciences, Nova Southeastern University, Fort Lauderdale, FL, United States * These authors contributed equally to this work 5 College of Medical Sciences, Nova Southeastern University, Fort Lauderdale, FL, United States * These authors contributed equally to this work. The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation Vitali Bialevich1,2,, Dhiraj Sinha1,2,, Katsiaryna Shamayeva1, Alena Guzanova3, David Řeha1,2, Eva Csefalvay1, Jannette Carey1,4, Marie Weiserova3 and Rüdiger H. Ettrich1,2,5 Vitali Bialevich1,2,, Dhiraj Sinha1,2,, Katsiaryna Shamayeva1, Alena Guzanova3, David Řeha1,2, Eva Csefalvay1, Jannette Carey1,4, Marie Weiserova3 and Rüdiger H. Ettrich1,2,5 Subjects Biochemistry, Biophysics, Computational Biology, Molecular Biology Keywords E. coli, Multisubunit enzyme complex, Molecular modeling, Domain interactions, DNA restriction enzymes INTRODUCTION Copyright 2017 Bialevich et al. Type I restriction-modification enzymes (RMs) are multisubunit, multifunctional molecular machines that recognize specific, typically asymmetric, DNA target sequences of ∼13–14 bp (Murray, 2002). In sharp contrast to the straightforward mechanisms of the Type II RMs (Loenen et al., 2014), the multisubunit organization of Type I RM Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS systems underlies their multifunctionality. Depending on the methylation status of adenine residues in the target sequence, three Type I subunits either act together as a typical methyltransferase, or recruit a pair of endonuclease motor subunits that initiate translocation of DNA through the enzyme and eventually cleave non-specifically at apparently random sites (Janscak et al., 1999). The protein complex remains bound at the target sequence while up to thousands of bp are pumped through the enzyme by tracking along the duplex helical pitch at rates of up to hundreds of bp per second (Bickle, Brack & Yuan, 1978; Studier & Bandyopadhyay, 1988; Szczelkun et al., 1996; Ellis et al., 1999; Seidel et al., 2004). Translocation is driven by RecA-helicase-like motor subunits that consume ∼1 ATP per ∼1 bp (Seidel et al., 2008) without separating the duplex strands (Stanley et al., 2006). The endonuclease activity of the motor subunits is unmasked under conditions that suggest a role for DNA topology in control of cleavage activity (Yuan, 1981; Janscak et al., 1999; Janscak & Bickle, 2000). The X-ray crystal structure of the first Type I R-M motor subunit was solved in 2009 for EcoR124I from the E. coli plasmid pTrcR124 (Lapkouski et al., 2009). EcoR124I is the archetype of Type I R-M family IC and is composed of three distinct subunit types, HsdS, HsdM, and HsdR, which are encoded by the hsd (host specificity of DNA) genes. The fully assembled enzyme complex is a pentamer with R2M2S1 stoichiometry (Janscak, Dryden & Firman, 1998; Mernagh et al., 1998). Small-angle neutron scattering experiments revealed the locations of HsdS and HsdM (Taylor et al., 2010), and electron microscopy and neutron and small-angle X-ray scattering (Kennaway et al., 2012) indicate that DNA binding triggers compaction of the R2M2S1 enzyme from a conformation that has been described as more relaxed, loose, or open. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 INTRODUCTION The motor subunit HsdR is composed of four distinct structural and functional domains: an endonuclease domain at the N-terminus with characteristic motifs of the RecB nuclease superfamily (Obarska-Kosinska et al., 2008; Sisáková et al., 2008a; Sisáková et al., 2008b); a C-terminal α-helical domain proposed to contact methyltransferase (Davies et al., 1999); and two RecA-like helicase domains situated between the endonuclease and C-terminal domains (Obarska-Kosinska et al., 2008; Lapkouski et al., 2009). Like other members of the SF2 translocase family, EcoR124I translocates on dsDNA (McClelland & Szczelkun, 2004; Firman & Szczelkun, 2000; Seidel et al., 2004) and lacks strand separation activity (Stanley et al., 2006). It has been proposed that helicase domain 1 contacts the 3′–5′ dsDNA strand tightly during translocation, while helicase domain 2 is proposed to be responsible for DNA advancement through large and rapid conformational changes (Lewis et al., 2008). Because both the translocase and endonuclease functions are part of the array of integrated structural domains in the large HsdR subunit of 1038 residues, a large number of interdomain contacts might be expected. However, the crystal structure of EcoR124I HsdR (Lapkouski et al., 2009) reveals a surprisingly sparse number of contacts particularly between the helical domain and helicase domain 2. The contact interface between the two domains is only 859 Å2. Only a tiny fraction of it, 15 Å2, is hydrophobic, and only two potential hydrogen-bonding contacts are found. One is centrally located between the two domains, a bifurcated contact in which the K527 sidechain atom Nζ is at a distance of Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 2/23 2.9 Å from the D796 carboxylate Oδ atom and 2.7 Å from the backbone carbonyl oxygen atom of N794. A second hydrogen bond is possible close to the short loop (K731-T734) that links the two domains, where the phenol O of Y736 is at a distance of 2.6 Å from the Oε2 atom of the E730 carboxylate (Fig. 1). These two hydrogen bonds constitute the only contacts detected at the domain interface. This finding led to a search for sequence similarities in the HsdR subunits of other Type I R-M enzymes that might support the significance of these contacts. The sequence alignment in Fig. INTRODUCTION 1C suggests that K527 may be part of a new motif that can be defined as (Q/D)KT that is also identified in the HsdR subunits of the EcoKI and EcoAI enzymes that typify Type I R-M families IA and IB. This proposal represents the first extension to all three Type I R-M families of a sequence alignment beyond the conserved DEAD-box motifs of the helicase active sites. As helicase domain 2 is proposed to be the key player in DNA advancement, undergoing large conformational changes during the translocation cycle (Dürr et al., 2005), the question arises whether these two contacts to the helical domain have a role in the translocation cycle. If so, the helical domain itself could have a previously undescribed role in the translocation cycle beyond its presumed role in interacting with methyltransferase. In the present work, the relevance of these two potential hydrogen-bonding interactions for the translocation activity of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results suggest a role for the helical domain in DNA translocation. MATERIALS AND METHODS Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Mutagenesis, expression and protein puriﬁcation Secondary structures of Eco124I HsdR from the crystal strutcure (pdb code: 2w00) are shown on top. Colors and symbols below the sequences indicate residue similarity: ‘’ fully conserved residues; ‘:’ highly similar residues; and ‘.’ somewhat similar residues. Figure 1 Interdomain interactions at the helical-helicase 2 domain interface in the HsdR subunit of EcoR124I restriction-modification complex. (A) The structure of HsdR consisting of four domains: the endonuclease domain is in yellow, the helicase 1 and the helicase 2 domains are depicted in cyan and ma- genta, respectively. The C-terminal helical domain is shown in green. ATP is represented in elemental col- ors as a skeletal model with cyan carbons, and magnesium is indicated by a yellow sphere. The modelled parts of the structure (not resolved in the crystal structure) are in orange and the modeled 180s loop com- prising residues 182–189 that is close to the catalytic site on the endonuclease domain is in red just below ATP (coordinates borrowed from K220A mutant HsdR crystal structure ((PDB id: 4BEC, Csefalvay et al., 2015)). The short linker between helicase 2 domain and the C-terminal helical domain is in grey at the lower right corner of the black rectangle (B). Enlargement of the region enclosed by the black rectangle in (A), showing residues involved in interactions at the interface between the helical domain and helicase do- main 2. Side chains are shown as skeletal models in atomic colors with carbon atoms colored according to their subunit shown in (A). Hydrogen bonding interactions discussed in the text are indicated with black dotted lines. (C) Alignment of the interface region sequence of EcoR124I from family IC of with sequences from HsdR of the archetypical members of the other two type I R-M families, EcoKI from family IA and EcoAI from family IB. Secondary structures of Eco124I HsdR from the crystal strutcure (pdb code: 2w00) are shown on top. Colors and symbols below the sequences indicate residue similarity: ‘’ fully conserved residues; ‘:’ highly similar residues; and ‘.’ somewhat similar residues. E. coli BL21(DE3) Gold competent cells were transformed with plasmid DNA containing the desired mutation. Overnight cultures were diluted 1:100 into 0.5 l of fresh LB medium in a 3-l flask supplemented to a final concentration of 100 µg ml−1 with ampicillin and incubated at 180 rpm at 37 ◦C until OD600 was about 0.5–0.6. Mutagenesis, expression and protein puriﬁcation Mutagenesis, expression and protein puriﬁcation Plasmid pTrcR124 (Janscak, Abadjieva & Firman, 1996) carrying the hsdR gene was used for site-directed mutagenesis. Oligonucleotide primers were used in the polymerase chain reaction (forward primer sequence is given, reverse primer is its complement; mutated codon underlined, changed nucleotides in bold): 5′-TTTCCGCCAG GCAACCCACCGTACC-3′ for K527A, 5′-GAGTTATACAGAGGCTATGGAAGGC-3′ for Y736A, and 5′-GCAAAACTATGCTGAATTTGCCACG-3′ for D796A. The double mutant K527A_Y736A was obtained by introducing a second point mutation into the mutant plasmid Y736A. The polymerase chain reaction was performed in 200 µl PCR tubes in an Eppendorf (Hamburg, Germany) mastercycler. The 20-µl reaction mixture contained 1.5 U Expand Long Range polymerase (Roche, Basel, Switzerland); 1X Expand Long Range Buffer with MgCl2; 100 ng of plasmid DNA pTrcR124; 0.3 µM each of the forward and reverse primers; 200 µM dNTPs; and 3 (v/v)% or 6 (v/v)% DMSO. PCR cycles were: 2 min at 96 ◦C;30×(30s at 96 ◦C, 90s at 55 ◦C, 8 min at 68 ◦C); 7 min at 68 ◦C. DpnI (20 U) was used to degrade methylated parental plasmid. The reaction mixture was transformed into E. coli DH5α competent cells grown on LB-agar plates containing 100 µg/ml of ampicillin. Plasmid DNA was isolated using a Zyppy plasmid miniprep kit (Zymo Research, Irvine, CA, USA) and sequenced. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 3/23 Figure 1 Interdomain interactions at the helical-helicase 2 domain interface in the HsdR subunit of EcoR124I restriction-modification complex. (A) The structure of HsdR consisting of four domains: the endonuclease domain is in yellow, the helicase 1 and the helicase 2 domains are depicted in cyan and ma- genta, respectively. The C-terminal helical domain is shown in green. ATP is represented in elemental col- ors as a skeletal model with cyan carbons, and magnesium is indicated by a yellow sphere. The modelled parts of the structure (not resolved in the crystal structure) are in orange and the modeled 180s loop com- prising residues 182–189 that is close to the catalytic site on the endonuclease domain is in red just below ATP (coordinates borrowed from K220A mutant HsdR crystal structure ((PDB id: 4BEC, Csefalvay et al., 2015)). The short linker between helicase 2 domain and the C-terminal helical domain is in grey at the lower right corner of the black rectangle (B). Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Mutagenesis, expression and protein puriﬁcation Enlargement of the region enclosed by the black rectangle in (A), showing residues involved in interactions at the interface between the helical domain and helicase do- main 2. Side chains are shown as skeletal models in atomic colors with carbon atoms colored according to their subunit shown in (A). Hydrogen bonding interactions discussed in the text are indicated with black dotted lines. (C) Alignment of the interface region sequence of EcoR124I from family IC of with sequences from HsdR of the archetypical members of the other two type I R-M families, EcoKI from family IA and EcoAI from family IB. Secondary structures of Eco124I HsdR from the crystal strutcure (pdb code: 2w00) are shown on top. Colors and symbols below the sequences indicate residue similarity: ‘’ fully conserved residues; ‘:’ highly similar residues; and ‘.’ somewhat similar residues. Figure 1 Interdomain interactions at the helical-helicase 2 domain interface in the HsdR subunit of EcoR124I restriction-modification complex. (A) The structure of HsdR consisting of four domains: the endonuclease domain is in yellow, the helicase 1 and the helicase 2 domains are depicted in cyan and ma- genta, respectively. The C-terminal helical domain is shown in green. ATP is represented in elemental col- ors as a skeletal model with cyan carbons, and magnesium is indicated by a yellow sphere. The modelled parts of the structure (not resolved in the crystal structure) are in orange and the modeled 180s loop com- prising residues 182–189 that is close to the catalytic site on the endonuclease domain is in red just below ATP (coordinates borrowed from K220A mutant HsdR crystal structure ((PDB id: 4BEC, Csefalvay et al., 2015)). The short linker between helicase 2 domain and the C-terminal helical domain is in grey at the lower right corner of the black rectangle (B). Enlargement of the region enclosed by the black rectangle in (A), showing residues involved in interactions at the interface between the helical domain and helicase do- main 2. Side chains are shown as skeletal models in atomic colors with carbon atoms colored according to their subunit shown in (A). Hydrogen bonding interactions discussed in the text are indicated with black dotted lines. (C) Alignment of the interface region sequence of EcoR124I from family IC of with sequences from HsdR of the archetypical members of the other two type I R-M families, EcoKI from family IA and EcoAI from family IB. Mutagenesis, expression and protein puriﬁcation IPTG was added to a final concentration of 1 mM and the culture was incubated for an additional 4 h at 37 ◦C and 30 ◦C for WT and mutant HsdR subunits, respectively. Harvested cells were washed in sodium chloride–Tris-EDTA buffer, pH 8.0. WT and mutant HsdR subunits were purified as described previously (Janscak, Abadjieva & Firman, 1996). EcoR124I Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 4/23 methyltransferase was expressed from pAC15M (Holubová et al., 2004) and pJS491 (Patel et al., 1992) plasmids encoding HsdM and HsdS subunits, respectively, and purified as described previously (Taylor et al., 1992). Purified methyltransferase and HsdR subunits were mixed together in a 1:6 ratio to reconstitute the EcoR124I enzyme complex in vitro. In vivo restriction activity assay y y E.coli strain JM109(DE3) (Yanisch-Perron, Vieira & Messing, 1985) served for com- plementation analysis of restriction function in vivo. For negative complementation the pTrcR124 plasmid expressing WT or mutants HsdR was transformed into E. coli JM109(DE3)[pKF650], a restricting (r+m+) host containing all three hsd genes of EcoR124II. For positive complementation pTrcR124 was transformed into E.coli JM109(DE3)[pACMS] expressing only the EcoR124II methyltransferase (r−m+) (Patel et al., 1992). Soft agar at 45 ◦C was mixed with 0.5 ml of each fresh overnight culture, gently mixed, and immediately poured onto agar plates with appropriate antibiotics. The virulent mutant of phage λ was used for testing of restriction phenotype (Jacob & Wollman, 1954). Solidified soft agar was spotted with 30 µl each of tenfold serial dilutions of λvir.0 phage lysate at 102 to 106 plaque-forming units/ml. The spots were dried at room temperature and the plates incubated overnight at 37 ◦C (Colson et al., 1965). Phage buffer, complex LB medium and in vivo restriction assays were as described (Hubácek, Holubová & Weiserová, 1998; Sisáková et al., 2008b). The solid medium is LB with agar added at 1.5 (w/v)%. Soft agar overlay is LB with agar added at 0.6 (w/v)%. Antibiotics were used at the following concentrations: ampicillin; 100 mg ml−1, chloramphenicol; 50 mg ml−1. The efficiency of plating was determined as the number of plaques on the tested strains compared to the number of plaques on the non-restricting control strain E. coli JM109(DE3) (Yanisch-Perron, Vieira & Messing, 1985). Values in the range 0.0001–0.01 correspond to the restriction-competent (r+) phenotype, those in the range 0.01–0.1 to the mixed-competence (r±) phenotype, and those in the range 0.1–1 to the restriction-deficient (r−) phenotype. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 DNA Translocation assay—steady state analysis Linear DNA for the triple-helix displacement assay was generated by ApaI digestion of pLKS5 plasmid DNA followed by phenol/chloroform extraction, chloroform extraction and isopropanol precipitation. The triplex was formed by incubating 50 nM linear and 25 nM 5′-end Tetramethylrhodamine (TAMRA) labeled oligonucleotide 5′- In vitro cleavage activity assay Cleavage activity was assayed in vitro using supercoiled plasmid DNA substrate pRK (Taylor et al., 1992) carrying a single recognition site for EcoR124I. Cleavage activity was assayed (Janscak, Abadjieva & Firman, 1996) at 37 ◦C in restriction buffer (50 mM Tris–HCl, pH 8.0, 1 mM DTT, 10 mM MgCl2 and 50 mM NaCl) containing 15 nM of the circular pRK plasmid DNA, 15 nM methyltransferase and 90 nM HsdR. After 1 min of pre-incubation the reaction was started by addition of ATP and SAM to a final concentration 4 mM and 0.2 mM, respectively. The reaction was stopped by adding 0.25 vol of stop reagent (3 (w/v)% SDS; 0.15 M EDTA; 10 (w/v)% glycerol; 0.1 (w/v)% bromophenol blue) and heating at 65 ◦C for 5 min. Samples were loaded into 1 (w/v)% agarose gels in Tris-acetate-EDTA buffer and run at 5 V/cm for 130 min. Gels were stained with 2 µg/ml ethidium bromide, destained in water and photographed under UV illumination. The relative amounts of DNA were evaluated by densitometry using ImageJ software (Abramoff, Magalhaes & Ram, 2004). Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 5/23 5/23 Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 DNA Translocation assay—steady state analysis DNA Translocation assay—steady state analysis The method utilizes a fluorescently labeled triplex forming oligonucleotide (TFO) attached at a specific distance from the EcoR124I binding site on plasmid DNA. Translocation by EcoR124I leads to the dissociation of the triplex, which can be monitored by the change in fluorescence upon displacement (Firman & Szczelkun, 2000; McClelland, Dryden & Szczelkun, 2005). Plasmid pLKS5 carrying a DNA triplex binding site 1,517 bp downstream of the EcoR124I recognition site was used (Stanley & Szczelkun, 2006). Linear DNA for the triple-helix displacement assay was generated by ApaI digestion of pLKS5 plasmid DNA followed by phenol/chloroform extraction, chloroform extraction and isopropanol precipitation. The triplex was formed by incubating 50 nM linear and 25 nM 5′-end Tetramethylrhodamine (TAMRA) labeled oligonucleotide 5′- ‘TTTCTTCTTCTTTTCTTTTCTT-3’ (Eurofins MWG Operon) in buffer (10 mM MES pH 5.5, 12.5 mM MgCl2) at 23 ◦C overnight. Following overnight incubation, the triplex was stored on ice, and then diluted 1/10 in reaction buffer (50 mM Tris–HCl, pH 8.0, 1 mM DTT, 10 mM MgCl2 and 50 mM NaCl) before use. 5 nM Triplex was pre-incubated with 40 nM MTase and 120 nM wt or mutant HsdR at RT ◦C for 5 min. Following Simons & Szczelkun (2011) the required concentration of the HsdR subunit for R2M2S1 complex formation is twice that of the DNA under the given conditions. A 120 nM concentration of HsdR (methyltransferase (M2S1) to HsdR ratio 1:3) was enough to saturate both methyltransferase binding sites under experimental conditions. Translocation reaction was initiated with 4 mM ATP and after 10 min triplex displacement was measured. Steady state anisotropy for the bound (triplex) and free forms of TAMRA labeled TFO was measured using a Tecan Safire 2 Multi-detection Microplate Reader. Excitation and emission wavelengths used were: 544 and 576 nm, bandwidths were kept <10 nm. Data are expressed as the means of three independent experiments. The method utilizes a fluorescently labeled triplex forming oligonucleotide (TFO) attached at a specific distance from the EcoR124I binding site on plasmid DNA. Translocation by EcoR124I leads to the dissociation of the triplex, which can be monitored by the change in fluorescence upon displacement (Firman & Szczelkun, 2000; McClelland, Dryden & Szczelkun, 2005). Plasmid pLKS5 carrying a DNA triplex binding site 1,517 bp downstream of the EcoR124I recognition site was used (Stanley & Szczelkun, 2006). Molecular dynamics simulations The WT crystal structure of the motor subunit HsdR from the restriction-modification system EcoR124I (PDB entry 2W00) and a recent crystal structure of HsdR mutant Lys220Ala (PDB entry 4BEC) were used for preparing the structural model for all simulations. Three missing segments from the WT crystal structure (residues 142–147, 585–590, and 859–869) were built using standard loop modeling in YASARA (Krieger, Koraimann & Vriend, 2002; Konagurthu et al., 2006) and added to the WT crystal structure. The WT crystal structure of the motor subunit HsdR from the restriction-modification system EcoR124I (PDB entry 2W00) and a recent crystal structure of HsdR mutant Lys220Ala (PDB entry 4BEC) were used for preparing the structural model for all simulations. Three missing segments from the WT crystal structure (residues 142–147, 585–590, and 859–869) were built using standard loop modeling in YASARA (Krieger, Koraimann & Vriend, 2002; Konagurthu et al., 2006) and added to the WT crystal structure. The missing segment from residue 182 to 189 is resolved in the Lys220Ala mutant crystal structure and thus was built into the WT structure by adding the coordinates from the mutant to the above modeled structure, followed by steepest-descent energy minimization. All classical MD simulations were performed using GROMACS 4.64 (Berendsen, Van der Spoel & Van Drunen, 1995; Van der Spoel et al., 2005; Pronk et al., 2013) with the AMBER99SB forcefield (Hornak et al., 2006). ATP was parameterized by applying the standard RESP procedure using Antechamber (Wang et al., 2004), where charges for free MgATP were derived from HF/6-31G calculation in Gaussian03 (Frisch et al., 2004). Histidine was assumed to be charged, with the ND and NE atoms protonated; arginine and lysine residues were assumed to be protonated. Mutants were prepared in silico by replacing the respective side-chain in YASARA followed by short minimization of 100 ps with freezing of all residues except those close to each point mutation, to avoid any local crashes in the side-chain and then simulated for 100ns using classical molecular dynamics simulations and analyzed to evaluate possible conformational changes. All systems were solvated in explicit TIP3P water (Jorgensen et al., 1983) in a cubic box with a margin of 10 Å and neutralized by adding sodium counterion. ATPase activity assay in vitro ATPase activity was assayed using a radioactivity-based ATPase assay. In a first step cellulose plates were prepared by marking positions for sample drops with a pencil: 1.5 cm from the bottom of the plate and 1 cm in between dots. A total of 50 ml of running buffer (0.4 M LiCl, 1 M Formic acid) or water was carefully poured in the glass chamber, avoiding drops on the walls. At first each cellulose plate was pre-run in running buffer and dried. Then, the same steps were performed with destilled water. Prepared plates can be stored at room temperature up to one week. ATPase activity itself was assayed in NEB2 buffer containing 10 mM Tris–HCl, pH 7.9, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT; 15 nM methyltransferase, 90 nM HsdR, and 6 molar excess (90 nM) of covalently closed circular DNA pRK containing single recognition site for EcoR124I to prevent DNA cleavage. Excess of DNA concentration over enzyme was used (6:1 ratio) following Janscak, Abadjieva & Firman (1996) and 6-folds excess in HsdR subunit concentration ensures efficient ATPase activity as was described by Janscak, Dryden & Firman (1998). The HsdR subunit does not turn over after cleavage of supercoiled DNA that might occur, nevertheless, ATPase activity is efficient under tested conditions (Simons & Szczelkun, 2011). Reactions (40 µL) were started by addition of ATP mixture containing Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 6/23 0.16 µCi (0.0013 mM) [γ 32P]-ATP to a final concentration of 2 mM and incubated at 37 ◦C. Aliquots (4 µL) were taken at the indicated time points and stopped by adding 1 (w/v)% SDS. The hydrolyzed 32Pi was separated from [γ 32P]-ATP by cellulose TLC in 0.4 M LiCl2, 1M formic acid (Randerath & Randerath, 1964) and the distribution of radioactivity between 32Pi and ATP was visualized using a Fujitsu 9000 scanner (Marini & Krejci, 2012). Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Restriction activity in vivo The restriction activity of wild-type (WT) HsdR and all mutants was measured in vivo by monitoring the ability of cells expressing WT HsdS-HsdM2 methyltransferase and WT or mutated HsdR to restrict the growth of unmodified bacteriophage λvir.0. Positive and negative complementation tests (Sisáková et al., 2008b), respectively, were used to distinguish between defects in DNA cleavage and defects in the interaction of mutant HsdR subunits with methyltransferase. Positive complementation uses a restriction-deficient (r−) host lacking WT HsdR to test if a mutant HsdR subunit functions in DNA cleavage. Negative complementation uses a restriction-competent (r+) host expressing WT HsdR to test if a mutant HsdR subunit that is defective in DNA cleavage is competent for assembly with methyltransferase, allowing it to compete with WT HsdR subunits and thus reduce their restriction activity. This effect is called a trans-dominant effect. Wildtype HsdR complements restriction in the r−host in the positive complementation test (Table 1, r− host), reducing λvir.0 infectivity as expected (value 0.001, within the range 0.0001–0.01 expected for the restriction-proficient phenotype REF). All four mutants, K527A, Y736A, K527A_Y736A, and D796A, fail to complement restriction in the r−host (all values within the range 0.1–1 expected for the restriction-deficient phenotype) and are therefore restriction-deficient. In the negative complementation test the reduced restriction activity in the r+ host (Table 1, r+ host) indicates that all four mutant subunits are fully competent for assembly with methyltransferase to form the endonuclease complex. The in vivo results thus support a role for the helical domain in enzyme activity. Molecular dynamics simulations The particle-mesh Ewald method (Darden et al., 1993) was applied to calculate long-range electrostatic interactions with a cutoff distance of 10 Å and a Lennard-Jones 6–12 potential was used to evaluate van der Waals interactions within 10 Å cutoff distance. The LINCS algorithm of fourth order expansion was used to constrain bond lengths (Hess et al., 1997). After solvation and neutralization each system was energy minimized for 10,000 step using steepest descent optimization method to remove poor van der Waals contacts in the initial geometry. After minimization two stages of equilibration were conducted. Firstly the system was equilibrated for 1 ns with position restraints of 10,000 kJ/mol on all heavy atoms. A constant temperature of 300 K was maintained using the V-rescale algorithm (Bussi, Donadio & Parrinello, 2007) with a coupling time of 0.1 ps and separate baths for the solute and the solvent. The pressure was kept constant at 1 bar using the Parrinello-Rahman pressure coupling scheme (Parrinello & Rahman, 1981) with a time constant of 2 ps. Initial velocities were generated Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 7/23 randomly using a Maxwell–Boltzmann distribution corresponding to 300 K. Neighbor lists were updated every 10 fs using a group cut-off scheme. Finally the production run was performed for 100 ns without restraints at 300 K in the isothermal–isobaric ensemble. Principal-component analysis (Amadei, Linnssen & Berendsen, 1993) was used using g_covar and g_anaeig tools in the GROMACS package to identify the global motions of the system using backbone atom only. The g_rms tool of the GROMACS package was used to calculate root mean square deviations (RMSD) during the trajectories taking the minimized crystal structure as reference. Root mean square fluctuations (RMSF) of the backbone of each residue were calculated by g_rmsf while atomic distances were measured by g_dist. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 DNA cleavage activity in vitro DNA cleavage activity is tested in vitro on a covalently closed circular plasmid DNA containing a single EcoR124I recognition site using the EcoR124I RM complex reconstituted with either WT or mutant HsdR. DNA cleavage in vitro proceeds in two steps (Janscak, Abadjieva & Firman, 1996). When translocation is impeded the enzyme first cleaves one strand of the DNA duplex resulting in a nicked DNA product, which is then linearized in a second step. Although cleavage is non-specific and distant from the single recognition sequence, the resulting linearized DNAs are all of the same length. For efficient DNA cleavage a 1:1 ratio of reconstituted EcoR124I R2M2S1 complex to Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 8/23 Table 1 Effect of mutations on the restriction phenotype of EcoR124I. HsdR Restrictiona Ability of cleavage Ability of assembly r−hostb r+ hostc WT 0.0012 ± 0.0005SD 0.0011 ± 0.0004 Yes Yes K527A 0.4388 ± 0.3009 0.1092 ± 0.0857 No Yes Y736A 0.3197 ± 0.2147 0.2307 ± 0.0694 No Yes K527A + Y736A 0.4230 ± 0.1953 0.1619 ± 0.0666 No Yes D796A 0.3962 ± 0.0885 0.1965 ± 0.1335 No Yes Notes. aRestriction activity was determined as the efficiency of plating of λvir.0 on tested strains relative to the efficiency of plating of λvir.0 on E. coli JM109(DE3) indicator (nonrestricting) strain. bThe positive complementation was tested in r−host E. coli JM109(DE3)[pACMS] (r−m+). cNegative complementation (transdominant effect) in r+ host E. coli JM109(DE3)[ pKF650] (r+m+). SD, standard deviation. DNA and 6:1 ratio of HsdR subunits to methyltransferase (M2S1-complex) was used. An excess of HsdR subunit over methyltransferase is required for successful formation of restriction-proficient complexes with stoichiometry of R2M2S1 because the second HsdR subunit binds to methyltransferase with at least two orders of magnitude lower affinity than the first one (Janscak, Dryden & Firman, 1998; Mernagh et al., 1998). With reconstituted WT enzyme in 30 min of incubation, essentially all the circular substrate with a single EcoR124I recognition site is cleaved on one or both strands, generating approximately 90% linear product and 10% nicked DNA (‘‘open circle’’ on Fig. 2). Mutants K527A, Y736A, and the double mutant K527A_Y736A are unable to cleave this circular DNA. Mutant D796A exhibits measurable but significantly decreased cleavage activity compared to WT, with ∼47 % of the DNA remaining circular, ∼35 % cleaved completely, and ∼18 % nicked. DNA cleavage activity in vitro To quantify the relative cleavage rates of different mutant proteins, rate constants for the disappearance of supercoiled DNA were estimated for WT and mutant D796A by fitting the data on Fig. 2 with an exponential function. The best-fit value of the rate constant λ for the reconstituted WT enzyme is 0.0238 s−1, whereas the rate constant λ for mutant D796A is approximately two thirds the WT value, 0.0154 s−1. The difference between WT and mutant values is in a similar range as found previously for other mutants that display a restriction-deficient phenotype in vivo (Csefalvay et al., 2015). Thus the in vivo results can be considered a more sensitive indication of enzyme activity than the in vitro results. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Translocation activity in vitro Translocation of duplex DNA by reconstituted WT and mutant enzymes was measured using a triplex displacement assay. In this assay, a fluorescently-labeled oligonucleotide forms a DNA triplex 1517 bp downstream of the recognition sequence and is displaced when this region of the DNA passes through the enzyme during translocation (McClelland, Dryden & Szczelkun, 2005). Complete displacement of the oligonucleotide by the enzyme results in a fluorescence signal similar to that of the free oligonucleotide. At the DNA to methyltransferase ratio of 8:1 and saturating concentration of HsdR subunits used in this Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 9/23 Figure 2 Cleavage of supercoiled substrate. (A) Supercoiled DNA substrate plasmid pRK carrying sin- gle EcoR124I recognition site is cleaved by EcoR124I R-M complex in vitro. EcoR124I was reconstituted from HsdS1HsdM2 methyltransferase and (A) WT HsdR or mutant HsdRs (B) K527A, (C) Y736A, (D) K527A_Y736A and (E) D796A. Aliquots were quenched at the time points indicated in seconds and re- solved on agarose gels stained with ethidium bromide. On (A) open circle and linear products are denoted as OC and L, respectively, and supercoiled substrate as SC; control (plasmid pRK linearized by HindIII restriction enzyme) is denoted as C; DNA molecular weight marker (M) with marked band size in kb on (A). (B) Quantification. DNA species were quantified using ImageJ software (Abramoff, Magalhaes & Ram, 2004). Gels were scanned under UV illumination, and the image converted to grey scale and then in- verted. The band density in the lane at time zero (before initiation of the reaction with ATP) is taken as 100% of supercoiled substrate. The three indicated DNA species OC, open circular product (▲); L, linear product ( ); SC, supercoiled substrate (■) were quantified individually from densities of bands in the gels on the left panels. Error bars represent standard deviations calculated from at least three independent ex- periments for each enzyme. Rates for the decrease of supercoiled DNA substrate were derived by fitting an exponential decay function to the data in SigmaPlot (solid lines); dashed lines connect the points only to guide the eye and do not represent fits to the data. Figure 2 Cleavage of supercoiled substrate. (A) Supercoiled DNA substrate plasmid pRK carrying sin- gle EcoR124I recognition site is cleaved by EcoR124I R-M complex in vitro. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Translocation activity in vitro Triplex-forming oligonucleotide (TFO) fluorescently labeled with TAMRA is hybridized to linearized DNA and translocation is initiated by addi- tion of reconstituted enzyme and ATP as described in Methods. Fluorescence intensities after 10 min reac- tion time are shown. Bound (triplex) and free (displaced) TFO are indicated by lower and upper horizon- tal lines, respectively. From left to right, enzyme reconstituted with: wildtype HsdR, mutant K527A HsdR, mutant Y736A HsdR, double mutant K527A_Y736A HsdR, mutant D796A HsdR, unreacted triplex only, and free TFO. No triplex displacement is observed in the absence of ATP or HsdR (not shown). Average values of fluorescence intensities calculated from four or more independent experimental replicates for each individual WT or mutant enzyme species are shown. Figure 3 DNA translocation analysis by triplex displacement. Triplex-forming oligonucleotide (TFO) fluorescently labeled with TAMRA is hybridized to linearized DNA and translocation is initiated by addi- tion of reconstituted enzyme and ATP as described in Methods. Fluorescence intensities after 10 min reac- tion time are shown. Bound (triplex) and free (displaced) TFO are indicated by lower and upper horizon- tal lines, respectively. From left to right, enzyme reconstituted with: wildtype HsdR, mutant K527A HsdR, mutant Y736A HsdR, double mutant K527A_Y736A HsdR, mutant D796A HsdR, unreacted triplex only, and free TFO. No triplex displacement is observed in the absence of ATP or HsdR (not shown). Average values of fluorescence intensities calculated from four or more independent experimental replicates for each individual WT or mutant enzyme species are shown. assay, the single recognition site on the circular DNA substrate is expected to be fully bound by the pentameric complex of EcoR124I with stoichiometryR2M2S1 (McClelland, Dryden & Szczelkun, 2005). In control experiments no triplex displacement is observed in the absence of ATP or HsdR. Only the enzyme complex reconstituted with WT HsdR results in fluorescence intensity similar to that of the free oligonucleotide. All four mutant enzymes, K527A, Y736A, D796A, and K527A_Y736A, showed fluorescence intensities similar to that of the initial DNA triplex with bound oligonucleotide (Fig. 3), indicating that these enzymes do not displace the oligonucleotide and thus have lost the ability to translocate duplex DNA. These results demonstrate that these point mutations in the helical domain affect translocation. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Translocation activity in vitro EcoR124I was reconstituted from HsdS1HsdM2 methyltransferase and (A) WT HsdR or mutant HsdRs (B) K527A, (C) Y736A, (D) K527A_Y736A and (E) D796A. Aliquots were quenched at the time points indicated in seconds and re- solved on agarose gels stained with ethidium bromide. On (A) open circle and linear products are denoted as OC and L, respectively, and supercoiled substrate as SC; control (plasmid pRK linearized by HindIII restriction enzyme) is denoted as C; DNA molecular weight marker (M) with marked band size in kb on (A). (B) Quantification. DNA species were quantified using ImageJ software (Abramoff, Magalhaes & Ram, 2004). Gels were scanned under UV illumination, and the image converted to grey scale and then in- verted. The band density in the lane at time zero (before initiation of the reaction with ATP) is taken as 100% of supercoiled substrate. The three indicated DNA species OC, open circular product (▲); L, linear product ( ); SC, supercoiled substrate (■) were quantified individually from densities of bands in the gels on the left panels. Error bars represent standard deviations calculated from at least three independent ex- periments for each enzyme. Rates for the decrease of supercoiled DNA substrate were derived by fitting an exponential decay function to the data in SigmaPlot (solid lines); dashed lines connect the points only to guide the eye and do not represent fits to the data. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 10/23 Figure 3 DNA translocation analysis by triplex displacement. Triplex-forming oligonucleotide (TFO) fluorescently labeled with TAMRA is hybridized to linearized DNA and translocation is initiated by addi- tion of reconstituted enzyme and ATP as described in Methods. Fluorescence intensities after 10 min reac- tion time are shown. Bound (triplex) and free (displaced) TFO are indicated by lower and upper horizon- tal lines, respectively. From left to right, enzyme reconstituted with: wildtype HsdR, mutant K527A HsdR, mutant Y736A HsdR, double mutant K527A_Y736A HsdR, mutant D796A HsdR, unreacted triplex only, and free TFO. No triplex displacement is observed in the absence of ATP or HsdR (not shown). Average values of fluorescence intensities calculated from four or more independent experimental replicates for each individual WT or mutant enzyme species are shown. Figure 3 DNA translocation analysis by triplex displacement. ATP hydrolysis in vitro DNA-dependent ATPase activities were measured using the circular DNA substrate containing a single EcoR124I recognition site after reconstitution of the purified WT and mutant HsdR subunits with HsdS-HsdM2 methyltransferase. Control assays omitting either DNA or methyltransferase indicate negligible ATP hydrolysis. A relative ATPase activity of 100% was assigned to the enzyme reconstituted with WT HsdR. The relative ATPase activity of reconstituted mutant enzymes Y736A, K527A, and double mutant Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 11/23 Figure 4 DNA-dependent ATPase activity. EcoR124I reconstituted from methyltransferase and HsdRs WT (black), or mutant K527A (red), Y736A (blue), D796A (green) or K527A_Y736A (dark cyan) was incubated at a final concentration of 15 nM with 90 nM circular plasmid DNA containing one recogni- tion site and 2 mM ATP containing 0.16 µCi g-32P-ATP. At the indicated time points ATP and inorganic phosphate were resolved on cellulose TLC, autoradiographed, and scanned to quantify the extent of hy- drolysis. The amount of ATP degraded is plotted as a function of time. Average values and standard devia- tions are calculated from at least three independent replicates for each mutant enzyme and six for WT en- zyme. Data for mutants are normalized to the average value for WT at 900 s taken as 100%. Figure 4 DNA-dependent ATPase activity. EcoR124I reconstituted from methyltransferase and HsdRs WT (black), or mutant K527A (red), Y736A (blue), D796A (green) or K527A_Y736A (dark cyan) was incubated at a final concentration of 15 nM with 90 nM circular plasmid DNA containing one recogni- tion site and 2 mM ATP containing 0.16 µCi g-32P-ATP. At the indicated time points ATP and inorganic phosphate were resolved on cellulose TLC, autoradiographed, and scanned to quantify the extent of hy- drolysis. The amount of ATP degraded is plotted as a function of time. Average values and standard devia- tions are calculated from at least three independent replicates for each mutant enzyme and six for WT en- zyme. Data for mutants are normalized to the average value for WT at 900 s taken as 100%. K527A_Y736A is ∼25%, 16%, and 8%, respectively (Fig. 4), indicating that these enzymes hydrolyze ATP very poorly in vitro, consistent with their inability to translocate duplex DNA in vitro. However, reconstituted mutant enzyme D796A exhibits a relative ATPase activity of ∼88%, in apparent contradiction to its lack of translocation activity in the triplex-displacement experiments. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Molecular dynamics simulations The initial structure of the HsdR subunit used in the simulations presented here (Sinha et al., 2014) is based on the published crystal structure (PDB id: 2W00, Lapkouski et al., 2009) with modeled missing loops (residues 142–147, 585–590, and 859–869) built using standard loop modeling in YASARA (Krieger, Koraimann & Vriend, 2002; Konagurthu et al., 2006). This structure has been shown previously to be stable in MD simulations, and the resulting trajectories achieve equilibrium after 50 ns of simulation (Sinha et al., 2014) allowing simulations of 100 ns, which is sufficient to test the effect of point mutations. Structures of single mutants K527A, D796A, and Y736A, and double mutant K527A_Y736A were prepared by swapping each individual residue for the WT residue of the starting structure in YASARA. The trajectories of WT and mutant HsdR subunits reveal that in all cases the secondary structure is maintained throughout and the fold is preserved. In all simulations the root mean square deviations (RMSD) from the starting structure reach a plateau of ∼3 to 3.5 Å after 50 ns, comparable to equilibration times and RMSD values reported previously for the WT HsdR system with modeled missing loops (Sinha et al., 2014) and acceptable for a system of this size having multiple domains. The initial structure of the HsdR subunit used in the simulations presented here (Sinha et al., 2014) is based on the published crystal structure (PDB id: 2W00, Lapkouski et al., 2009) with modeled missing loops (residues 142–147, 585–590, and 859–869) built using standard loop modeling in YASARA (Krieger, Koraimann & Vriend, 2002; Konagurthu et al., 2006). Global motions during the simulations were analyzed by principal components analysis (PCA). Equilibrated trajectories for the final 20 ns of each individual WT or mutant simulation were extracted and joined into one combined trajectory to allow calculation of the first eigenvector that identifies the motion with the largest change (Fig. 5). The global collective motion described by the first eigenvector for mutants K527A, Y736, and K527A_Y736A is a partial opening of the cleft between helicase domains 1 and 2, due mainly to movement of helicase domain 2. Mutant D796A and WT HsdR maintain the starting conformation of the WT subunit, in which the cleft remains closed. Projections along the first eigenvector were analyzed to quantify these structural changes (Fig. 6). Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 ATP hydrolysis in vitro The finding that mutant D796A retains ATPase activity but lacks translocation activity suggests that the correlation between these two activities may be weaker than has been previously assumed (Abadjieva & Firman, 1996). Although ATPase activity has been considered an indicator of translocation activity and is a necessary condition for translocation, ATPase activity is only one component of translocation activity, and in this case it is not sufficient for translocation. The decoupling of ATPase activity from translocation in the D796A mutant may reflect structural or dynamic changes that cannot be further described at present. Alternatively or additionally, the different temperatures for the translocation and ATPase assays might be related to the inability to translocate efficiently despite ATPase activity. Under the conditions tested here, although the D796A mutant was able to partially cleave the supercoiled DNA substrate in vitro, the rate of DNA cleavage was insufficient for cells to survive phage infection in vivo. Nevertheless, the inefficient ATP hydrolysis by three of the enzymes reconstituted with mutant HsdR subunits indicates an unexpected role for the helical domain in ATP hydrolysis. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 12/23 Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Molecular dynamics simulations These projections describe the extent to which the motion described by the first eigenvector is present in the simulation of each protein. WT HsdR shows no major change along the first eigenvector and its projection value oscillates around zero, as expected because the initial WT structure represents an energy-minimized and equilibrated structure. Like WT HsdR, mutant D796A shows no change along the first eigenvector and its projection oscillates around zero, indicating no opening of the cleft between the two helicase subunits. In contrast, K527A, Y736A, and double mutant K527A_Y736A move ∼10 +/−2 Å along the first eigenvector, indicating considerable movement of helicase domain 2 away from helicase domain 1 to open the cleft compared with the initial crystal structure. These global structural changes of helicase domain 2 during the simulations are also reflected in the rmsd values calculated for C-alpha atoms by superimposing the last snapshot of each simulated structure on the WT crystal structure. During 100ns of WT MD simulation helicase domain 2 stays in a position almost identical to that in the crystal, with a maximum rmsd of ∼0.2 Å. Significant structural changes occur during the simulations of mutants Y736A, K527A, and double mutant K527A_Y736A, with maximum rmsd values of ∼4.1 Å, ∼3.8 Å and ∼2.8 Å, respectively. Mutant D796A shows a maximum rmsd of ∼0.6 Å, indicating only a minor conformational change similar to that observed in WT. The interdomain distances between centers of mass of the two helicase domains Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Figure 5 Overlay of WT and mutant structures. Helicase domains 1 and 2 are shown in tube representa- tion with the WT conformation in gray. Mutant conformations are overlaid on helicase domain 1, which is thus identical in all cases and therefore shown only once for better visualization. The position of each helicase 2 domain is depicted in color for each mutant (K527A, green; Y736A, blue; K527A_Y736A, red, and D796A, cyan). Mutant D796A domain 2 overlays substantially with WT, whereas the other three mu- tants are shifted due to the cleft opening discussed in the text. Figure 5 Overlay of WT and mutant structures. Helicase domains 1 and 2 are shown in tube representa- tion with the WT conformation in gray. Mutant conformations are overlaid on helicase domain 1, which is thus identical in all cases and therefore shown only once for better visualization. Molecular dynamics simulations The position of each helicase 2 domain is depicted in color for each mutant (K527A, green; Y736A, blue; K527A_Y736A, red, and D796A, cyan). Mutant D796A domain 2 overlays substantially with WT, whereas the other three mu- tants are shifted due to the cleft opening discussed in the text. Figure 5 Overlay of WT and mutant structures. Helicase domains 1 and 2 are shown in tube representa- tion with the WT conformation in gray Mutant conformations are overlaid on helicase domain 1 which Figure 5 Overlay of WT and mutant structures. Helicase domains 1 and 2 are shown in tube representa- tion with the WT conformation in gray. Mutant conformations are overlaid on helicase domain 1, which is thus identical in all cases and therefore shown only once for better visualization. The position of each helicase 2 domain is depicted in color for each mutant (K527A, green; Y736A, blue; K527A_Y736A, red, and D796A, cyan). Mutant D796A domain 2 overlays substantially with WT, whereas the other three mu- tants are shifted due to the cleft opening discussed in the text. confirm the helicase cleft opening in mutants K527A, Y736A, and K527A_Y736A, whereas in mutant D796A the two domains are somewhat closer to each other than in WT (Table 2). These results suggest that loss of the limited interdomain contacts between the helical domain and helicase domain 2 leads to substantial structural changes in mutants K527A, Y736A and double mutant K527A_Y736A, characterized by opening of the cleft between the two helicase domains located ∼25 Å away from the site of these mutations. Such a long-range effect is further indication of an integral role for the helical domain in HsdR function. In contrast, in WT and mutant D796A HsdR, helicase domain 2 largely maintains the conformation observed in the WT crystal structure. This result suggests that in the D796A mutant K527 may be able to maintain at least partial contact with the helical domain even though D796 is no longer available as a partner. To further evaluate this possibility a more subtle interaction analysis of the simulation results was carried out. The persistence of the interdomain contacts observed in the crystal structure was analyzed in the equilibrated part of each simulation. Although neither H atoms nor hydrogen bonds are detectable in crystal structures, H atoms are included in the calculations based on Bialevich et al. Molecular dynamics simulations (2017), PeerJ, DOI 10.7717/peerj.2887 14/23 Figure 6 Projection of the first eigenvector of the joined trajectories in the final 20 ns of the equili- brated part of each individual simulation. For analysis the 20 ns segments from the simulation of each protein were joined to make the composite trajectory shown. Data for the WT simulation are represented from 0 to 20 ns, for K527A from 20 to 40 ns, for Y736A from 40–60 ns, for K527A_Y736A from 60 to 80 ns, and for D796A from 80 to 100 ns. Figure 6 Projection of the first eigenvector of the joined trajectories in the final 20 ns of the equili- brated part of each individual simulation. For analysis the 20 ns segments from the simulation of each protein were joined to make the composite trajectory shown. Data for the WT simulation are represented from 0 to 20 ns, for K527A from 20 to 40 ns, for Y736A from 40–60 ns, for K527A_Y736A from 60 to 80 ns, and for D796A from 80 to 100 ns. Figure 6 Projection of the first eigenvector of the joined trajectories in the final 20 ns of the equili- brated part of each individual simulation. For analysis the 20 ns segments from the simulation of each protein were joined to make the composite trajectory shown. Data for the WT simulation are represented from 0 to 20 ns, for K527A from 20 to 40 ns, for Y736A from 40–60 ns, for K527A_Y736A from 60 to 80 ns, and for D796A from 80 to 100 ns. predicted covalent geometries (Berendsen, Van der Spoel & Van Drunen, 1995). Hydrogen bonds are observed between the K527 amino group and two acceptors, the backbone carbonyl O atom of N794 and the carboxylate Oδ atom of D796, and between the hydroxyl group of Y736 and carboxylate Oε atom of E730 in the short loop linking the two domains. Figure 7A reveals that during simulation of WT HsdR the hydrogen bond between the K527 amino group and the backbone carbonyl O atom of N794 is lost completely after 20 ns, whereas the hydrogen bond between the amino group of K527 and the carboxylate Oδ atom of D796 is maintained. The hydrogen bond between the Y736 hydroxyl and the E730 carboxylate is maintained throughout the simulation in WT (Fig. 7B). Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Molecular dynamics simulations In mutant D796A the hydrogen bond between the K527 amino group and the D796 carboxylate is no longer possible. The contact between the K527 amino group and the N794 backbone carbonyl O atom is maintained after the initial equilibration time (Fig. 7C), as is the interaction between the Y736 hydroxyl and the E730 carboxylate (Fig. 7D). The resulting conformation of mutant D796A is thus very similar to WT throughout the simulation, showing no large-scale domain movement although its interactions at the helical-helicase interface differ locally. Although mutant K527A is unable to hydrogen bond with either N794 or D796 as a result of the point mutation, the Y736-E730 contact close to the short loop linking the two domains persists throughout the simulation. Mutant Y736A does not form the hydrogen bond close to the short loop linking the two domains, and the interaction between the K527 amino group and the carboxylate Oδ atom of D796 is also lost completely during equilibration. Only a contact between the K527 amino group and the backbone carbonyl O atom of N794 is persistent. Finally, analysis of H-bond numbers at the interface between Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Table 2 Interdomain distances and H-bonding between helicase I and helicase II domains. terdomain distances and H-bonding between helicase I and helicase II domains. Table 2 Interdomain distances and H-bonding between helicase I and helicase II domains. Avg. Inter-domain distance (Å) Avg. Number of Interactions (H-bond) WT 29.30 ± .14 13 ± 1.40 K527A 29.77 ± .18 10 ± 1.05 Y736A 29.90 ± .17 09 ± 1.17 K527A_Y736A 30.30 ± .21 09 ± 1.05 D796A 29.01 ± .13 17 ± 2.11 Figure 7 Atomic distances during the 100 ns simulations. (A) WT K527(Nz)-N794(O) in black, WT K527(Nz)-D796(CG) in red; (B) WT Y736(OH)-E730(CD); (C) mutant D796A K527(Nz)-N794(O); (D) mutant D796A Y736(OH)-E730(CD). Figure 7 Atomic distances during the 100 ns simulations. (A) WT K527(Nz)-N794(O) in black, WT K527(Nz)-D796(CG) in red; (B) WT Y736(OH)-E730(CD); (C) mutant D796A K527(Nz)-N794(O); (D) mutant D796A Y736(OH)-E730(CD). Figure 7 Atomic distances during the 100 ns simulations. (A) WT K527(Nz)-N794(O) in black, WT K527(Nz)-D796(CG) in red; (B) WT Y736(OH)-E730(CD); (C) mutant D796A K527(Nz)-N794(O); (D) mutant D796A Y736(OH)-E730(CD). helicase domains 1 and 2 during the simulations suggests a correlation with helicase cleft opening (Table 2). DISCUSSION The structurally characterized homologs of HsdR are the RecA-like SF2 helicases for its translocation activity and the Type II restriction endonucleases for its endonuclease activity. Both these homologs carry out their enzyme activity as isolated proteins with no additional functional domains nor assembly with other subunits required. The HsdR subunit in contrast is a single polypeptide chain that represents a symbiotic fusion of two RecA-like helicase domains with folds characteristic of SF2 helicases, an endonuclease domain characteristic of the Type II enzymes, and an auxiliary C-terminal helical domain. Based on the results presented here, the role of the helical domain can now be said to include participation in all the individual activities of the HsdR motor subunit: DNA cleavage, DNA translocation, and ATP hydrolysis. Because the observed interdomain interactions contribute to enzymatic function as described in this work, they may be conserved in other Type1 restriction-modification systems in which single HsdR subunits have multiple functions. Sequence alignment suggests that the main interaction partner identified here in helicase domain 2, K527, is conserved throughout Type I R-M families IA, IB, IC as part of a proposed new (Q/D)KT motif (Fig. 1C). A similar three-dimensional arrangement and function can be suggested for this region of these other Type I R-M systems. Other related translocases from the SF2 superfamily such as Rad54 display low sequence identity in this region, suggesting that the (Q/D)KT motif may be conserved specifically in the Type I R-M systems, which require interdomain contact between helicase domain 2 and the helical domain to integrate the functions of the domains. The present work demonstrates a previously unsuspected role for the helical C-terminal domain of the HsdR motor subunit in DNA translocation, cleavage, and ATPase activities. This domain has been assumed to be involved in assembly with the HsdS/M methylase (Dryden et al., 2001; Obarska-Kosinska et al., 2008). A role involving all three of the subunit’s activities implicates the C-terminal domain in controlling enzymatic function. Thus a controlling function resides at a nexus of intersubunit communication. Molecular dynamics simulations Thus, double mutant K527A_Y736A with the largest interdomain distance displays only up to nine interdomain H-bonds compared to ∼13 in WT HsdR, whereas closure of the cleft in mutant D796A is correlated with up to 18 H-bonds at the interdomain interface. helicase domains 1 and 2 during the simulations suggests a correlation with helicase cleft opening (Table 2). Thus, double mutant K527A_Y736A with the largest interdomain distance displays only up to nine interdomain H-bonds compared to ∼13 in WT HsdR, whereas closure of the cleft in mutant D796A is correlated with up to 18 H-bonds at the interdomain interface. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 16/23 ACKNOWLEDGEMENTS The authors wish to thank Dr. Ladislav Cséfalvay for his assistance with ATPase activity assays. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Competing Interests Rudiger H. Ettrich is an Academic Editor for PeerJ. Author Contributions Author Contributions • Vitali Bialevich and Dhiraj Sinha performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Vitali Bialevich and Dhiraj Sinha performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Katsiaryna Shamayeva performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, reviewed drafts of the paper. • Alena Guzanova performed the experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper. • David Řeha analyzed the data, prepared figures and/or tables, reviewed drafts of the paper. • Eva Csefalvay conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, reviewed drafts of the paper. • Jannette Carey conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper. • Marie Weiserova conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Rüdiger H. Ettrich conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. Data Availability The following information was supplied regarding data availability: The raw data of experimental work is included in the manuscript as gels in Figs. 2A–2D, as data points in Figs. 3 and 4, and as the efficiency of plating in Table 2. The computational models of WT and the mutants have been supplied as a Supplemental File. Funding This study was supported by the Czech Science Foundation (P207/12/2323). Access to modeling facilities was supported by the Czech research infrastructure for systems biology C4SYS (project no. LM2015055). Additionally, RE was supported by a Fulbright-Masaryk Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: Czech Science Foundation: P207/12/2323. Czech Science Foundation: P207/12/2323. Czech research infrastructure for systems biology C4SYS: LM2015055. Fulbright-Masaryk Scholarship. Supplemental Information pp Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.2887#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.2887#supplemental-information. peerj.2887#supplemental-information. peerj.2887#supplemental-information. Bialevich et al. (2017), PeerJ, DOI 10.7717/peerj.2887 18/23 REFERENCES Abadjieva A, Firman K. 1996. 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https://openalex.org/W4386476398	https://angeo.copernicus.org/preprints/angeo-2023-21/angeo-2023-21.pdf	English	null	Reply on RC2	null	2,023	cc-by	15,274	On mechanisms for HF pump-enhanced optical emissions at 557.7 and 630.0 nm from atomic oxygen in the high-latitude F-region ionosphere The emissions at 557.7 nm are attributed to electron acceleration by upper hybrid waves of meter-scale wavelengths that can be excited with f0 = 5.423 MHz ≲4fe. for the HF pump frequency f0 = 6.200 MHz ≈4.6fe to I5577/I6300 ≈0.5 when f0 = 5.423 MHz ≲4fe, where fe is the 5 ionospheric electron gyro frequency. The observations are interpreted in terms of decreased electron heating efﬁciency and thereby weaker enhancement at 630.0 nm for f0 = 5.423 MHz ≲4fe. The emissions at 557.7 nm are attributed to electron acceleration by upper hybrid waves of meter-scale wavelengths that can be excited with f0 = 5.423 MHz ≲4fe. On mechanisms for HF pump-enhanced optical emissions at 557.7 and 630.0 nm from atomic oxygen in the high-latitude F-region ionosphere Thomas B. Leyser1, Tima Sergienko2, Urban Brändström2, Björn Gustavsson3, and Michael T. Rietveld4 1Swedish Institute of Space Physics, Uppsala, Sweden. 2Swedish Institute of Space Physics, Kiruna, Sweden. 3Department of Physics and Technology, The Arctic University of Norway, Tromsø, Norway. 4Scientist emeritus, EISCAT Scientiﬁc Association, Ramfjordmoen, Norway. Correspondence: Thomas Leyser (thomas.leyser@irfu.se) Thomas B. Leyser1, Tima Sergienko2, Urban Brändström2, Björn Gustavsson3, and Michael T. Rietveld4 1Swedish Institute of Space Physics, Uppsala, Sweden. 2Swedish Institute of Space Physics, Kiruna, Sweden. 3Department of Physics and Technology, The Arctic University of Norway, Tromsø, Norway. 4Scientist emeritus, EISCAT Scientiﬁc Association, Ramfjordmoen, Norway. Correspondence: Thomas Leyser (thomas.leyser@irfu.se) Abstract. The EISCAT Heating facility was used to transmit powerful high frequency (HF) electromagnetic waves into the F-region ionosphere to enhance optical emissions at 557.7 and 630.0 nm from atomic oxygen. The emissions were imaged by three stations of the Auroral Large Imaging System in northern Sweden and the EISCAT UHF incoherent scatter radar was used to obtain plasma parameter values. The ratio of the 557.7 to 630.0 nm column emission rates changed from I5577/I6300 ≈0.2 for the HF pump frequency f0 = 6.200 MHz ≈4.6fe to I5577/I6300 ≈0.5 when f0 = 5.423 MHz ≲4fe, where fe is the 5 ionospheric electron gyro frequency. The observations are interpreted in terms of decreased electron heating efﬁciency and thereby weaker enhancement at 630.0 nm for f0 = 5.423 MHz ≲4fe. The emissions at 557.7 nm are attributed to electron acceleration by upper hybrid waves of meter-scale wavelengths that can be excited with f0 = 5.423 MHz ≲4fe. Abstract. The EISCAT Heating facility was used to transmit powerful high frequency (HF) electromagnetic waves into the F-region ionosphere to enhance optical emissions at 557.7 and 630.0 nm from atomic oxygen. The emissions were imaged by three stations of the Auroral Large Imaging System in northern Sweden and the EISCAT UHF incoherent scatter radar was used to obtain plasma parameter values. The ratio of the 557.7 to 630.0 nm column emission rates changed from I5577/I6300 ≈0.2 for the HF pump frequency f0 = 6.200 MHz ≈4.6fe to I5577/I6300 ≈0.5 when f0 = 5.423 MHz ≲4fe, where fe is the 5 ionospheric electron gyro frequency. The observations are interpreted in terms of decreased electron heating efﬁciency and thereby weaker enhancement at 630.0 nm for f0 = 5.423 MHz ≲4fe. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. 1 Introduction Powerful high frequency (HF) electromagnetic waves transmitted into the ionosphere from the ground may enhance optical 10 emissions from atmospheric constituents, notably atomic oxygen and molecular nitrogen. Such emissions can be detected on the ground and are studied to get information on a variety of phenomena related to plasma energization by HF pumping. Following the ﬁrst unambiguous observations of HF pump-enhanced optical emissions at high latitudes (Brändström et al., 1999; Kosch et al., 2002b), a number of interesting results have been obtained. Here we limit ourselves to HF pumping with Powerful high frequency (HF) electromagnetic waves transmitted into the ionosphere from the ground may enhance optical 10 emissions from atmospheric constituents, notably atomic oxygen and molecular nitrogen. Such emissions can be detected on the ground and are studied to get information on a variety of phenomena related to plasma energization by HF pumping. Following the ﬁrst unambiguous observations of HF pump-enhanced optical emissions at high latitudes (Brändström et al., 1999; Kosch et al., 2002b), a number of interesting results have been obtained. Here we limit ourselves to HF pumping with Powerful high frequency (HF) electromagnetic waves transmitted into the ionosphere from the ground may enhance optical 10 emissions from atmospheric constituents, notably atomic oxygen and molecular nitrogen. Such emissions can be detected on the ground and are studied to get information on a variety of phenomena related to plasma energization by HF pumping. Following the ﬁrst unambiguous observations of HF pump-enhanced optical emissions at high latitudes (Brändström et al., 1999; Kosch et al., 2002b), a number of interesting results have been obtained. Here we limit ourselves to HF pumping with the frequency f0 below or near the ionospheric critical frequency and with left-handed circular polarization (often referred to 15 as ordinary mode) which gives the strongest effects. The spatial distribution of optical emissions enhanced above background levels has shown that the strongest coupling be- tween an HF beam and the ionospheric F-region plasma at high latitudes and long time scales occurs for a beam in the direction of geomagnetic zenith. Even with a vertical beam, the most intense optical emissions occur towards magnetic zenith within the beam. 1 Introduction By comparing the obtained altitude and temporal variations of the optical emissions with modelled emissions of excitation of O(1D) by a maxwellian electron velocity distribution, the authors predicted interactions, as found in experiments both at EISCAT and HAARP (Kosch et al., 2004, 2007). 25 Imaging of enhanced optical emissions from several stations with the Auroral Large Imaging System (ALIS) in northern Sweden and HF transmissions with EISCAT Heating provided for the ﬁrst tomography-like reconstruction of the volume emission at 630.0 nm (Gustavsson et al., 2001). By comparing the obtained altitude and temporal variations of the optical emissions with modelled emissions of excitation of O(1D) by a maxwellian electron velocity distribution, the authors predicted the source distribution for the emissions to be maxwellian at low energies but with a depletion above 1.96 eV (which is the 30 threshold for the excited state). Observation of optical emissions simultaneously at several wavelengths, each requiring different minimum electron energy to be enhanced, provides the possibility to study electron energization in the excited plasma turbulence. Gustavsson et al. (2002) obtained nearly simultaneous images of enhancements at 557.7 and 630.0 nm in experiments with EISCAT Heating. the source distribution for the emissions to be maxwellian at low energies but with a depletion above 1.96 eV (which is the 30 threshold for the excited state). Observation of optical emissions simultaneously at several wavelengths, each requiring different minimum electron energy to be enhanced, provides the possibility to study electron energization in the excited plasma turbulence. Gustavsson et al. (2002) obtained nearly simultaneous images of enhancements at 557.7 and 630.0 nm in experiments with EISCAT Heating. The 557.7 to 630.0 nm intensity ratio of 0.3–0.4 was relatively high which implies that the optical enhancements were caused 35 by a nonthermal tail in the electron velocity distribution. A similarly high ratio has been observed in experiments with HAARP (Pedersen et al., 2003). Gustavsson and Eliasson (2008) observed emissions at 427.8, 557.7, 630.0 and 844.6 nm, together with incoherent scatter radar measurements of the ion temperature, electron temperature and concentration at EISCAT. By using a two-stream electron The 557.7 to 630.0 nm intensity ratio of 0.3–0.4 was relatively high which implies that the optical enhancements were caused 35 by a nonthermal tail in the electron velocity distribution. A similarly high ratio has been observed in experiments with HAARP (Pedersen et al., 2003). 1 Introduction The pump- small-scale geomagnetic ﬁeld-aligned density striations with the CUTLASS coherent scatter radar. They found a signiﬁcant 45 reduction in the optical enhancement and the radar backscatter when f0 ≈3fe., indicating that the 630.0 nm emissions are linked to upper hybrid turbulence and ﬁlamentary striations. Further, Gustavsson et al. (2006) performed EISCAT experiments with f0 stepping near sfe (s = 3, 4) and optical imaging at 630.0, 557.7 and 427.8 nm together with incoherent scatter radar measurements of the electron temperature. The pump- small-scale geomagnetic ﬁeld-aligned density striations with the CUTLASS coherent scatter radar. They found a signiﬁcant 45 reduction in the optical enhancement and the radar backscatter when f0 ≈3fe., indicating that the 630.0 nm emissions are linked to upper hybrid turbulence and ﬁlamentary striations. Further, Gustavsson et al. (2006) performed EISCAT experiments with f0 stepping near sfe (s = 3, 4) and optical imaging at 630 0 557 7 and 427 8 nm together with incoherent scatter radar measurements of the electron temperature The pump- at 630.0, 557.7 and 427.8 nm together with incoherent scatter radar measurements of the electron temperature. The pump enhanced emissions as well as electron temperature were all minimum for f0 ≈4fe. Whereas the enhancement of the emission 50 intensity at 630.0 nm and electron temperature were roughly symmetric for f0 around 4fe, the emissions at 427.8 nm were markedly stronger for f0 of few tens of kilohertz above sfe. This suggests that there are different electron energization processes underlying the emissions at 630.0 nm and 427.8 nm. The observations at 427.8 nm were the ﬁrst direct evidence of pump- induced ionization of thermospheric N2. The experimental results are consistent with theory that predict electron acceleration enhanced emissions as well as electron temperature were all minimum for f0 ≈4fe. Whereas the enhancement of the emission 50 intensity at 630.0 nm and electron temperature were roughly symmetric for f0 around 4fe, the emissions at 427.8 nm were markedly stronger for f0 of few tens of kilohertz above sfe. This suggests that there are different electron energization processes underlying the emissions at 630.0 nm and 427.8 nm. The observations at 427.8 nm were the ﬁrst direct evidence of pump- induced ionization of thermospheric N2. The experimental results are consistent with theory that predict electron acceleration enhanced emissions as well as electron temperature were all minimum for f0 ≈4fe. 1 Introduction Gustavsson and Eliasson (2008) observed emissions at 427.8, 557.7, 630.0 and 844.6 nm, together with incoherent scatter radar measurements of the ion temperature, electron temperature and concentration at EISCAT. By using a two-stream electron transport code they could estimate the electron ﬂux between 1.9 and 100 eV that is needed to account for the observations. 40 The electron energy distribution was found to be depleted approximately in the range 2–4 eV, probably due to excitation of vibrational states in N2, and have a nonthermal tail at higher energies. Experimental results on pump-enhanced optical emissions have been presented for varying f0 near an harmonic s (s = 3, 4) of the ionospheric electron gyro frequency fe. Kosch et al. (2002a) observed simultaneously emissions at 630.0 nm and transport code they could estimate the electron ﬂux between 1.9 and 100 eV that is needed to account for the observations. 40 The electron energy distribution was found to be depleted approximately in the range 2–4 eV, probably due to excitation of vibrational states in N2, and have a nonthermal tail at higher energies. Experimental results on pump-enhanced optical emissions have been presented for varying f0 near an harmonic s (s = 3, 4) of the ionospheric electron gyro frequency fe. Kosch et al. (2002a) observed simultaneously emissions at 630.0 nm and transport code they could estimate the electron ﬂux between 1.9 and 100 eV that is needed to account for the observations. 40 The electron energy distribution was found to be depleted approximately in the range 2–4 eV, probably due to excitation of vibrational states in N2, and have a nonthermal tail at higher energies. Experimental results on pump-enhanced optical emissions have been presented for varying f0 near an harmonic s (s = 3, 4) of the ionospheric electron gyro frequency fe. Kosch et al. (2002a) observed simultaneously emissions at 630.0 nm and small-scale geomagnetic ﬁeld-aligned density striations with the CUTLASS coherent scatter radar. They found a signiﬁcant 45 reduction in the optical enhancement and the radar backscatter when f0 ≈3fe., indicating that the 630.0 nm emissions are linked to upper hybrid turbulence and ﬁlamentary striations. Further, Gustavsson et al. (2006) performed EISCAT experiments with f0 stepping near sfe (s = 3, 4) and optical imaging at 630.0, 557.7 and 427.8 nm together with incoherent scatter radar measurements of the electron temperature. 1 Introduction This was observed in emissions at 630.0 nm from the O(1D) excited state as obtained with the EISCAT (European Inco- 20 herent Scatter association) Heating facility in Norway (Kosch et al., 2000), with the Sura facility in Russia (Grach et al., 2007; Shindin et al., 2015), as well as with the HAARP (High Frequency Active Auroral Research Program) facility in Alaska, USA beam. This was observed in emissions at 630.0 nm from the O(1D) excited state as obtained with the EISCAT (European Inco- 20 herent Scatter association) Heating facility in Norway (Kosch et al., 2000), with the Sura facility in Russia (Grach et al., 2007; Shindin et al., 2015), as well as with the HAARP (High Frequency Active Auroral Research Program) facility in Alaska, USA 1 1 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. (Pedersen and Carlson, 2001), and also at 557.7 nm from O(1S) (Pedersen et al., 2003, 2008). In addition, optical emissions show evidence of complex HF beam re-organization during pumping and associated nonlinear effects in the pump–plasma interactions, as found in experiments both at EISCAT and HAARP (Kosch et al., 2004, 2007). 25 Imaging of enhanced optical emissions from several stations with the Auroral Large Imaging System (ALIS) in northern Sweden and HF transmissions with EISCAT Heating provided for the ﬁrst tomography-like reconstruction of the volume emission at 630 0 nm (Gustavsson et al 2001) By comparing the obtained altitude and temporal variations of the optical (Pedersen and Carlson, 2001), and also at 557.7 nm from O(1S) (Pedersen et al., 2003, 2008). In addition, optical emissions show evidence of complex HF beam re-organization during pumping and associated nonlinear effects in the pump–plasma interactions, as found in experiments both at EISCAT and HAARP (Kosch et al., 2004, 2007). 25 (Pedersen and Carlson, 2001), and also at 557.7 nm from O(1S) (Pedersen et al., 2003, 2008). In addition, optical emissions show evidence of complex HF beam re-organization during pumping and associated nonlinear effects in the pump–plasma interactions, as found in experiments both at EISCAT and HAARP (Kosch et al., 2004, 2007). 25 Imaging of enhanced optical emissions from several stations with the Auroral Large Imaging System (ALIS) in northern Sweden and HF transmissions with EISCAT Heating provided for the ﬁrst tomography-like reconstruction of the volume emission at 630.0 nm (Gustavsson et al., 2001). 1 Introduction 70 In addition to these results for f0 ≳3fe, a number of interesting experiments and theories have been presented for f0 near 2fe, but those are outside the scope of the present paper, as the relevant dispersion properties of electron Bernstein and upper hybrid modes are different for s = 2 and s ≥3. Here we are concerned with f0 near 4fe and higher. 1 Introduction (2015) observed emissions at 557.7 and 630.0 nm at the Sura facility for f0 near 4fe, an ERP of about 100 MW and the HF beam directed either vertically or tilted 12◦south from the vertical in the magnetic meridional plane towards magnetic zenith (19◦south). Simultaneous observations of the frequency spectrum of stimulated electromagnetic emissions were used to determine where f0 was relative to 4fe. The pump-enhanced 630.0 nm emissions did not exhibit a dependence on 65 f0 near 4fe. And no minimum in the optical emission intensities was observed when f0 ≈4fe, which is contrary to previous measurements by others as mentioned above. The authors attribute this to natural variations in the ionospheric interaction altitudes during the experiments. Further, the authors found that for a vertical HF beam, 557.7 nm emissions occurred for f0 To further analyze these experimental results, Sergienko et al. (2012) performed Monte Carlo simulations of the transport of energized electrons into the ambient thermosphere. The observed 630.0 nm emissions could be accounted for by predomi- nantly thermal electrons (> 70 % of the emission intensity), with accelerated electrons playing a minor role for the emissions. 60 However, to explain the emission intensity at 427.8 nm, electrons must be accelerated to 60 eV or more. Shindin et al. (2015) observed emissions at 557.7 and 630.0 nm at the Sura facility for f0 near 4fe, an ERP of about 100 MW and the HF beam directed either vertically or tilted 12◦south from the vertical in the magnetic meridional plane towards magnetic zenith (19◦south). Simultaneous observations of the frequency spectrum of stimulated electromagnetic emissions were used to determine where f0 was relative to 4fe. The pump-enhanced 630.0 nm emissions did not exhibit a dependence on 65 f0 near 4fe. And no minimum in the optical emission intensities was observed when f0 ≈4fe, which is contrary to previous measurements by others as mentioned above. The authors attribute this to natural variations in the ionospheric interaction altitudes during the experiments. Further, the authors found that for a vertical HF beam, 557.7 nm emissions occurred for f0 about 5–15 kHz below 4fe and also at about 220–280 kHz above 4fe. With the HF beam directed towards magnetic zenith, pump-enhanced emissions were observed for f0 about 15–20 kHz above 4fe. 2 Experiment setup The EISCAT Heating facility (Rietveld et al., 2016) was used to transmit HF waves in the cycle of 150 s on/85 s off with the 75 beam pointing in geomagnetic zenith (∼78◦elevation south) and with left-handed circularly polarization (ordinary mode), on 16 February 2015. From 16:00:00 to 16:49:30 UT, f0 = 6.200 MHz and the effective radiative power (ERP) was approximately 138 MW. To keep f0 below the decreasing ionospheric ordinary-mode critical frequency after sunset, f0 = 5.423 MHz from 16:50:55 to 17:09:05 UT and the ERP was 116 MW. Local time was UT+1 hour. The EISCAT Heating facility (Rietveld et al., 2016) was used to transmit HF waves in the cycle of 150 s on/85 s off with the 75 beam pointing in geomagnetic zenith (∼78◦elevation south) and with left-handed circularly polarization (ordinary mode), on 16 February 2015. From 16:00:00 to 16:49:30 UT, f0 = 6.200 MHz and the effective radiative power (ERP) was approximately 138 MW. To keep f0 below the decreasing ionospheric ordinary-mode critical frequency after sunset, f0 = 5.423 MHz from 16:50:55 to 17:09:05 UT and the ERP was 116 MW. Local time was UT+1 hour. The EISCAT Heating facility (Rietveld et al., 2016) was used to transmit HF waves in the cycle of 150 s on/85 s off with the 75 beam pointing in geomagnetic zenith (∼78◦elevation south) and with left-handed circularly polarization (ordinary mode), on 16 February 2015. From 16:00:00 to 16:49:30 UT, f0 = 6.200 MHz and the effective radiative power (ERP) was approximately 138 MW. To keep f0 below the decreasing ionospheric ordinary-mode critical frequency after sunset, f0 = 5.423 MHz from 16:50:55 to 17:09:05 UT and the ERP was 116 MW. Local time was UT+1 hour. Optical emissions were observed with the ALIS stations in Abisko, Kiruna and Tjautjas. The cameras had a ﬁeld of view 80 of 60◦and gave an image size of 512 × 512 pixels with a temporal resolution of 7 s. Emissions were imaged at 427.8 nm from N+ 2 (1NG) with a threshold of 18.6 eV (Holma et al., 2006), 557.7 nm from O(1S) with the threshold 4.17 eV (Haslett and Megill, 1974), 630.0 nm from O(1D) with the threshold 1.96 eV (Haslett and Megill, 1974), and 844.6 nm from O(3p3P) with the threshold 10.99 eV (Gustavsson et al., 2005). Kvammen et al. 1 Introduction Whereas the enhancement of the emission 50 intensity at 630.0 nm and electron temperature were roughly symmetric for f0 around 4fe, the emissions at 427.8 nm were markedly stronger for f0 of few tens of kilohertz above sfe. This suggests that there are different electron energization processes underlying the emissions at 630.0 nm and 427.8 nm. The observations at 427.8 nm were the ﬁrst direct evidence of pump- induced ionization of thermospheric N2. The experimental results are consistent with theory that predict electron acceleration by upper hybrid oscillations localized in cylindrical density depletions and formation of a suprathermal tail in the electron 55 velocity distribution (Istomin and Leyser, 2003; Najmi et al., 2017). The acceleration efﬁciency is the largest for f0 slightly above sfe for s ≥3. by upper hybrid oscillations localized in cylindrical density depletions and formation of a suprathermal tail in the electron 55 velocity distribution (Istomin and Leyser, 2003; Najmi et al., 2017). The acceleration efﬁciency is the largest for f0 slightly above sfe for s ≥3. by upper hybrid oscillations localized in cylindrical density depletions and formation of a suprathermal tail in the electron 55 velocity distribution (Istomin and Leyser, 2003; Najmi et al., 2017). The acceleration efﬁciency is the largest for f0 slightly above sfe for s ≥3. 2 2 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. To further analyze these experimental results, Sergienko et al. (2012) performed Monte Carlo simulations of the transport of energized electrons into the ambient thermosphere. The observed 630.0 nm emissions could be accounted for by predomi- nantly thermal electrons (> 70 % of the emission intensity), with accelerated electrons playing a minor role for the emissions. 60 However, to explain the emission intensity at 427.8 nm, electrons must be accelerated to 60 eV or more. To further analyze these experimental results, Sergienko et al. (2012) performed Monte Carlo simulations of the transport of energized electrons into the ambient thermosphere. The observed 630.0 nm emissions could be accounted for by predomi- nantly thermal electrons (> 70 % of the emission intensity), with accelerated electrons playing a minor role for the emissions. 60 However, to explain the emission intensity at 427.8 nm, electrons must be accelerated to 60 eV or more. Shindin et al. 2 Experiment setup Figure 1 shows the geometry of the experiment with the relative positions of the EISCAT facilities at the Ramfjordmoen site outside Tromsø, Norway, and the ALIS stations in the Kiruna region, Sweden. Stimulated electromagnetic emission were received on the ground in Kroken (Tromsø), but were weak and only rarely exhibited spectral structure. Ionograms from the EISCAT Dynasonde show elevated D-region absorption. altitude resolution of approximately 3 km. Figure 1 shows the geometry of the experiment with the relative positions of the EISCAT facilities at the Ramfjordmoen site outside Tromsø, Norway, and the ALIS stations in the Kiruna region, Sweden. Stimulated electromagnetic emission were received on the ground in Kroken (Tromsø), but were weak and only rarely altitude resolution of approximately 3 km. Figure 1 shows the geometry of the experiment with the relative positions of the EISCAT facilities at the Ramfjordmoen site outside Tromsø, Norway, and the ALIS stations in the Kiruna region, Sweden. Stimulated electromagnetic emission were received on the ground in Kroken (Tromsø), but were weak and only rarely exhibited spectral structure. Ionograms from the EISCAT Dynasonde show elevated D-region absorption. 2 Experiment setup (2019) analyzed all four emission lines for this same experiment and presented tomography-like reconstructions of the three-dimensional distribution of the optical volume emission 85 rates. However, in the present treatment we focus on the 557.7 and 630.0 nm lines which are the strongest and for which we have the most data. As the emissions were imaged from three sites, we too employed tomography-like inversion to get the volume emission rates for the two spectral lines, along the lines described by Gustavsson et al. (2001). experiment and presented tomography-like reconstructions of the three-dimensional distribution of the optical volume emission 85 rates. However, in the present treatment we focus on the 557.7 and 630.0 nm lines which are the strongest and for which we have the most data. As the emissions were imaged from three sites, we too employed tomography-like inversion to get the volume emission rates for the two spectral lines, along the lines described by Gustavsson et al. (2001). The EISCAT UHF incoherent scatter radar was operated with a meridional scan pattern to measure background plasma parameter values at different angles to the geomagnetic ﬁeld, using the beata program with a temporal resolution of 5 s and 90 The EISCAT UHF incoherent scatter radar was operated with a meridional scan pattern to measure background plasma parameter values at different angles to the geomagnetic ﬁeld, using the beata program with a temporal resolution of 5 s and 90 3 3 Altitude (km) Tromsø North (km) Tromsø East (km) Figure 1. Illustration of the relative positions of the EISCAT facilities at the Ramfjordmoen site outside Tromsø, Norway, and the ALIS stations in the Kiruna region, Sweden. The black line shows the direction of magnetic zenith. The ALIS station at Silkkimuotka was not used in the experiments. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 1. Illustration of the relative positions of the EISCAT facilities at the Ramfjordmoen site outside Tromsø, Norway, and the ALIS stations in the Kiruna region, Sweden. The black line shows the direction of magnetic zenith. The ALIS station at Silkkimuotka was not used in the experiments. altitude resolution of approximately 3 km. 3 Experimental results 95 Figure 2 shows an overview of the time development of the altitude proﬁle of the pump-enhanced optical emissions at 630.0 nm above background as observed from the three ALIS sites. The column emission rate weakened as f0 was changed from 6.200 MHz to 5.423 MHz at about 16:50 UT. For the second HF pulse at f0 = 5.423 MHz observed at the Abisko station, the relatively short duration shown in the enhancement is due to lacking synchronization between the pump wave and optical ﬁlter wheel sequence. The slow growth of the emission intensity and extension towards higher altitudes during the pump pulses 100 can be seen. Also, the emissions occurred at lower altitudes for the lower f0. Figure 3 displays similarly an overview of the emissions at 557.7 nm for the same time period. The emission intensity did not weaken as f0 was decreased at about 16:50 UT. The images from the Kiruna site show even an increase in the emissions as f0 was decreased, despite a lower ERP. But again the altitude region of the emissions decreased as f0 was lowered. It is interesting that Kvammen et al. (2019) observed a 4 4 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 2. Overview of the column emissions at 630.0 nm from 16:00:00 to 17:09:05 UT on 16 February 2015. From 16:00:00 to 16:49:30 UT, f0 = 6.200 MHz, and from 16:50:55 to 17:09:05 UT, f0 = 5.423 MHz. Figure 2. Overview of the column emissions at 630.0 nm from 16:00:00 to 17:09:05 UT on 16 February 2015. From 16:00:00 to 16:49:30 UT, f0 = 6.200 MHz, and from 16:50:55 to 17:09:05 UT, f0 = 5.423 MHz. clear intensity increase in the 844.6 nm line which has a threshold of 10.99 eV, which similarly to the threshold of 4.17 eV of 105 the 557.7 nm line is several times above the threshold of 1.96 eV for 630.0 nm. Figure 4 shows the altitude proﬁles of the electron density, electron temperature and ion temperature obtained with the EISCAT UHF radar in geomagnetic zenith. 3 Experimental results 95 Similar to Fig. 2, but for the emissions at 557.7 nm. of the volume emission rate for the third pump pulse at f0 = 5.423 MHz. In this case, the maximum of the emissions at 557.7 nm (right) occurred about 7–10 km below that at 630.0 nm (left). Also, by comparing Figs. 5 and 6, we see that the emission https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 3. Similar to Fig. 2, but for the emissions at 557.7 nm. Figure 3. Similar to Fig. 2, but for the emissions at 557.7 nm. of the volume emission rate for the third pump pulse at f0 = 5.423 MHz. In this case, the maximum of the emissions at 557.7 nm (right) occurred about 7–10 km below that at 630.0 nm (left). Also, by comparing Figs. 5 and 6, we see that the emission rates for both lines grow slower with f0 = 5.423 MHz. 20 of the volume emission rate for the third pump pulse at f0 = 5.423 MHz. In this case, the maximum of the emissions at 557.7 nm (right) occurred about 7–10 km below that at 630.0 nm (left). Also, by comparing Figs. 5 and 6, we see that the emission t f b th li l ith f 5 423 MH 20 rates for both lines grow slower with f0 = 5.423 MHz. 120 Figure 7 summarizes the temporal evolution of the column emission rates at 630.0 nm (left) and 557.7 nm (right) during the growth phase following pump-on for f0 = 6.200 MHz (red) and f0 = 5.423 MHz (blue), for the same data as in Figs. 5 and 6. The 630.0 nm emissions are seen to be weaker for f0 = 5.423 MHz than for f0 = 6.200 MHz. However, the emission intensity at 557.7 nm is approximately the same for the two f0 towards the end of the pump pulses. The intensity ratio towards the end of the plotted time interval is I5577/I6300 ≈0.2 for f0 = 6.200 MHz and we observe I5577/I6300 ≈0.5 for f0 = 5.423 125 MHz, which agrees with Kvammen et al. 3 Experimental results 95 Clear pump-induced enhancements in the electron temperature can be seen which are slowly conducted upward in the ionosphere with time, along the geomagnetic ﬁeld where the thermal conductivity is the clear intensity increase in the 844.6 nm line which has a threshold of 10.99 eV, which similarly to the threshold of 4.17 eV of 105 the 557.7 nm line is several times above the threshold of 1.96 eV for 630.0 nm. Figure 4 shows the altitude proﬁles of the electron density, electron temperature and ion temperature obtained with the EISCAT UHF radar in geomagnetic zenith. Clear pump-induced enhancements in the electron temperature can be seen which are slowly conducted upward in the ionosphere with time, along the geomagnetic ﬁeld where the thermal conductivity is the clear intensity increase in the 844.6 nm line which has a threshold of 10.99 eV, which similarly to the threshold of 4.17 eV of 05 the 557.7 nm line is several times above the threshold of 1.96 eV for 630.0 nm. highest. The electron density and ion temperature did not exhibit notable pump-induced modulations. However, the electron 110 density slowly decreased with time as the experiments occurred shortly after sunset. Figure 5 shows the altitude proﬁles of the volume emission rates at 630.0 nm (left) and 557.7 nm (right) for the last pump cycle at f0 = 6.200 MHz, as obtained by tomography-like reconstruction of the emission regions. The maximum volume emission rates occurred at approximately the same height for the two spectral lines, near hmax ≈239 km. The altitude proﬁle for the 557.7 nm emission is wider than that for 630.0 nm, which. is consistent with that the excitation threshold for the former 115 (4.17 eV) is higher, and the electrons having a longer mean free path, compared to the latter (1.96 eV). The times for the plotted proﬁles are determined by the sequence in the ﬁlter wheels for the ALIS cameras. Figure 6 shows similarly the height proﬁles for the 557.7 nm emission is wider than that for 630.0 nm, which. is consistent with that the excitation threshold for the former 115 (4.17 eV) is higher, and the electrons having a longer mean free path, compared to the latter (1.96 eV). The times for the plotted proﬁles are determined by the sequence in the ﬁlter wheels for the ALIS cameras. Figure 6 shows similarly the height proﬁles 5 ebruary 2015 Figure 3. 3 Experimental results 95 (2019) for the same experiment. We do not have data for the remaining part of the pump pulse of 150 s because of the used sequence for the ﬁlter wheels. 6 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 4. Overview of the electron density (top), electron temperature (center) and ion temperature (bottom) as obtained with the EISCAT UHF radar directed in magnetic zenith, from 16:00:00 to 17:09:05 UT on 16 February 2015. From 16:00:00 to 16:49:30 UT, f0 = 6.200 MHz and from 16:50:55 to 17:09:05 UT, f0 = 5.423 MHz. Figure 4. Overview of the electron density (top), electron temperature (center) and ion temperature (bottom) as obtained with the EISCAT UHF radar directed in magnetic zenith, from 16:00:00 to 17:09:05 UT on 16 February 2015. From 16:00:00 to 16:49:30 UT, f0 = 6.200 MHz and from 16:50:55 to 17:09:05 UT, f0 = 5.423 MHz. 4 Emission growth times To obtain a measure of the growth time of the optical emissions after pump-on, we ﬁt a theoretical growth time to the ex- perimental data for the emission from a given excited state. The temporal evolution of an emission can be described by the 130 continuity equation for the number density nα of atoms in the excited state α (O(1D), O(1S)). nα is related to the observed column emission rate Iλ through nα(t) = Iλ(t)/Aα/Dλ(t), where Aα is the Einstein coefﬁcient(s) for the emission and Dλ is the observed spatial scale of the imaged optical region at the wavelength λ. The number density nα is described by the equation 130 dnα dt + ∇· (nαv) = Qα(r,t) −Lα(t)nα 135 (1) 7 Volume emission rate (cm s ) −3 −1 Altitude (km) 630.0 nm 557.7 nm Figure 5. Temporal evolution of the height proﬁle of the volume emission rates in magnetic zenith at 630.0 nm (left) and 557.7 nm (right) for f0 = 6.200 MHz. The accuracy of the height determination is approximately ±1 km. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. Volume emission rate (cm s ) −3 −1 Altitude (km) 630.0 nm 557.7 nm Figure 5. Temporal evolution of the height proﬁle of the volume emission rates in magnetic zenith at 630.0 nm (left) and 557.7 nm (right) for f0 = 6.200 MHz. The accuracy of the height determination is approximately ±1 km. Volume emission rate (cm s ) −3 −1 Altitude (km) 630.0 nm 557.7 nm Figure 5. Temporal evolution of the height proﬁle of the volume emission rates in magnetic zenith at 630.0 nm (left) and 557.7 nm (right) for f0 = 6.200 MHz. The accuracy of the height determination is approximately ±1 km. on rate (cm s ) −3 −1 557.7 nm Volume emission rate (cm s ) −3 −1 Altitude (km) 630.0 nm 557.7 nm Figure 6. The same as Fig. 5, but for f0 = 5.423 MHz. 557.7 nm Volume emission rate (cm s ) −3 −1 Figure 6. The same as Fig. 5, but for f0 = 5.423 MHz. 4 Emission growth times Temporal evolution of pump-enhanced column emission rates at 630.0 nm (left) and 557.7 nm (right) after pump-on at time t = 0 s, for the same pump pulses as in Figs. 5 and 6. The solid lines connect the data points shown by markers (red ∗for f0 = 6.200 MHz and blue + for f0 = 5.423 MHz). The dashed lines are the ﬁts with Eq. (3) to obtain τobs. Time afte Intensity (R) 630.0 nm er pump-on (s) Intensity (R) 557.7 nm Figure 7. Temporal evolution of pump-enhanced column emission rates at 630.0 nm (left) and 557.7 nm (right) after pump-on at time t = 0 s, for the same pump pulses as in Figs. 5 and 6. The solid lines connect the data points shown by markers (red ∗for f0 = 6.200 MHz and blue + for f0 = 5.423 MHz). The dashed lines are the ﬁts with Eq. (3) to obtain τobs. Table 1. Emission growth times τobs after pump-on obtained by ﬁtting Eq. (3) with the observed column emission rates at 630.0 nm (I6300) and 557.7 nm (I5577). Table 1. Emission growth times τobs after pump-on obtained by ﬁtting Eq. (3) with the observed column emission rates at 630.0 nm (I6300) and 557.7 nm (I5577). Iλ f0 (MHz) τα (s) τobs (s) I6300 6.200 40 39 I5577 6.200 1.3 5.4 I6300 5.423 25 74 I5577 5.423 1.3 11 altitudes of the experiments. Further, for the experiment conditions, the term ∇· (vN) in Eq. (1) can be neglected because of the low velocity v of the neutrals. For the purpose of the present analysis, the spatial dependencies of Qα and Lα are neglected 145 as we are interested only in a relatively small spatial region around the emission maximum, not in the entire emitting volume. For our case of HF pump-enhanced emissions, the loss is constant with Lα = 1/τα, where τα is the effective lifetime of the excited state α. For the O(1S) state, τO(1S) equals the radiative lifetime so that τO(1S) = 1.3 s, because this state is not quenched by collisions. For the O(1D) state, τO(1D) is affected by quenching and we take τO(1D) for the relevant altitudes from Gustavsson et al. (2001). 150 For the purpose of our analysis, we take the source term Qα to be constant. The solution to Eq. 4 Emission growth times where Qα(t) is the excitation rate which includes the electron impact excitation (thermal and accelerated) and chemical reac- tions. The loss rate Lα(t) is given by Lα(t) = X i (qini) − X j Aj (2) Lα(t) = X i (qini) − X j Aj (2) where qi is the rate coefﬁcient for collisional de-excitation (quenching) by collisions with other species with the number density 140 ni. The last term is the sum of the Einstein coefﬁcients for all emissions originating from the excited state in question. For 140 the 630.0 nm emission, LO(1D) includes the sum of three Einstein coefﬁcients and qi is for reactions with the main neutrals in the thermosphere at the heights of the F-region ionosphere (N2, O2 and O) as well as with thermal electrons. For the 557.7 nm line, LO(1S) is the sum of the Einstein coefﬁcients only since the O(1S) state is not quenched by collisions at the F-region ni. The last term is the sum of the Einstein coefﬁcients for all emissions originating from the excited state in question. For 140 the 630.0 nm emission, LO(1D) includes the sum of three Einstein coefﬁcients and qi is for reactions with the main neutrals in the thermosphere at the heights of the F-region ionosphere (N2, O2 and O) as well as with thermal electrons. For the 557.7 nm line, LO(1S) is the sum of the Einstein coefﬁcients only since the O(1S) state is not quenched by collisions at the F-region 8 8 Time after pump-on (s) Intensity (R) Intensity (R) 630.0 nm 557.7 nm Figure 7. Temporal evolution of pump-enhanced column emission rates at 630.0 nm (left) and 557.7 nm (right) after pump-on at time t = 0 s, for the same pump pulses as in Figs. 5 and 6. The solid lines connect the data points shown by markers (red ∗for f0 = 6.200 MHz and blue + for f0 = 5.423 MHz). The dashed lines are the ﬁts with Eq. (3) to obtain τobs. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. Time after pump-on (s) Intensity (R) Intensity (R) 630.0 nm 557.7 nm Figure 7. 4 Emission growth times The theoretical lifetime τO(1D) ≈40 s in the 160 third column from the left, is for the altitude hmax ≈239 km (Gustavsson et al., 2001) where the 630.0 nm volume emission rate is maximum for f0 = 6.200 MHz (Fig. 5). Similarly, τO(1D) ≈25 s is for hmax ≈225 km (Gustavsson et al., 2001) with f0 = 5.423 MHz (Fig. 6). 160 f0 ( g ) With f0 = 6.200 MHz, τobs for the 630.0 nm line is almost equal to τO(1D). For the 557.7 nm line, τobs is approximately four With f0 = 6.200 MHz, τobs for the 630.0 nm line is almost equal to τO(1D). For the 557.7 nm lin times larger than τO(1S). These results suggest that for f0 = 6.200 MHz, the excitation sources Qα for both emissions reach 165 their maximum faster than τO(1D) ≈40 s but slower than τO(1S) ≈1.3 s, and probably the source growth rates are different for the two emissions. On the other hand, with f0 = 5.423 MHz, τobs for b oth emissions is several times larger than τα. In addition, τobs for both emissions is larger than with f0 = 6.200 MHz. This suggests that for f0 = 5.423 MHz, the source Qα increases during both emissions is larger than with f0 = 6.200 MHz. This suggests that for f0 = 5.423 MHz, the source Qα increases during pumping, and slower than for f0 = 6.200 MHz. A relatively slow growth of emissions at 557.7 nm has also been observed in 170 experiments at the HAARP facility with f0 = 7.8 MHz and an ERP of approximately 160 MW (Pedersen et al., 2003). The 557.7 to 630.0 nm intensity ratio in that case was approximately 1:3. pumping, and slower than for f0 = 6.200 MHz. A relatively slow growth of emissions at 557.7 nm has also been observed in 170 experiments at the HAARP facility with f0 = 7.8 MHz and an ERP of approximately 160 MW (Pedersen et al., 2003). The 557.7 to 630.0 nm intensity ratio in that case was approximately 1:3. 5 Discussion Excitation of the O(1D) state, the source of the 630.0 nm line, has been attributed to mainly electron heating from a maxwellian electron distribution (Mantas, 1994; Mantas and Carlson, 1996), however, taking into account collisional de-excitation by 175 collisions with molecular oxygen and nitrogen in the atmosphere (Gustavsson et al., 2001). The association of the 630.0 nm emissions with electron heating is consistent with our results for the optical (Fig. 2) and electron temperature enhancements (Fig. 4). Both decreased when f0 was changed from 6.200 to 5.423 MHz at about 16:51 UT. HF pump-enhanced optical emissions have been connected to upper hybrid waves because the emission intensities are HF pump-enhanced optical emissions have been connected to upper hybrid waves because the emission intensities are sensitive to f0 near sfe (Kosch et al., 2002a; Gustavsson et al., 2006). The response of ionospheric F-region plasma to HF 180 pumping is asymmetric around sfe (Leyser et al., 1989; Stubbe et al., 1994; Honary et al., 1995; Gustavsson et al., 2006). This is related to asymmetries in the dispersion characteristics of upper hybrid and electron Bernstein modes for frequencies near sfe and s ≥3. Theories of the localization of upper hybrid oscillations in density depletions of ﬁlamentary density striations along the geomagnetic ﬁeld predict an asymmetry in the trapping mechanism with deeper depletions for f0 ≳sfe than for sensitive to f0 near sfe (Kosch et al., 2002a; Gustavsson et al., 2006). The response of ionospheric F-region plasma to HF 180 pumping is asymmetric around sfe (Leyser et al., 1989; Stubbe et al., 1994; Honary et al., 1995; Gustavsson et al., 2006). This is related to asymmetries in the dispersion characteristics of upper hybrid and electron Bernstein modes for frequencies near sfe and s ≥3. Theories of the localization of upper hybrid oscillations in density depletions of ﬁlamentary density striations along the geomagnetic ﬁeld predict an asymmetry in the trapping mechanism with deeper depletions for f0 ≳sfe than for f0 ≲sfe (Mjølhus, 1993). Therefore stronger pump-induced effects are expected for f0 ≳sfe than for f0 ≲sfe which gives a 185 corresponding asymmetry also in the anomalous absorption of HF waves by scattering on the striations (Honary et al., 1995; Gurevich et al., 1996) and electron acceleration by the localized upper hybrid oscillations (Istomin and Leyser, 2003). 4 Emission growth times (1) is nα(t) = τQα 1 −exp −t τ nα(t) = τQα 1 −exp −t τ (3) nα(t) = τQα 1 −exp −t τ (3) (3) (3) With Qα constant, the growth time is τ = τα. However, for the general case when Qα is time dependent, τ is determined by both the lifetime τα and Qα(t). Therefore, if ﬁtting the experimental data by Eq. (3) for which Qα is constant, gives an observed growth time τobs very different from τα, this implies that the source Qα is not constant. 155 by both the lifetime τα and Qα(t). Therefore, if ﬁtting the experimental data by Eq. (3) for which Qα is constant, gives an observed growth time τobs very different from τα, this implies that the source Qα is not constant. 155 observed growth time τobs very different from τα, this implies that the source Qα is not constant. 155 9 9 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. To obtain τobs from the measurements, we recall that nα(t) = Iλ(t)/Aα/Dλ(t), so that the observed intensity Iλ(t) ∝nα(t) for a ﬁxed Dλ. Thus, Iλ(t) is ﬁtted with a quantity proportional to Eq. (3) for the images at the different t = ti of the data. Iλ(ti) is averaged over a small square (15 × 15 pixels) around the maximum intensity in the images. The ﬁtting is done by the least-squares Levenberg–Marquardt algorithm. Table 1 summarizes τobs for the column emission rates at 557.7 and 630.0 nm. The theoretical lifetime τO(1D) ≈40 s in the 160 third column from the left, is for the altitude hmax ≈239 km (Gustavsson et al., 2001) where the 630.0 nm volume emission rate is maximum for f0 = 6.200 MHz (Fig. 5). Similarly, τO(1D) ≈25 s is for hmax ≈225 km (Gustavsson et al., 2001) with f0 = 5.423 MHz (Fig. 6). With f0 = 6.200 MHz, τobs for the 630.0 nm line is almost equal to τO(1D). For the 557.7 nm line, τobs is approximately four Table 1 summarizes τobs for the column emission rates at 557.7 and 630.0 nm. 5 Discussion However, for the fourth HF pulse 200 at f0 = 5.423 MHz, a weak broad upshifted maximum (BUM) can be identiﬁed. In Fig. 4 it can be seen that the electron temperature is slightly more enhanced in the fourth (and last) HF pulse than in the preceding pulses at f0 = 5.423 MHz, which indicates a stronger plasma excitation in the last pulse. For s = 4, the BUM has been observed in the range 4fe −20 kHz ≲f0 ≲4fe + 120 kHz (Frolov et al., 1998), thus mainly at f0 ≳4fe. As fe decreases with increasing altitude, the weak BUM could have been excited at higher altitudes where f0 ≳4fe compared to where the optical emissions were enhanced. Also, we 205 note that the height proﬁles in Figs. 5 and 6 are for the third HF pulse for which the quality of the optical data is higher than for the fourth pulse. The pump–plasma interaction altitudes may have changed slightly between the two pulses. Gustavsson and Eliasson (2008) discussed pump-enhanced optical emissions at 427.8, 557.7, 630.0 and 844.6 nm for f0 = 5.423 MHz and cycling the pump wave 4 min on/2 min off. By ﬁtting observed optical emissions and modelled en- hanced electron ﬂuxes they obtained estimates of the volume emission rates for the different optical lines. They found that the 210 maximum volume emission rate for all spectral lines occurred at approximately the same altitude (their ﬁgure 7), which agrees with our observations for f0 = 6.200 MHz (Fig. 5). Also, the ratio of the column emission rates for the emissions at 557.7 and 630.0 nm varied in the range I5577/I6300 ≈0.1–0.4 during the experiment (from their ﬁgure 2), which agrees with our ratio for the same emission lines I5577/I6300 ≈0.2, but again at f0 = 6.200 MHz. In our case with f0 = 5.423 MHz, the maximum hanced electron ﬂuxes they obtained estimates of the volume emission rates for the different optical lines. They found that the 210 maximum volume emission rate for all spectral lines occurred at approximately the same altitude (their ﬁgure 7), which agrees with our observations for f0 = 6.200 MHz (Fig. 5). 5 Discussion These phenomena are expected to be correlated with electron temperature enhancements which are the source of optical emissions at f0 ≲sfe (Mjølhus, 1993). Therefore stronger pump-induced effects are expected for f0 ≳sfe than for f0 ≲sfe which gives a 185 corresponding asymmetry also in the anomalous absorption of HF waves by scattering on the striations (Honary et al., 1995; Gurevich et al., 1996) and electron acceleration by the localized upper hybrid oscillations (Istomin and Leyser, 2003). These phenomena are expected to be correlated with electron temperature enhancements which are the source of optical emissions at 10 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. 630.0 nm and which therefore too are expected to be stronger for f0 ≳sfe than for f0 ≲sfe. With f0 ≈sfe, upper hybrid and electron Bernstein phenomena are suppressed because of the linear dispersion characteristics. 0 630.0 nm and which therefore too are expected to be stronger for f0 ≳sfe than for f0 ≲sfe. With f0 ≈sfe, upper hybrid and electron Bernstein phenomena are suppressed because of the linear dispersion characteristics. 190 In order to ﬁnd out the relation between f0 = 5.423 MHz and 4fe in our experiment, we use the IGRF model (International Geomagnetic Reference Field, 13th generation) for the altitude variation of the geomagnetic ﬁeld in the pump–ionosphere interaction region. We ﬁnd that 4fe ≈f0 = 5.423 MHz occurred at the height of h ≈235 km for the date of our experiment. This altitude is above that for the maximum volume emission rates at both 630.0 and 557.7 nm. Figure 6 shows this height to be electron Bernstein phenomena are suppressed because of the linear dispersion characteristics. 190 In order to ﬁnd out the relation between f0 = 5.423 MHz and 4fe in our experiment, we use the IGRF model (International Geomagnetic Reference Field, 13th generation) for the altitude variation of the geomagnetic ﬁeld in the pump–ionosphere interaction region. We ﬁnd that 4fe ≈f0 = 5.423 MHz occurred at the height of h ≈235 km for the date of our experiment. This altitude is above that for the maximum volume emission rates at both 630.0 and 557.7 nm. Figure 6 shows this height to be electron Bernstein phenomena are suppressed because of the linear dispersion characteristics. 5 Discussion 190 In order to ﬁnd out the relation between f0 = 5.423 MHz and 4fe in our experiment, we use the IGRF model (International Geomagnetic Reference Field, 13th generation) for the altitude variation of the geomagnetic ﬁeld in the pump–ionosphere interaction region. We ﬁnd that 4fe ≈f0 = 5.423 MHz occurred at the height of h ≈235 km for the date of our experiment. This altitude is above that for the maximum volume emission rates at both 630.0 and 557.7 nm. Figure 6 shows this height to be hmax ≈227 km for 630.0 nm and hmax ≈219 km for 557.7 nm. Since fe increases with decreasing altitude, we conclude that 195 f0 < 4fe in the regions with maximum volume emission rates. The IGRF model gives for the 630.0 nm emissions, fe ≈1.360 MHz at hmax ≈227 km, so that ∆f ≡f0 −4fe ≈−17 kHz. For the emissions at 557.7 nm, fe ≈1.364 MHz at hmax ≈219 km, giving ∆f ≈−33 kHz. hmax ≈227 km for 630.0 nm and hmax ≈219 km for 557.7 nm. Since fe increases with decreasing altitude, we conclude that 195 f0 < 4fe in the regions with maximum volume emission rates. The IGRF model gives for the 630.0 nm emissions, fe ≈1.360 MHz at hmax ≈227 km, so that ∆f ≡f0 −4fe ≈−17 kHz. For the emissions at 557.7 nm, fe ≈1.364 MHz at hmax ≈219 km, giving ∆f ≈−33 kHz. Stimulated electromagnetic emissions can also be used to estimate the vicinity of f0 to sfe (Leyser, 2001), but in our Stimulated electromagnetic emissions can also be used to estimate the vicinity of f0 to experiment the emissions were generally too weak for spectral structure to be identiﬁed. However, for the fourth HF pulse 200 at f0 = 5.423 MHz, a weak broad upshifted maximum (BUM) can be identiﬁed. In Fig. 4 it can be seen that the electron temperature is slightly more enhanced in the fourth (and last) HF pulse than in the preceding pulses at f0 = 5.423 MHz, which indicates a stronger plasma excitation in the last pulse. For s = 4, the BUM has been observed in the range 4fe −20 kHz ≲f0 ≲4fe + 120 kHz (Frolov et al., 1998), thus mainly at f0 ≳4fe. As fe decreases with increasing altitude, the weak BUM experiment the emissions were generally too weak for spectral structure to be identiﬁed. 5 Discussion Also, the ratio of the column emission rates for the emissions at 557.7 and 630.0 nm varied in the range I5577/I6300 ≈0.1–0.4 during the experiment (from their ﬁgure 2), which agrees with our ratio for the same emission lines I5577/I6300 ≈0.2, but again at f0 = 6.200 MHz. In our case with f0 = 5.423 MHz, the maximum of the emissions at 557.7 nm occurred about 7–10 km below that at 630.0 nm (Fig. 6). Further, the ratio I5577/I6300 ≈0.5 is 215 slightly higher than that for the same emission lines presented by Gustavsson and Eliasson (2008) for f0 = 5.423 MHz. Gustavsson and Eliasson (2008) found the maximum volume emission rate for 630.0 and 557.7 nm at hmax ≈220 km and higher, depending on time in the experiment (their ﬁgure 7). According to the IGRF model for the date of their experiments, 4fe ≈5.414 MHz at 220 km and lower at increasing altitudes. Thus, in their case f0 ≳4fe in the height region with the main pump-enhanced optical emissions. In view of the similarities between the results of Gustavsson and Eliasson (2008) and our 220 case with f0 = 6.200 MHz, together with that in our case f0 = 5.423 MHz ≲4fe at the altitudes of maximum volume emission rates as discussed above, we conjecture that f0 = 5.423 MHz ≲4fe in the regions where the optical emissions were enhanced in our experiment. pump-enhanced optical emissions. In view of the similarities between the results of Gustavsson and Eliasson (2008) and our 220 case with f0 = 6.200 MHz, together with that in our case f0 = 5.423 MHz ≲4fe at the altitudes of maximum volume emission rates as discussed above, we conjecture that f0 = 5.423 MHz ≲4fe in the regions where the optical emissions were enhanced in our experiment. 11 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. Further, the dispersion characteristics of upper hybrid waves (Leyser et al., 1989; Istomin and Leyser, 1995; Mishin et al., 2005) are similar for the case of Gustavsson and Eliasson (2008) with f0 = 5.423 MHz ≳4fe and our case for f0 = 6.200 225 MHz ≈4.6fe. In both cases, upper hybrid waves can have a wide range of wave numbers, which facilitates large electron temperature enhancements and emissions at 630.0 nm. 5 Discussion 4), ρe ≈0.023 m, which gives k⊥max ≈5 m−1 or upper hybrid wavelengths longer than about 1 m. For electron acceleration by upper hybrid oscillations localized in the density depletion of a striation pumped by a left-handed 240 circularly polarized electromagnetic wave, that gives a power-law tail in the electron velocity distribution. From their equation (40) we obtain an estimate of the maximum possible upper hybrid wave number perpendicular to the geomagnetic ﬁeld for ∆f ≈−33 kHz (557.7 nm) as k⊥maxρe ≈0.1, where ρe is the thermal electron gyro radius. With an electron temperature of 2500 K (Fig. 4), ρe ≈0.023 m, which gives k⊥max ≈5 m−1 or upper hybrid wavelengths longer than about 1 m. For ∆f ≈−17 kHz (630.0 nm), k⊥max is even smaller which implies an even smaller range of possible upper hybrid wavelengths. 245 For wave numbers k⊥≳k⊥max, upper hybrid waves have negative group dispersion for which the waves cannot be localized in density depletions and therefore can only be relatively weakly excited. As the 557.7 nm column emission rate in our case was approximately the same or even increased as f0 was decreased, we conclude that this emission is excited through electron acceleration by relatively long upper hybrid wavelengths, about 1 m or ∆f ≈−17 kHz (630.0 nm), k⊥max is even smaller which implies an even smaller range of possible upper hybrid wavelengths. 245 For wave numbers k⊥≳k⊥max, upper hybrid waves have negative group dispersion for which the waves cannot be localized in density depletions and therefore can only be relatively weakly excited. As the 557.7 nm column emission rate in our case was approximately the same or even increased as f0 was decreased, we conclude that this emission is excited through electron acceleration by relatively long upper hybrid wavelengths, about 1 m or longer that can be excited for f0 = 5.423 MHz ≲4fe as well as for f0 = 6.200 MHz. This assumes that the pump-induced 250 electron energization occurred approximately at the altitudes where the optical volume emission rates are the largest. The slow growth of the 557.7 nm line relative to the lifetime of the O(1S) state (Table 1) is consistent with the fact that upper hybrid waves need to be localized in the slowly growing density depletions of striations for efﬁcient electron acceleration. 5 Discussion On the other hand, for f0 ≲4fe, as in our case for f0 = 5.423 MHz, upper hybrid waves with positive group dispersion are limited to relatively small wave numbers only which is expected to give less electron heating and lower emission levels at 630.0 nm. Therefore, our result of I5577/I6300 ≈0.5 appears consistent with I5577/I6300 ≈0.1–0.4 as observed by Gustavsson and Eliasson (2008), both with f0 = 5.423 MHz but the former with 230 f0 ≲4fe and the latter with f0 ≳4fe. The fact that the column emission rate at 630.0 nm in our case decreased when f0 was lowered from f0 = 6.200 to f0 = 5.423 MHz is consistent with the theoretically predicted asymmetry in pump-induced effects for f0 around 4fe, with weaker enhancements for f0 ≲4fe and the largest excitations for f0 ≳4fe. For f0 = 6.200 MHz ≈4.6fe, the excitation level should f0 ≲4fe and the latter with f0 ≳4fe. The fact that the column emission rate at 630.0 nm in our case decreased when f0 was lowered from f0 = 6.200 to f0 = 5.423 MHz is consistent with the theoretically predicted asymmetry in pump-induced effects for f0 around 4fe, with weaker enhancements for f0 ≲4fe and the largest excitations for f0 ≳4fe. For f0 = 6.200 MHz ≈4.6fe, the excitation level should be somewhere in between these two cases, that is, stronger than for f0 = 5.423 MHz ≲4fe, as observed. However, we note 235 that no such asymmetry in the 630.0 nm emission has been observed in previous experiments (Gustavsson et al., 2006; Shindin et al., 2015), although the emission intensity is minimum for f0 ≈sfe. Emissions at 557.7 nm have been interpreted in terms of electron acceleration giving a non-thermal tail in the electron distribution, rather than electron heating (Gustavsson et al., 2002, 2005). Istomin and Leyser (2003) presented a model of , g ( , , ) y ( ) p electron acceleration by upper hybrid oscillations localized in the density depletion of a striation pumped by a left-handed 240 circularly polarized electromagnetic wave, that gives a power-law tail in the electron velocity distribution. From their equation (40) we obtain an estimate of the maximum possible upper hybrid wave number perpendicular to the geomagnetic ﬁeld for ∆f ≈−33 kHz (557.7 nm) as k⊥maxρe ≈0.1, where ρe is the thermal electron gyro radius. With an electron temperature of 2500 K (Fig. 5 Discussion Their results can therefore not be used for a comparison of electron energization efﬁciency of f0 ≲4fe and f0 ≳4fe as would be relevant for the present study. But the importance of electron heating for f0 not near sfe is consistent with our result of f However, with f0 = 4.01fe in their simulations, the frequency shift of f0 above 4fe is less than the lower hybrid frequency 280 they used. This implies that decay products of parametric interaction between upper hybrid oscillations at f0 and lower hybrid oscillations have frequencies below 4fe, so that the interaction involves upper hybrid waves both slightly above and below 4fe. Their results can therefore not be used for a comparison of electron energization efﬁciency of f0 ≲4fe and f0 ≳4fe as would be relevant for the present study. But the importance of electron heating for f0 not near sfe is consistent with our result of enhanced 630.0 nm emissions and electron temperatures for f0 = 6.200 MHz. 285 Shindin et al. (2015) detected enhancements at 557.7 nm only for f0 about 15–20 kHz above 4fe in experiments at the Sura facility, with the HF beam tilted 12◦south from vertical in the magnetic meridional plane and the photometers directed in magnetic zenith (19◦south). For a vertical HF beam, emissions were observed with f0 about 5–15 kHz below 4fe and also at about 220–280 kHz above 4fe. Our suggestion that the emission was enhanced for f0 ≲4fe with the Heating beam in magnetic enhanced 630.0 nm emissions and electron temperatures for f0 = 6.200 MHz. 285 Shindin et al. (2015) detected enhancements at 557.7 nm only for f0 about 15–20 kHz above 4fe in experiments at the Sura facility, with the HF beam tilted 12◦south from vertical in the magnetic meridional plane and the photometers directed in magnetic zenith (19◦south). For a vertical HF beam, emissions were observed with f0 about 5–15 kHz below 4fe and also at about 220–280 kHz above 4fe. Our suggestion that the emission was enhanced for f0 ≲4fe with the Heating beam in magnetic zenith thus agrees with Shindin et al. (2015) for a vertical Sura beam, but disagrees with their observations for the Sura beam 290 tilted towards magnetic zenith. However, we do not ﬁnd it clear whether Shindin et al. 5 Discussion With f0 sufﬁciently near 4fe, regardless of wether f0 ≲4fe or f0 ≳4fe, upper hybrid and electron Bernstein oscillations would have only relatively long wavelengths, which are suggested to be involved in the electron acceleration that enhances emissions at 557.7 nm, (and 844.6 nm as studied by Kvammen et al. (2019)). and f0 ≳4fe at higher altitudes, there would be a region with f0 ≈4fe in between with minimum pump–plasma interaction 270 involving upper hybrid and electron Bernstein modes, therefore minimum electron heating and enhancements at 630.0 nm. With f0 sufﬁciently near 4fe, regardless of wether f0 ≲4fe or f0 ≳4fe, upper hybrid and electron Bernstein oscillations would have only relatively long wavelengths, which are suggested to be involved in the electron acceleration that enhances emissions at 557.7 nm, (and 844.6 nm as studied by Kvammen et al. (2019)). Najmi et al. (2017) presented results from Vlasov and test-particle simulations that indicate the physics of electron energiza- 275 tion by wave modes perpendicular to the ambient magnetic ﬁeld and trapped in the density depletion of a striation in the upper hybrid resonance region. When f0 is between 3fe and 4fe, bulk electron heating occurs by electron Bernstein waves which leads to an essentially thermal electron velocity distribution. For f0 ≳4fe, resonant interaction with the upper hybrid mode was found to dominate which leads to electron acceleration and the formation of a suprathermal tail of energetic electrons. Najmi et al. (2017) presented results from Vlasov and test-particle simulations that indicate the physics of electron energiza- 275 tion by wave modes perpendicular to the ambient magnetic ﬁeld and trapped in the density depletion of a striation in the upper hybrid resonance region. When f0 is between 3fe and 4fe, bulk electron heating occurs by electron Bernstein waves which leads to an essentially thermal electron velocity distribution. For f0 ≳4fe, resonant interaction with the upper hybrid mode was found to dominate which leads to electron acceleration and the formation of a suprathermal tail of energetic electrons. However, with f0 = 4.01fe in their simulations, the frequency shift of f0 above 4fe is less than the lower hybrid frequency 280 they used. This implies that decay products of parametric interaction between upper hybrid oscillations at f0 and lower hybrid oscillations have frequencies below 4fe, so that the interaction involves upper hybrid waves both slightly above and below 4fe. 5 Discussion Conversely, the 260 larger enhancements of both the 630.0 nm intensity and the electron temperature for f0 = 6.200 MHz are attributed to mainly the wider range of upper hybrid wavelengths that are available compared to that for f0 = 5.423 MHz ≲4fe, in addition to the slightly higher ERP. Kvammen et al (2019) concluded for the same experiment that f 5 423 MHz ≳4f by comparing their observed increase Kvammen et al. (2019) concluded for the same experiment that f0 = 5.423 MHz ≳4fe by comparing their observed increase in the enhancement at 844.6 nm (10.99 eV threshold) for f0 = 5.423 MHz to Gustavsson et al. (2006) who observed larger 265 enhancements at 427.8 nm (18.75 eV threshold) for f0 ≳4fe than for f0 ≲4fe. We note that the ERP in the experiments by Gustavsson et al. (2006) was four to ﬁve times higher than in the present experiment, implying that a comparison between the experiments may not be obvious. In view of that f0 = 5.423 MHz ≲4fe at the altitudes of maximum volume emission rates we suggest the possibility that f0 ≲4fe in the excitation regions of the optical emissions. With f0 ≲4fe. at lower altitudes Kvammen et al. (2019) concluded for the same experiment that f0 5.423 MHz ≳4fe by comparing their observed increase in the enhancement at 844.6 nm (10.99 eV threshold) for f0 = 5.423 MHz to Gustavsson et al. (2006) who observed larger 265 enhancements at 427.8 nm (18.75 eV threshold) for f0 ≳4fe than for f0 ≲4fe. We note that the ERP in the experiments by Gustavsson et al. (2006) was four to ﬁve times higher than in the present experiment, implying that a comparison between the experiments may not be obvious. In view of that f0 = 5.423 MHz ≲4fe at the altitudes of maximum volume emission rates we suggest the possibility that f0 ≲4fe in the excitation regions of the optical emissions. With f0 ≲4fe. at lower altitudes and f0 ≳4fe at higher altitudes, there would be a region with f0 ≈4fe in between with minimum pump–plasma interaction 270 involving upper hybrid and electron Bernstein modes, therefore minimum electron heating and enhancements at 630.0 nm. 5 Discussion The fact that both the 630.0 and 557.7 nm emissions exhibited a much longer growth time than the lifetime of the corresponding excited states is consistent with the relatively weak level of upper hybrid phenomena that is expected for f0 ≲4fe where only relatively 255 long wavelengths can be excited. The emission intensity at 557.7 nm for f0 = 5.423 MHz was relatively high despite the lower ERP and the expected limited wavelength range of upper hybrid oscillations. This can be understood by the assumption that the anomalous absorption of the states is consistent with the relatively weak level of upper hybrid phenomena that is expected for f0 ≲4fe where only relatively 255 long wavelengths can be excited. The emission intensity at 557.7 nm for f0 = 5.423 MHz was relatively high despite the lower ERP and the expected limited wavelength range of upper hybrid oscillations. This can be understood by the assumption that the anomalous absorption of the The emission intensity at 557.7 nm for f0 = 5.423 MHz was relatively high despite the lower ERP and the expected limited wavelength range of upper hybrid oscillations. This can be understood by the assumption that the anomalous absorption of the 12 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. pump wave (and associated electron temperature enhancements) decreased as f0 was lowered from 6.200 MHz to 5.423 MHz, thereby giving stronger pumping of the long-wavelength upper hybrid oscillations for electron acceleration. Conversely, the 260 f pump wave (and associated electron temperature enhancements) decreased as f0 was lowered from 6.200 MHz to 5.423 MHz, pump wave (and associated electron temperature enhancements) decreased as f0 was lowered from 6.200 MHz to 5.423 MHz, thereby giving stronger pumping of the long-wavelength upper hybrid oscillations for electron acceleration. Conversely, the 260 larger enhancements of both the 630.0 nm intensity and the electron temperature for f0 = 6.200 MHz are attributed to mainly the wider range of upper hybrid wavelengths that are available compared to that for f0 = 5.423 MHz ≲4fe, in addition to the slightly higher ERP. thereby giving stronger pumping of the long-wavelength upper hybrid oscillations for electron acceleration. 5 Discussion One difference between these two experiments that may have inﬂuenced the excitation efﬁciency for the optical emissions is that Gustavsson et al. (2002) used a vertically transmitted pump beam while Kosch et al. (2005) transmitted the beam in magnetic zenith where maximum optical effects are expected (Kosch et al., 2000; Pedersen and Carlson, 2001; 310 Pedersen et al., 2008). This could have contributed to the high ratio I5577/I6300 for f0 ≳3fe observed by Kosch et al. (2005) despite the lower ERP. 5 Discussion (2015) actually obtained conditions with f0 ≲4fe and the HF beam tilted towards magnetic zenith, so that a comparison with the observed 557.7 nm enhancements for f0 ≳4fe could actually be made. Further, although Shindin et al. (2015) by superposed epoch analysis convincingly bring zenith thus agrees with Shindin et al. (2015) for a vertical Sura beam, but disagrees with their observations for the Sura beam 290 tilted towards magnetic zenith. However, we do not ﬁnd it clear whether Shindin et al. (2015) actually obtained conditions with f0 ≲4fe and the HF beam tilted towards magnetic zenith, so that a comparison with the observed 557.7 nm enhancements for f0 ≳4fe could actually be made. Further, although Shindin et al. (2015) by superposed epoch analysis convincingly bring 13 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. CC BY 4.0 License. forth the pump-enhanced 557.7 nm emission, their data remains noisy with variations in the natural background airglow. The experimental conditions in our case were better and the optical emissions were enhanced well above background. Another 295 point is that even with f0 about 15–20 kHz above 4fe in some altitude region, enhancement of optical emissions could also occur at some ten kilometers or more lower altitudes at which f0 ≲4fe. 295 f0 ≲3fe in the early part of the experiment which changed to f0 ≳3fe in the later part because of the slowly increasing pump 300 reﬂection height during the evening. We note that the largest ratio I5577/I6300 ≈0.4 was observed in the early part of the experiment (their ﬁgure 2), when f0 ≲3fe, which is consistent with our results with f0 = 5.423 MHz ≲4fe. Kosch et al. (2005) presented experimental results from the HAARP facility and found I5577/I6300 ≈0.2–0.4, but for f0 ≳ 3fe (their ﬁgure 1). We note that the largest ratio I5577/I6300 ≈0.4 was observed in the ﬁrst displayed pump pulse when f0 was the closest to 3fe. The ERP was 47.8 MW. However, no results were obtained for f0 ≲3fe to compare with. Thus, Kosch 305 et al. (2005) observed the largest ratio I5577/I6300 ≈0.4 for f0 ≳3fe, while Gustavsson et al. (2002) observed their largest ratio I5577/I6300 ≈0.4 for f0 ≲3fe. In addition, the ERP was larger in the experiment by Gustavsson et al. (2002) than for Kosch et al. (2005). 6 Conclusions The EISCAT Heating facility was used to excite ionospheric F-region plasma by HF pumping 150 s on/85 s off and the beam The EISCAT Heating facility was used to excite ionospheric F-region plasma by HF pumping 150 s on/85 s off and the beam pointing in geomagnetic zenith, with f0 = 6.200 MHz followed by f0 = 5.423 MHz. Optical emissions imaged at 557.7 and 315 630.0 nm from three ALIS stations were analyzed and plasma parameter values were obtained with the EISCAT UHF inco- herent scatter radar. From tomography-like reconstruction of the altitude distribution of the optical volume emission rates, we conclude that f0 = 6.200 MHz ≈4.6fe and f0 = 5.423 MHz ≲4fe in the height regions with the largest optical enhance- ments. The ratio of the column emission rates was I5577/I6300 ≈0.2 with f0 = 6.200 MHz. As f0 was decreased to 5.423 pointing in geomagnetic zenith, with f0 = 6.200 MHz followed by f0 = 5.423 MHz. Optical emissions imaged at 557.7 and 315 630.0 nm from three ALIS stations were analyzed and plasma parameter values were obtained with the EISCAT UHF inco- herent scatter radar. From tomography-like reconstruction of the altitude distribution of the optical volume emission rates, we conclude that f0 = 6.200 MHz ≈4.6fe and f0 = 5.423 MHz ≲4fe in the height regions with the largest optical enhance- ments. The ratio of the column emission rates was I5577/I6300 ≈0.2 with f0 = 6.200 MHz. As f0 was decreased to 5.423 MHz, the pump-induced enhancements of both the 630.0 nm emissions and electron temperature decreased. This is consistent 320 with that the O(1D) state is excited mainly by electron heating. On the other hand, the emission intensity at 557.7 nm was approximately the same or even increased slightly as f0 was decreased to 5.423 MHz, so that I5577/I6300 ≈0.5. The 557.7 nm enhancement is suggested to depend primarily on upper hybrid waves with wavelengths of 1 m or longer that can be excited for f0 = 5.423 MHz ≲4fe. The fact that the growth times of both emissions in this case were several times longer than the lifetimes of the respective excited states, indicates that both emissions depend on the formation of small-scale density 325 striations, which is expected to be slower for f0 ≲4fe than for f0 not near sfe (s ≥3). 14 https://doi.org/10.5194/angeo-2023-21 Preprint. Discussion started: 20 July 2023 c⃝Author(s) 2023. 6 Conclusions CC BY 4.0 License. Data availability. Access to the raw data may be provided upon reasonable request to the authors. Author contributions. TBL organized the experiment, interpreted the results and wrote the paper. TS analysed the data, did the theoretical modeling and provided the plots for the ﬁgures. All took part in the experiment and contributed to the paper. Competing interests. The authors declare that they have no conﬂict of interest. 330 Acknowledgements. 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https://openalex.org/W4361207172	https://digitalcommons.wustl.edu/cgi/viewcontent.cgi?article=2934&context=oa_4	English	null	Median Nerve Stimulation for Treatment of Tics: A 4-Week Open Trial with Ecological Momentary Assessment	Journal of clinical medicine	2,023	cc-by	11,053	Washington University School of Medicine Washington University School of Medicine Digital Commons@Becker Digital Commons@Becker 2020-Current year OA Pubs Open Access Publicati 3-28-2023 Median nerve stimulation for treatment of tics: A 4-week open Median nerve stimulation for treatment of tics: A 4-week open trial with ecological momentary assessment trial with ecological momentary assessment Ann M Iverson Amanda L Arbuckle David Y Song Emily C Bihun Kevin J Black Open Access Publications Open Access Publications Keywords: clinical trial; median nerve stimulation; open label; persistent motor tic disorder; tic disorders; Tourette syndrome Citation: Iverson, A.M.; Arbuckle, A.L.; Song, D.Y.; Bihun, E.C.; Black, K.J. Median Nerve Stimulation for Treatment of Tics: A 4-Week Open Trial with Ecological Momentary Assessment. J. Clin. Med. 2023, 12, 2545. https://doi.org/10.3390/ jcm12072545 Median nerve stimulation for treatment of tics: A 4-week open Median nerve stimulation for treatment of tics: A 4-week open trial with ecological momentary assessment trial with ecological momentary assessment Ann M Iverson Amanda L Arbuckle David Y Song Emily C Bihun Kevin J Black Follow this and additional works at: https://digitalcommons.wustl.edu/oa_4 Please let us know how this document benefits you. Follow this and additional works at: https://digitalcommons.wustl.edu/oa_4 Please let us know how this document benefits you. Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Article verson 1 , Amanda L. Arbuckle 2 , David Y. Song 3, Emily C. Bihun 2 and Kevin J. Black 4,* Ann M. Iverson 1 , Amanda L. Arbuckle 2 , David Y. Song 3, Emily C. Bihun 2 and Kevin 1 Washington University School of Medicine, Washington University in St. Louis, St. Louis, MO 63110, USA 2 Department of Psychiatry, Washington University in St. Louis, St. Louis, MO 63110, USA 3 University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA 4 Departments of Psychiatry, Neurology, Radiology and Neuroscience, Washington University in St. Louis, St. Louis, MO 63110, USA 1 Washington University School of Medicine, Washington University in St. Louis, St. Louis, MO 63110, USA 2 Department of Psychiatry Washington University in St Louis St Louis MO 63110 USA * Correspondence: kevin@wustl.edu Abstract: Median nerve stimulation (MNS) at 10–12 Hz was recently proposed as a treatment for Tourette syndrome and other chronic tic disorders (TS/CTD). We report on 31 participants ages 15–64 with TS/CTD in an open-label, comparative (within-group, several time points) study of MNS (ClinicalTrials.gov registration number NCT05016765). Participants were recruited from completers of a randomized controlled trial (RCT) of MNS and were given a transcutaneous electrical nerve stimulation (TENS) unit to use as desired for 12 Hz MNS for 4 weeks. Participants were instructed to complete surveys regarding tic symptoms and stimulation discomfort before and after stimulation, as well as twice daily when randomly prompted by text message. Participants also completed an extensive ﬁnal survey. Twenty-seven participants completed the study. Median device use was 1.5 days per week and 50 min per day used. Tic frequency improved during MNS (mean improvement: 1.0 on a 0–5 scale, p < 0.001), as did tic intensity (mean improvement: 0.9, p < 0.001). Mean discomfort was mild (1.2 on a 3-point scale). In total, 21 participants (78%) planned to continue using the device. Participants’ results in this study did not correlate signiﬁcantly with their results in the blinded RCT. We found MNS to improve tic frequency and intensity with minimal side effects. 1. Introduction New treatments are needed for chronic tic disorders (CTD), including Tourette syn- drome (TS), which affect approximately 1.4 million children and adults in the United States [1,2]. Patients with these disorders have one or more vocal or motor tics (or both in the case of Tourette syndrome) which have persisted for at least one year [3]. Co- morbid conditions are also common in this population, speciﬁcally ADHD and OCD [4]. Whereas a variety of treatments can be used for tic disorders, including various pharma- cological and behavioral therapies, many patients desire new treatment options [5]. For pharmacologic agents in particular, there is a relative lack of conclusive evidence as to the optimal treatment regimen [6]. The development of new treatments is made more difﬁcult by the fact that the etiology and pathophysiology of TS/CTD is still not well understood. Neuroimaging studies of these patients have demonstrated impaired function of the cortical-striatal-thalamic-cortical circuit and abnormal neurotransmitter function, but further research is needed in this area [7]. Academic Editor: Sylvie Raoul Received: 22 February 2023 Revised: 21 March 2023 Accepted: 25 March 2023 Published: 28 March 2023 Received: 22 February 2023 Revised: 21 March 2023 Accepted: 25 March 2023 Published: 28 March 2023 2. Materials and Methods We followed the CONSORT reporting suggestions adapted to non-randomized trials (see supplementary ﬁgure and table) [11]. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Morera Maiquez and colleagues from Stephen Jackson’s lab at the University of Not- tingham recently proposed a completely novel treatment idea involving indirect rhythmic electrical stimulation of the brain via peripheral stimulation of the median nerve [8]. The hypothesis was that increasing EEG power in a frequency range associated with motor inhibition would reduce tics. They demonstrated that 12 Hz stimulation of the median https://www.mdpi.com/journal/jcm J. Clin. Med. 2023, 12, 2545. https://doi.org/10.3390/jcm12072545 J. Clin. Med. 2023, 12, 2545 2 of 19 nerve (MNS) evoked synchronous contralateral EEG activity over the primary sensorimotor cortex and created small but statistically signiﬁcant effects on the initiation of voluntary movements. Blinded video ratings showed signiﬁcant reduction in tic number and tic intensity during 1-min stimulation blocks, and participants reported a signiﬁcantly lower urge to tic. We recently replicated these results and extended them with longer stimulation blocks and a comparison condition intended to serve as an active placebo [9]. These results suggest that MNS may be a promising treatment for Tourette syndrome, but the real-world practicality of such a treatment has not been tested, as no studies have been published outside of the laboratory setting. In day-to-day use of such a device, patients would not only be exposed to much longer treatment duration but might also identify practical barriers to its use. We therefore conducted an open-label outpatient study of 12 Hz MNS with a portable TENS (transcutaneous electrical nerve stimulation) device to assess the tolerability, practicality and efﬁcacy of MNS outside of a controlled setting. Whereas a randomized trial is technically possible, using the non-randomized format allows us to collect preliminary information on tolerability and practicality much more quickly. We included ecological momentary assessments (EMAs) of tic intensity and frequency, i.e., brief surveys of tic severity and device usage several times a day, to minimize recall bias and improve response validity [10]. 2.2. Companion Randomized Controlled Trial The open-label study is an extension of a randomized controlled trial (RCT), of which the results are published separately [9]. Brieﬂy, the RCT was a double-blind, randomized, crossover study that compared rhythmic MNS to arrhythmic MNS in people ages 15–64 with TS. Stimulation occurred in a laboratory setting in two sessions approximately one week apart. Participants were randomized to receive either rhythmic or arrhythmic stim- ulation on the ﬁrst visit and the other on the second visit. Stimulation was delivered in both one-minute and ﬁve-minute blocks, interspersed with blocks without stimulation. In the RCT, participants were video recorded during stimulation sessions, and tic frequency and intensity were later rated by a trained rater blind to presence of stimulation, type of stimulation, block order, and visit number. 2.1. Ethics Approval The Washington University Human Research Protection Ofﬁce (IRB) approved this study (approval # 202109160). All participants provided written informed consent (those under age 18 provided written documentation of assent and a parent consented). The study used a device cleared by the FDA for pain relief, 501(k) premarket notiﬁcation number K080661, and the IRB deemed this a non-signiﬁcant-risk device study. 2.4. Participants Participants were invited from the 32 people who completed an in-person RCT, of which the results are reported separately [9]. Brieﬂy, inclusion criteria for the controlled trial were age 15–64 inclusive at initial screening visit, current DSM-5 Tourette’s Disorder or Persistent (Chronic) Tic Disorder, and at least 1 tic per minute (on average) during the 5-min baseline video session on the ﬁrst visit. Exclusion criteria included, among others, an implanted device that could be affected by electrical current, signiﬁcant neurologic disease not counting TS (exceptions included febrile seizures or uncomplicated migraine), and any recent or planned change in somatic or psychotherapeutic treatment. All inclusion and exclusion criteria appear in the published RCT study protocol [13]. In total, 31 of the 32 RCT participants consented to participate in this open follow-on study. Participants provided permission for their baseline information from the RCT to be utilized for this study well. These baseline measures included the Yale Global Tic Severity Scale (YGTSS) [14], Diagnostic Conﬁdence Index [15], Adult Tic Questionnaire (ATQ) tic severity rating [16,17], Premonitory Urge for Tics Scale (PUTS) [18], the ADHD Rating Scale [19], and a self-report version [20] of the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) Scale [21,22]. In addition, participants provided information about past medical and family history, as well as current treatments. 2.3. Study Design The open-label study protocol was published prior to enrolling the ﬁrst study par- ticipant, with trivial changes published by enrollment of the third participant [12]. Over a period of 4 weeks, participants selected whether and when to apply MNS, and they reported on tic intensity each time the device was turned on or off and at random times twice daily when prompted by a text message. The planned sample size for the RCT from which participants for the open-label study were invited was 32, chosen for practical reasons of enrollment and cost, with the expectation that 32 participants would reasonably reﬂect the experience of adults and older teenagers who were willing to participate in a RCT for treatment of tics. The study’s goals included determining the real-world usage and apparent utility of stimulation in people with chronic tics, determining momentary self-rated efﬁcacy and side J. Clin. Med. 2023, 12, 2545 3 of 19 3 of 19 effects of stimulation, and testing whether a participant’s RCT results predicted their results in open use. We hypothesized that MNS stimulation would be effective in the treatment of tics and that results would correlate with those found in the RCT. Participants were recruited between November 2021 and April 2022. All data collection was completed by June 2022. 2.5. Intervention Participants in the study were provided a transcutaneous electrical nerve stimula- tion (TENS) unit (TENS-7000, Roscoe Medical, Inc., Middleburg Heights, OH) and 1- inch-diameter circular adhesive carbon ﬁlm electrodes (Syrtenty® part number TSYR1000, Walpole, MA). Participants were instructed on proper use of the device and proper elec- trode attachment (anode centered over the median nerve with the distal edge at the distal wrist crease, cathode approximately 30 mm proximal center-to-center; see also [23]). Par- ticipants could select either wrist for MNS, but almost all chose the non-dominant hand. Simulation consisted of 200 µs square-wave pulses at 12 Hz. Participants were instructed to use the device as frequently as desired for as long as desired. At the ﬁrst study visit, the stimulation threshold for each participant was identiﬁed as the minimum current setting at which a visible twitch of the abductor pollicis brevis was observed. Suprathreshold stimulation was encouraged, with an explanation that previous physiological and efﬁcacy data used suprathreshold MNS, but participants selected the stimulation current each time they turned on the device. 2.6. Outcome Measures The pre-speciﬁed primary outcome measures included time spent using the device (minutes per day and number of days per week used over the four-week period), how many participants self-reported they planned to continue using the stimulator after the study period ended, comparison of tic frequency and of tic intensity between “turning ON” and “turning OFF” surveys [14], mean discomfort while using stimulator (0–3 scale adapted from the CGI-I Efﬁcacy Index [24]), and relationship to the results from the blinded study (correlation of change in tic frequency and intensity during this study with change in tic frequency and intensity respectively during active, rhythmic stimulation during the RCT). Tic intensity ratings used the 0–5 scale of the tic intensity item on the YGTSS [14]. Tic frequency was adapted from the YGTSS tic frequency item, since the original scale reports on frequency over a 1-week period but the present study inquires about tic frequency over periods of minutes to hours. The anchor points provided for frequency ratings varied J. Clin. Med. 2023, 12, 2545 4 of 19 depending on the duration of stimulation (e.g., one cannot usefully answer questions about tic frequency over the past hour if stimulation lasted only 10 min; see the form in ref. [12] for details. All other outcome measures were secondary. Over the four-week study period, participants were asked to complete electronic ecological momentary assessment (EMA) surveys at each instance of three types of events: immediately prior to turning on the device, immediately after turning off the device, and in response to twice-daily texts at random times between 09:00 and 21:00. Surveys prior to turning on the device included current tic frequency and intensity, and surveys after turning off the device included the same questions in addition to questions regarding dis- comfort from stimulation, overall symptom improvements, and current device settings [14]. Text-prompted surveys were similar; if the stimulator was off when the text arrived, an additional question asked how long ago the stimulator was last used. q g g At the conclusion of the four-week study period, the participants completed a ﬁnal survey that included the Adult Tic Questionnaire (ATQ) tic severity rating [16,17], Pre- monitory Urge for Tics Scale (PUTS) [18], Clinical Global Impression–Efﬁcacy Index [24], their perception of duration of improvement after stimulation, and their plans to con- tinue using the device. The survey also solicited open-ended comments about MNS and study participation. 2.6. Outcome Measures All surveys were completed online via RedCap at the location of the participant’s choosing. 3. Results Individual participant data appear in ref. [12]. Individual participant data appear in ref. [12]. 2.7. Statistical Analysis Participant data were collected and stored using REDCap [25]. Descriptive data are provided as mean ± standard deviation or median and IQR, depending on the normality of the data. Paired t-tests, or paired samples Wilcoxon tests as appropriate, tested most primary and secondary outcomes. Correlation analysis with Pearson’s or Spearman’s coef- ﬁcients as appropriate was conducted for relevant comparisons. Sample size is provided for each analysis, as available sample size varied based on participant survey completion. Analysis was conducted using R Statistical Software [26,27]. 3.1. Participants The baseline characteristics of all participants are shown in Table 1. The baseline characteristics of all participants are shown in Table 1. Successful treatments = number of treatments attempted judged by the participant to provide at least minimal improvement. Family history items reﬂect the percentage of ﬁrst-degree relatives with the condition named. The baseline characteristics of all participants are shown in Table 1. Table 1. Baseline characteristics of all participants (n = 31). Mean SD Median IQR Age 34.5 16.6 35 16–45 Sex (male) 64.5% NA NA NA YGTSS Impairment 19.2 16.1 20 0–30 YGTSS Total Tic Score 24.9 9.1 25 20–29.5 DCI 60.9 20.2 56 44.5–78.5 ATQ 38.5 21.4 35 21.5–55 PUTS 21.7 7.2 23 15.5–27 Table 1. Baseline characteristics of all participants (n = 31). J. Clin. Med. 2023, 12, 2545 5 of 19 Table 1. Cont. Table 1. Cont. Mean SD Median IQR Distress/Impairment Last Week 61.3% NA NA NA Distress/Impairment Lifetime 100% NA NA NA Current Antipsychotic Use 22.6% NA NA NA Current Alpha-2 Agonist Use 9.7% NA NA NA Lifetime Antipsychotic Use 35.5% NA NA NA Lifetime Alpha-2 Agonist Use 51.6% NA NA NA Number of Treatments Tried 3.9 3.8 3 1.0–5.5 Successful Treatments 0.6 1.0 0 0–1 Adequate Behavior Therapy 9.7% NA NA NA % of Participants with Family History of Tics in First Degree Relatives 45.2% NA NA NA % of Participants with Family History of Tics, OCD, or ADHD in First Degree Relatives 67.7% NA NA NA Y-BOCS Obsession Subtotal 3.5 3.7 3 0–5.5 Y-BOCS Compulsion Subtotal 3.0 2.9 2 0–6 Y-BOCS Total 6.5 5.9 5 0–11 ADHD Rating Scale 13.2 13.3 7 2.5–20.5 Baseline characteristics were gathered at the ﬁrst study visit of the randomized controlled trial from which the open-label study participants were drawn. Y-BOCS = Yale-Brown Obsessive Compulsive Scale, PUTS = Premonitory Urge for Tics Scale, YGTSS = Yale Global Tic Severity Score, DCI = Diagnostic Conﬁdence Index, ATQ = Adult Tic Questionnaire, OCD = Obsessive-Compulsive Disorder, ADHD = Attention-Deﬁcit Hyperactivity Disorder. Successful treatments = number of treatments attempted judged by the participant to provide at least minimal improvement. Family history items reﬂect the percentage of ﬁrst-degree relatives with the condition named. 3.2. Outcomes Results for pre-speciﬁed primary and secondary outcomes measures are included in Tables 2 and 3 below as well as in Figures 1 5 Baseline characteristics were gathered at the ﬁrst study visit of the randomized controlled trial from which the open-label study participants were drawn. Y-BOCS = Yale-Brown Obsessive Compulsive Scale, PUTS = Premonitory Urge for Tics Scale, YGTSS = Yale Global Tic Severity Score, DCI = Diagnostic Conﬁdence Index, ATQ = Adult Tic Questionnaire, OCD = Obsessive-Compulsive Disorder, ADHD = Attention-Deﬁcit Hyperactivity Disorder. 3.2. Outcomes Results for pre-speciﬁed primary and secondary outcomes measures are included in Tables 2 and 3 below, as well as in Figures 1–5. J. Clin. Med. 2023, 12, 2545 6 of 19 Table 2. Outcomes. Table 2. Outcomes. Outcome Result 95% C.I. p Type Days per week device used (n = 31) 1.5, 1.4 (median, IQR) 1.1–2.3 — primary Minutes per day used (n = 31) 49.6, 93.0 (median, IQR) 45.5–150.3 — primary Number who plan to continue using device after 4 weeks (n = 27) 21 yes (77.8%), 6 no 62.1–93.4 — primary Change in tic frequency when turning device OFF versus previous ON (n = 25 for t-test) Turning off: 2.1 ± 1.1 (mean ± SD) Turning on: 3.1 ± 1.0 (mean ± SD) Difference: 1.0 (mean) 0.6–1.4 <0.001 (paired t test) primary Change in tic intensity when turning device OFF versus previous ON (n = 25 for t-test) Turning off: 2.0 ± 1.1 (mean ± SD) Turning on: 2.9 ± 0.8 (mean ± SD) Difference: 0.9 (mean) 0.6–1.9 <0.001 a primary Mean discomfort while using stimulator (n = 26) 1, 0.9 (median, IQR) 1.0–1.7 — primary Effect on tic frequency compared to in RCT (see Figure 5a) (n = 24) r = 0.21 −0.22–0.56 0.33 primary Effect on tic intensity compared to in RCT (see Figure 5b) (n = 25) r = 0.36 −0.05–0.66 0.08 primary Overall impact of stimulation on symptoms throughout study period (n = 27) 3.1 ± 0.9 (mean ± SD) 2.7–3.4 — secondary J. Clin. Med. 2023, 12, 2545 7 of 19 Table 2. Cont. Outcome Result 95% C.I. 3.2. Outcomes p Type Discomfort from ﬁnal survey (n = 27) 1.2 ± 1.0 (mean ± SD) 0.8–1.6 — secondary Therapeutic effect while using from ﬁnal survey (n = 27) 1.5 ± 0.9 (mean ± SD) 1.1–1.9 — secondary Difference in ATQ before and after study (n = 25) 0.7 ± 18.0 (mean ± SD) −6.7–8.2 0.84 secondary Difference in PUTS before and after study (n = 25) −0.9 ± 4.3 (mean ± SD) −2.8–1.0 0.35 secondary Correlation between tic frequency and stimulation amplitude (see Figure 2a) (n = 24) R = −0.16 Rho = −0.19 −0.5–0.26 0.44 0.37 secondary Correlation between tic intensity and stimulation amplitude (see Figure 2b) (n = 24) R = 0.05 Rho = 0.15 −0.36–0.45 0.80 0.49 secondary Correlation between tic frequency and stimulation discomfort (see Figure 2c) (n = 25) R = −0.33 Rho = −0.36 −0.64–0.08 0.11 0.08 secondary Correlation between tic intensity and stimulation discomfort (see Figure 2d) (n = 25) R = −0.31 Rho = −0.27 −0.63–0.09 0.13 0.19 secondary Correlation between stimulation amplitude and stimulation discomfort (see Figure 2e) (n = 24) R = 0.36 Rho = 0.31 −0.06–0.66 0.09 0.13 exploratory J. Clin. Med. 2023, 12, 2545 8 of 19 Table 2. Cont. Table 2. Cont. Outcome Result 95% C.I. p Type Change in tic frequency when device OFF for more than 60 min vs. less than 60 min (t test, n = 16) More than 60: 2.7 ± 0.9 (mean ± SD) Less than 60: 2.3, 2.1 (median, IQR) Difference: 0.3 (mean) −0.2–0.7 0.20 secondary Change in tic intensity when device OFF for more than 60 min vs. less than 60 min (t test, n = 16) More than 60: 2.5 ± 0.8 (mean ± SD) Less than 60: 2.3, 2.2 (median, IQR) Difference: 0.1 (mean) −0.4–0.5 0.77 secondary Participants’ perception of length of tic improvement (n = 21) 15, 35 (median, IQR) 15–60 — secondary Change in tic frequency when device OFF for more than 10 min vs. less than 10 min (n = 7 for t test) More than 10: 3, 2 (median, IQR) Less than 10: 2.2, 1.3 (median, IQR) Difference: 0.4 (mean) −0.5–1.3 0.30 secondary Change in tic intensity when device OFF for more than 10 min vs. 3.2. Outcomes less than 10 min (n = 7 for t test) More than 10: 3, 2.5 (median, IQR) Less than 10: 1.9, 1.3 (median, IQR) Difference: 0.3 (mean) −0.7–1.3 0.47 secondary Correlation between tic frequency and CGI-I (see Figure 4a) (n = 22) R= −0.58 Rho= −0.64 −0.80–−0.21 0.005 0.001 secondary Correlation between tic intensity and CGI-I (see Figure 4b) (n = 22) R= −0.41 Rho = −0.44 −0.71–0.01 0.06 0.04 secondary J. Clin. Med. 2023, 12, 2545 9 of 19 Table 2. Cont. Outcome Result 95% C.I. p Type Difference in days per week device used between RCT responders b (n = 13) and non-responders (n = 18) RCT responders: 133.4 ± 207.9 (mean, SD) RCT non-responders: 72.3 ± 62.0 (mean, SD) −66.9–189.0 0.32 exploratory Difference in minutes per day used between RCT responders b (n = 13) and non-responders (n = 18) RCT responders: 2.0 ± 2.3 (mean, SD) RCT non-responders: 1.4 ± 0.9 (mean, SD) −0.9–2.0 0.44 exploratory a Paired samples Wilcoxon test. b RCT responders deﬁned as those with CGI-I Score of 2 (much improved) or better on the rhythmic stimulation day. Table 3. Responders Analysis. Mean, Non-Responders (n = 9) Mean, Responders (n = 18) p Value Age 37.7 33.9 0.52 Y-BOCS Obsession Subtotal 2.7 4.2 0.45 Y-BOCS Compulsion Subtotal 2.9 3.3 0.90 Y-BOCS Total 5.6 7.6 0.51 PUTS 19.4 21.9 0.45 YGTSS Impairment 20.6 15.6 0.52 YGTSS Total Tic Score 22.9 25.8 0.12 DCI 52.7 64.3 0.20 ATQ 34.8 41.2 0.43 ADHD 9.1 14.4 0.20 Number of Treatments Tried 3.9 4 0.73 Successful Treatments 0.3 0.8 0.44 Family History of Tics (% of participants with history in ﬁrst degree relative) 55.6 27.8 0.22 Table 2. Cont. Outcome Result 95% C.I. p Type Difference in days per week device used between RCT responders b (n = 13) and non-responders (n = 18) RCT responders: 133.4 ± 207.9 (mean, SD) RCT non-responders: 72.3 ± 62.0 (mean, SD) −66.9–189.0 0.32 exploratory Difference in minutes per day used between RCT responders b (n = 13) and non-responders (n = 18) RCT responders: 2.0 ± 2.3 (mean, SD) RCT non-responders: 1.4 ± 0.9 (mean, SD) −0.9–2.0 0.44 exploratory a Paired samples Wilcoxon test. b RCT responders deﬁned as those with CGI-I Score of 2 (much improved) or better on the rhythmic stimulation day. a Paired samples Wilcoxon test. 3.2. Outcomes b RCT responders deﬁned as those with CGI-I Score of 2 (much improved) or better on the rhythmic stimulation day. Table 3. Responders Analysis. Mean, Non-Responders (n = 9) Mean, Responders (n = 18) p Value Age 37.7 33.9 0.52 Y-BOCS Obsession Subtotal 2.7 4.2 0.45 Y-BOCS Compulsion Subtotal 2.9 3.3 0.90 Y-BOCS Total 5.6 7.6 0.51 PUTS 19.4 21.9 0.45 YGTSS Impairment 20.6 15.6 0.52 YGTSS Total Tic Score 22.9 25.8 0.12 DCI 52.7 64.3 0.20 ATQ 34.8 41.2 0.43 ADHD 9.1 14.4 0.20 Number of Treatments Tried 3.9 4 0.73 Successful Treatments 0.3 0.8 0.44 Family History of Tics (% of participants with history in ﬁrst degree relative) 55.6 27.8 0.22 Table 3. Responders Analysis. J. Clin. Med. 2023, 12, 2545 10 of 19 10 of 19 Table 3. Cont. Mean, Non-Responders (n = 9) Mean, Responders (n = 18) p Value Family History Tics/OCD/ADHD (% of participants with history in ﬁrst degree relative) 77.8 55.6 0.41 Sex (% male) 66.7 72.2 1 Distress/Impairment Last Week (% yes) 66.6 66.6 1 Distress/Impairment Lifetime (% yes) 100 100 1 Adequate Behavior Therapy (% yes) 0 16.7 0.53 Comparison of responders vs. non-responders, where responders are those with a CGI-I score of 3 (minimally improved) or better. Y-BOCS = Yale-Brown Obsessive Compulsive Scale, PUTS = Premonitory Urge for Tics Scale, YGTSS = Yale Global Tic Severity Score, DCI = Diagnostic Conﬁdence Index, ATQ = Adult Tic Question- naire. Successful treatments = number of treatments attempted rated by participant to provide at least minimal improvement. Family history items displayed as percentage of ﬁrst-degree relatives with condition. 2023, 12, x FOR PEER REVIEW 8 a Paired samples Wilcoxon test. b RCT responders defined as those with CGI‐I Score of 2 (much proved) or better on the rhythmic stimulation day. (a) (b) Figure 1. Participant Usage Patterns. (a) Minutes per day used by each participant. Participant ordered by usage (n = 31). (b) Days per week for each participant. Participants are ordered by u (n = 31). Figure 1. Participant Usage Patterns. (a) Minutes per day used by each participant. Participants are ordered by usage (n = 31). (b) Days per week for each participant. Participants are ordered by usage (n = 31). Table 3. Cont. Table 3. Cont. 3.2. Outcomes Mean, Non-Responders (n = 9) Mean, Responders (n = 18) p Value Family History Tics/OCD/ADHD (% of participants with history in ﬁrst degree relative) 77.8 55.6 0.41 Sex (% male) 66.7 72.2 1 Distress/Impairment Last Week (% yes) 66.6 66.6 1 Distress/Impairment Lifetime (% yes) 100 100 1 Adequate Behavior Therapy (% yes) 0 16.7 0.53 Comparison of responders vs. non-responders, where responders are those with a CGI-I score of 3 (minimally improved) or better. Y-BOCS = Yale-Brown Obsessive Compulsive Scale, PUTS = Premonitory Urge for Tics Scale, YGTSS = Yale Global Tic Severity Score, DCI = Diagnostic Conﬁdence Index, ATQ = Adult Tic Question- naire. Successful treatments = number of treatments attempted rated by participant to provide at least minimal improvement. Family history items displayed as percentage of ﬁrst-degree relatives with condition. IEW 8 o Paired samples Wilcoxon test. b RCT responders defined as those with CGI‐I Score of 2 (much roved) or better on the rhythmic stimulation day. Comparison of responders vs. non-responders, where responders are those with a CGI-I score of 3 (minimally improved) or better. Y-BOCS = Yale-Brown Obsessive Compulsive Scale, PUTS = Premonitory Urge for Tics Scale, YGTSS = Yale Global Tic Severity Score, DCI = Diagnostic Conﬁdence Index, ATQ = Adult Tic Question- naire. Successful treatments = number of treatments attempted rated by participant to provide at least minimal improvement. Family history items displayed as percentage of ﬁrst-degree relatives with condition. aired samples Wilcoxon test. b RCT responders defined as those with CGI‐I Score of 2 (much oved) or better on the rhythmic stimulation day. (a) (b) Figure 1. Participant Usage Patterns. (a) Minutes per day used by each participant. Participants ordered by usage (n = 31). (b) Days per week for each participant. Participants are ordered by us (n = 31). Figure 1. Participant Usage Patterns. (a) Minutes per day used by each participant. Participants are ordered by usage (n = 31). (b) Days per week for each participant. Participants are ordered by usage (n = 31). (b) (a) (b) gure 1. Participant Usage Patterns. (a) Minutes per day used by each participant. Participants dered by usage (n = 31). (b) Days per week for each participant. Participants are ordered by us = 31). Figure 1. Participant Usage Patterns. (a) Minutes per day used by each participant. Participants are ordered by usage (n = 31). (b) Days per week for each participant. 3.2. Outcomes Participants are ordered by usage (n = 31). 2023, 12, 2545 11 n. Med. 2023, 12, x FOR PEER REVIEW 9 of 17 (a) (b) (c) (d) (e) Figure 2. Impact of Stimulation Amplitude and Discomfort on Tic Frequency and Intensity. (a) Mean improvement in tic frequency compared with mean stimulation amplitude for each participant (n = 24). (b) Mean improvement in tic intensity compared with mean stimulation amplitude for each participant (n = 24). (c) Mean improvement in tic frequency compared with mean stimulation dis‐ comfort for each participant (n = 25). (d) Mean improvement in tic intensity compared with mean Figure 2. Impact of Stimulation Amplitude and Discomfort on Tic Frequency and Intensity. (a) M improvement in tic frequency compared with mean stimulation amplitude for each partici (n = 24). (b) Mean improvement in tic intensity compared with mean stimulation amplitud each participant (n = 24). (c) Mean improvement in tic frequency compared with mean stimula discomfort for each participant (n = 25). (d) Mean improvement in tic intensity compared with m stimulation discomfort for each participant (n = 25). (e) Mean discomfort compared with m amplitude for each participant (exploratory analysis) (n = 24). J. Clin. Med. 2023, 12, 2545 J. Clin. Med. 2023, 12, x 11 of 19 17 (a) (b) (c) (d) (e) Figure 2. Impact of Stimulation Amplitude and Discomfort on Tic Frequency and Intensity. (a) Mean improvement in tic frequency compared with mean stimulation amplitude for each participant (n = 24). (b) Mean improvement in tic intensity compared with mean stimulation amplitude for each participant (n = 24). (c) Mean improvement in tic frequency compared with mean stimulation dis comfort for each participant (n = 25). (d) Mean improvement in tic intensity compared with mean Figure 2. Impact of Stimulation Amplitude and Discomfort on Tic Frequency and Intensity. (a improvement in tic frequency compared with mean stimulation amplitude for each part (n = 24). (b) Mean improvement in tic intensity compared with mean stimulation amplit each participant (n = 24). (c) Mean improvement in tic frequency compared with mean stim discomfort for each participant (n = 25). (d) Mean improvement in tic intensity compared wit stimulation discomfort for each participant (n = 25). (e) Mean discomfort compared with amplitude for each participant (exploratory analysis) (n = 24). (a) (b) (c) (d) (a) (b) (d) (b) (a) (c) (e) (c) (d) (e) Figure 2. 3.2. Outcomes 5 = Minimally worse—slightly worse but may not be clinically meaning- ful; may represent very little change in basic clinical status, level of care, or functional capacity. 6 = Much worse—clinically signiﬁcant increase in symptoms and diminished functioning. 7 = Very much worse—severe exacerbation of symptoms and loss of functioning. d. 2023, 12, x FOR PEER REVIEW 11 o worse—clinically significant increase in symptoms and diminished functioning. 7 = Very m worse—severe exacerbation of symptoms and loss of functioning. Figure 3. Final Participant Clinical Global Impression–Improvement Scores (n = 27). 1 = Ver improved—nearly all better; good level of functioning; minimal symptoms; represents a ve stantial change. 2 = Much improved—notably better with significant reduction of sympto crease in the level of functioning but some symptoms remain. 3 = Minimally improved— better with little or no clinically meaningful reduction of symptoms; represents very little ch basic clinical status, level of care, or functional capacity. 4 = No change—symptoms remain tially unchanged. 5 = Minimally worse—slightly worse but may not be clinically meaningf represent very little change in basic clinical status, level of care, or functional capacity. 6 = Figure 3. Final Participant Clinical Global Impression–Improvement Scores (n = 27). 1 = Very much improved—nearly all better; good level of functioning; minimal symptoms; represents a very substantial change. 2 = Much improved—notably better with signiﬁcant reduction of symptoms; increase in the level of functioning but some symptoms remain. 3 = Minimally improved—slightly better with little or no clinically meaningful reduction of symptoms; represents very little change in basic clinical status, level of care, or functional capacity. 4 = No change—symptoms remain essentially unchanged. 5 = Minimally worse—slightly worse but may not be clinically meaning- ful; may represent very little change in basic clinical status, level of care, or functional capacity. 6 = Much worse—clinically signiﬁcant increase in symptoms and diminished functioning. 7 = Very much worse—severe exacerbation of symptoms and loss of functioning. VIEW 11 worse—clinically significant increase in symptoms and diminished functioning. 7 = Very m worse—severe exacerbation of symptoms and loss of functioning. (a) (b) Figure 4. Correlation Between Participants’ EMA Responses During the Study and Their Repo Improvement During the Final Survey. 3.2. Outcomes Impact of Stimulation Amplitude and Discomfort on Tic Frequency and Intensity. (a) Mean improvement in tic frequency compared with mean stimulation amplitude for each participant (n = 24). (b) Mean improvement in tic intensity compared with mean stimulation amplitude for each participant (n = 24). (c) Mean improvement in tic frequency compared with mean stimulation dis‐ comfort for each participant (n = 25). (d) Mean improvement in tic intensity compared with mean Figure 2. Impact of Stimulation Amplitude and Discomfort on Tic Frequency and Intensity. (a) Mean improvement in tic frequency compared with mean stimulation amplitude for each participant (n = 24). (b) Mean improvement in tic intensity compared with mean stimulation amplitude for each participant (n = 24). (c) Mean improvement in tic frequency compared with mean stimulation discomfort for each participant (n = 25). (d) Mean improvement in tic intensity compared with mean stimulation discomfort for each participant (n = 25). (e) Mean discomfort compared with mean amplitude for each participant (exploratory analysis) (n = 24). J. Clin. Med. 2023, 12, 2545 12 of 19 nificantl and r = 12 of 19 nificantl and r = Figure 3. Final Participant Clinical Global Impression–Improvement Scores (n = 27). 1 = Very improved—nearly all better; good level of functioning; minimal symptoms; represents a very stantial change. 2 = Much improved—notably better with significant reduction of symptom crease in the level of functioning but some symptoms remain. 3 = Minimally improved—sl better with little or no clinically meaningful reduction of symptoms; represents very little chan basic clinical status, level of care, or functional capacity. 4 = No change—symptoms remain e tially unchanged. 5 = Minimally worse—slightly worse but may not be clinically meaningful represent very little change in basic clinical status, level of care, or functional capacity. 6 = Figure 3. Final Participant Clinical Global Impression–Improvement Scores (n = 27). 1 = Very much improved—nearly all better; good level of functioning; minimal symptoms; represents a very substantial change. 2 = Much improved—notably better with signiﬁcant reduction of symptoms; increase in the level of functioning but some symptoms remain. 3 = Minimally improved—slightly better with little or no clinically meaningful reduction of symptoms; represents very little change in basic clinical status, level of care, or functional capacity. 4 = No change—symptoms remain essentially unchanged. 3.2. Outcomes (a) Mean improvement in tic frequency from EMA sur compared with Clinical Global Impression–Improvement score for each participant (n = 22 Mean improvement in tic intensity from EMA surveys compared with Clinical Global Impress Improvement for each participant (n = 22). Figure 4. Correlation Between Participants’ EMA Responses During the Study and Their Report of Improvement During the Final Survey. (a) Mean improvement in tic frequency from EMA sur- veys compared with Clinical Global Impression–Improvement score for each participant (n = 22). (b) Mean improvement in tic intensity from EMA surveys compared with Clinical Global Impression– Improvement for each participant (n = 22). (b) (a) (b) (a) Figure 4. Correlation Between Participants’ EMA Responses During the Study and Their Repo mprovement During the Final Survey. (a) Mean improvement in tic frequency from EMA sur compared with Clinical Global Impression–Improvement score for each participant (n = 22 Mean improvement in tic intensity from EMA surveys compared with Clinical Global Impres mprovement for each participant (n = 22). Figure 4. Correlation Between Participants’ EMA Responses During the Study and Their Report of Improvement During the Final Survey. (a) Mean improvement in tic frequency from EMA sur- veys compared with Clinical Global Impression–Improvement score for each participant (n = 22). (b) Mean improvement in tic intensity from EMA surveys compared with Clinical Global Impression– Improvement for each participant (n = 22). 13 of 19 22). (b) ression– 13 of 19 22). (b) ression– J. Clin. Med. 2023, 12, 2545 (a) (a) (b) Figure 5. (a) Each participant’s mean improvement in tic frequency following stimulation sessions during this open‐label study was compared with his or her improvement in tic frequency (number of 10‐s tic‐free intervals) in the first 5‐minute block of rhythmic stimulation in the RCT (n = 24). (b) Each participant’s mean improvement in tic intensity following stimulation sessions during open‐ label study was compared with his or her improvement in tic intensity in the first 5‐minute block of rhythmic stimulation in the RCT (n = 25). 3 7 R d A l i Figure 5. (a) Each participant’s mean improvement in tic frequency following stimulation sessions during this open-label study was compared with his or her improvement in tic frequency (number of 10-s tic-free intervals) in the ﬁrst 5-min block of rhythmic stimulation in the RCT (n = 24). 3.4. Daily Survey Results Based on the results of surveys ﬁlled out immediately prior to and following stimula- tion sessions, the mean improvements in tic frequency and intensity were 1.0 (p < 0.001) and 0.9 (p < 0.001), respectively (Table 2). Tic frequency improved, on average, from “3 = PRETTY OFTEN” to “2 = SOMETIMES” (anchor description varied based on the relevant time interval). Mean tic intensity improved from 3 (moderate) to 2 (mild). y p Discomfort during stimulation was low, with a median score of 1 corresponding to “discomfort noticeable, but not severe enough to concern me or to turn it off.” There was no correlation between the stimulation amplitude or discomfort associated with stimulation and tic intensity or frequency (Table 2, Figure 2). However, the range of stimulation ampli- tudes used was limited, with the majority of participants choosing the same amplitude. An exploratory analysis compared discomfort and amplitude; there was a trend for those who used higher amplitude stimulation to experience more discomfort (Figure 2e). 3.2. Outcomes (b) Each participant’s mean improvement in tic intensity following stimulation sessions during open-label study was compared with his or her improvement in tic intensity in the ﬁrst 5-min block of rhythmic stimulation in the RCT (n = 25). (b) (b) (a) Figure 5. (a) Each participant’s mean improvement in tic frequency following stimulation sessions during this open‐label study was compared with his or her improvement in tic frequency (number of 10‐s tic‐free intervals) in the first 5‐minute block of rhythmic stimulation in the RCT (n = 24). (b) Each participant’s mean improvement in tic intensity following stimulation sessions during open‐ label study was compared with his or her improvement in tic intensity in the first 5‐minute block of rhythmic stimulation in the RCT (n = 25). Figure 5. (a) Each participant’s mean improvement in tic frequency following stimulation sessions during this open-label study was compared with his or her improvement in tic frequency (number of 10-s tic-free intervals) in the ﬁrst 5-min block of rhythmic stimulation in the RCT (n = 24). (b) Each participant’s mean improvement in tic intensity following stimulation sessions during open-label study was compared with his or her improvement in tic intensity in the ﬁrst 5-min block of rhythmic stimulation in the RCT (n = 25). 3.7. Responders Anal Pa ti i a t 3.3. Usage Patterns Participants who had a CGI‐I score of 3 (minimally improved) or better were catego‐ rized as responders to stimulation. Examining multiple factors, we did not find any sta‐ tistically significant differences between participants who responded to the stimulation and those who did not (Table 3). Median usage of the device was 1.5 days per week, with an average of 50 min of stimulation per day used (Table 2). However, usage varied widely among participants, with some participants reporting up to daily use many hours per day (Figure 1). Five participants in the study never ﬁlled out a survey indicating that they had turned the device on after the initial study visit at which they were trained on usage of the device and survey response, including one participant who did not ﬁll out any EMA surveys. 3.7. Responders Analysis Participants who had a CGI-I score of 3 (minimally improved) or better were cate- gorized as responders to stimulation. Examining multiple factors, we did not ﬁnd any statistically signiﬁcant differences between participants who responded to the stimulation and those who did not (Table 3). 3.8. Open-ended Comments and Adverse Events Participants were also asked a series of open-ended questions about the device. Full responses can be viewed in ref. [12]. The most common complaints were related to the form of the device. Many people felt that the TENS unit, which had long wires and adhesive pads that many participants removed after each use of the device, was too bulky or cumbersome to deal with on a regular basis. People frequently noted that they only used the device at home due to embarrassment or impracticality and suggested that a smaller design would improve their experience. There were also complaints that the amplitude dial was too sensitive, such that small position changes would produce big changes in the intensity of stimulation experienced. Some noted that correct positioning of the pads and of the wrist was crucial for good effect. g No serious adverse events occurred. In general, comments about discomfort ranged dramatically, from no discomfort to discomfort that heavily outweighed any perceived beneﬁt. Two participants reported skin irritation at the electrode site, possibly contact dermatitis due to the adhesive [28], and two people felt that muscle activation with active stimulation limited the dexterity of their hands. Overall, many people suggested that they planned to use the device, but only for settings in which their tics were particularly bad. On the other hand, one person classiﬁed the device as “a godsend”. 3.6. Final Survey Results In the ﬁnal survey (completed by 27 of 31 participants), a majority of participants (78%) indicated that they planned to continue using the stimulator. Two-thirds (66%) indicated that their symptoms overall were either much improved or minimally improved on the CGI-I rating scale, with only a single participant who indicated their symptoms worsened during the study period (Figure 3). The participants’ responses on the CGI-I scale were correlated with their frequency and intensity EMA scores during the study (r = −0.54 and r = −0.41) (Table 2, Figure 4). There were no signiﬁcant changes in participants’ PUTS or ATQ scores from the beginning of the study period to the end, with mean differences of 0.7 (p = 0.84) and −0.9 (p = 0.85), respectively (Table 2). Participants’ responses in this open-label study did not correlate signiﬁcantly with their results on the rhythmic stimulation day of the RCT: r = 0.21 (p = 0.33) and r = 0.36 (p = 0.08) (Table 2, Figure 5). 3.5. Duration of Improvement in Symptoms after Stimulation Ends Pre-speciﬁed analyses dictated comparing the results of participants’ responses to text surveys sent at random times. Surveys ﬁlled out within 60 min of the end of stimulation were compared with those ﬁlled out more than 60 min following the end of stimulation. There were no signiﬁcant differences in tic frequency or intensity between the two groups, with mean differences of 0.1 (p = 0.20) and 0.3 (p = 0.77), respectively (Table 2). At the conclusion of the study, participants were surveyed on how long they felt beneﬁts lasted after the conclusion of stimulation. The median response was 15 min (IQR 35), so a similar J. Clin. Med. 2023, 12, 2545 14 of 19 14 of 19 analysis to that listed above was conducted comparing responses received within 10 min (the nearest available time point in the survey) to those received after 10 min (Table 2). Again, there were no signiﬁcant differences in tic frequency or intensity between the two groups, with mean differences of 0.4 (p = 0.30) and 0.3 (p = 0.47), respectively (Table 2). However, the sample size for this analysis was quite small (n = 7), as many participants did not have a random text response within 15 min of the end of a stimulation session. Discussion 4. Discussion i u io In this pilot study, median nerve stimulation significantly improved tic frequ nd intensity with relatively low amounts of discomfort. Most participants planne ontinue using the device following the conclusion of the study period. Our result onsistent with the hypothesis that the device has promise as a treatment for TS/CTD In this pilot study, median nerve stimulation signiﬁcantly improved tic frequency and intensity with relatively low amounts of discomfort. Most participants planned to continue using the device following the conclusion of the study period. Our results are consistent with the hypothesis that the device has promise as a treatment for TS/CTD in a real-world setting. yp p eal‐world setting. Most participants did not use the device every day, with median usage of 1.5 d er week and 50 min per day of use. Participants’ open‐ended comments helped to exp his frequency, with many participants stating that the format of the device with wires and adhesive pads made it awkward to wear outside the home. Therefore, m d h h l d h d l d d h Most participants did not use the device every day, with median usage of 1.5 days per week and 50 min per day of use. Participants’ open-ended comments helped to explain this frequency, with many participants stating that the format of the device with long wires and adhesive pads made it awkward to wear outside the home. Therefore, many participants stated that they planned to use the device only during periods where their tics were particularly bothersome or when they especially did not want their tics to be noticed. articipants stated that they planned to use the device only during periods where cs were particularly bothersome or when they especially did not want their tics t oticed. Our study demographics were relatively representative of the general TS popula lthough the mean age was 34.5, while most people with TS who seek clinical care hildren and adolescents [2]. The broad age range for our study was chosen to allow udy of the device in teenagers and adults of a variety of ages, as participants of diffe ges may respond to stimulation differently. Younger children were excluded, as s hildren under age 15 had withdrawn from a previous study of median nerve stimula ue to the sensation associated with stimulation [8]. 3.9. Diurnal Variation of Tics As an exploratory analysis, given the lack of prior EMA studies in TS, we examined diurnal effects on tic severity. Tic frequency and intensity without stimulation were rel- atively constant at different points throughout the day, with little variation between the hours of 9 a.m. and 9 p.m. when most participants supplied data (Figure 6). J. Clin. Med. 2023, 12, 2545 15 of 19 between 15 of 19 between (a) (b) Figure 6. Tic Frequency and Intensity Throughout the Day. (a) Mean tic frequency per hour, w number of participants with results in each hour shown on right axis. Frequency is drawn f surveys filled out before turning on device and random surveys where device was not on. (b) M tic intensity per hour, with number of participants with results in each hour shown on right Intensity is drawn from surveys filled out before turning on device and random surveys w device was not on. Figure 6. Tic Frequency and Intensity Throughout the Day. (a) Mean tic frequency per hour, with number of participants with results in each hour shown on right axis. Frequency is drawn from surveys ﬁlled out before turning on device and random surveys where device was not on. (b) Mean tic intensity per hour, with number of participants with results in each hour shown on right axis. Intensity is drawn from surveys ﬁlled out before turning on device and random surveys where device was not on. (b) ( ) (b) (a) igure 6. Tic Frequency and Intensity Throughout the Day. (a) Mean tic frequency per hour, umber of participants with results in each hour shown on right axis. Frequency is drawn urveys filled out before turning on device and random surveys where device was not on. (b) M c intensity per hour, with number of participants with results in each hour shown on right ntensity is drawn from surveys filled out before turning on device and random surveys w evice was not on. Figure 6. Tic Frequency and Intensity Throughout the Day. (a) Mean tic frequency per hour, with number of participants with results in each hour shown on right axis. Frequency is drawn from surveys ﬁlled out before turning on device and random surveys where device was not on. (b) Mean tic intensity per hour, with number of participants with results in each hour shown on right axis. 3.9. Diurnal Variation of Tics Intensity is drawn from surveys ﬁlled out before turning on device and random surveys where device was not on. Discussion 4. Discussion Therefore, further study is neede p y y p y Our study demographics were relatively representative of the general TS population, although the mean age was 34.5, while most people with TS who seek clinical care are children and adolescents [2]. The broad age range for our study was chosen to allow for study of the device in teenagers and adults of a variety of ages, as participants of different ages may respond to stimulation differently. Younger children were excluded, as some children under age 15 had withdrawn from a previous study of median nerve stimulation due to the sensation associated with stimulation [8]. Therefore, further study is needed in children. However, tic severity in our study was representative of TS patients seeking clinical care, with a median YTGSS total tic score of 25 compared with a mean of 24 in a recent large sample that included clinical trial participants and outpatients at referral centers [29]. y hildren. However, tic severity in our study was representative of TS patients seek inical care, with a median YTGSS total tic score of 25 compared with a mean of 24 As noted above, results for intensity, frequency, and discomfort from surveys through- out the study period were encouraging, with mean improvements of approximately one point in intensity and frequency, and mean discomfort of one on a 0–3 scale. In addition, J. Clin. Med. 2023, 12, 2545 16 of 19 16 of 19 these results are fairly consistent with participant reports on similar questions asked dur- ing the ﬁnal surveys, and there was statistically signiﬁcant correlation between the EMA surveys during the study and the CGI-I score on the ﬁnal survey. This internal consistency is encouraging with regard to the validity of our results. ATQ and PUTS scores did not change from the beginning to the end of the study, which may relate to the fact that most patients used the device only intermittently and reported beneﬁt persisting for, at most, a few minutes after stimulation was turned off. The lack of signiﬁcant correlation between the RCT and open study results was surprising. It is possible that this result arises from differences in participant self-reporting versus the blinded, objective clinical rating that was completed in the RCT. Alternatively, details of ad lib usage chosen by individual participants (e.g., pad placement, left vs. Discussion 4. Discussion right forearm, amplitude) likely differed at times from the methods used in the RCT. We did not identify any characteristics that made a participant more likely to respond to stimulation, including the discomfort they experienced during stimulation or the amplitude they chose. A larger sample may be needed to detect such a difference; alternatively, different predictive factors may need to be examined. The ﬁrst MNS report in TS suggested that the beneﬁts of stimulation might extend past the active stimulation period [8]. We did not detect post-stimulation improvement in our study, comparing tic frequency and intensity at 15 and 60 min after the end of stimulation. However, since random texts only occasionally reached participants shortly after they turned off the device, power to detect such improvement was limited, and this conclusion must be viewed as tentative. To the best of our knowledge, this report also includes the ﬁrst EMA data in a clinical trial in TS. We did not observe variation in tic frequency throughout daytime hours, though the power to detect such effects is limited. More importantly, EMA surveys during the trial period correlated with the CGI-I score at its end, supporting the validity of the EMA survey severity items chosen. However, EMA participation was variable, with a few participants completing no prompted surveys, and many responses were delayed more than a few minutes after the intended time. A more integrated smart phone app may improve response speed or completion rate. 4.3. Clinical Signiﬁcance Wearable wristwatch-style stimulation devices are being developed, which may ad- dress some complaints from patients that the TENS units were cumbersome. Ultimately, a smaller, wearable device would appear to be a valuable treatment option for many patients, including those who have failed more conventional treatments or those who do not prefer pharmacologic or behavioral therapies. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm12072545/s1, Figure S1: CONSORT participant diagram; Table S1: CONSORT 2010 checklist of information to include when reporting non- randomized studies. Author Contributions: Conceptualization, A.M.I. and K.J.B.; software, D.Y.S.; formal analysis, A.M.I.; investigation, A.L.A., D.Y.S. and E.C.B.; data curation, A.L.A. and E.C.B.; writing—original draft preparation, A.M.I.; writing—review and editing, A.L.A., D.Y.S., E.C.B. and K.J.B.; visualization, A.M.I.; supervision, K.J.B.; project administration, A.L.A. and K.J.B.; funding acquisition, K.J.B. All authors have read and agreed to the published version of the manuscript. Funding: Supported by Innovation Funds from the Neuroimaging Laboratories Research Center, Mallinckrodt Institute of Radiology, at Washington University in St. Louis, and by the Washington University Yearlong Research Program Dean’s Fellowship to author A.M.I. REDCap is supported by Clinical and Translational Science Award (CTSA) NIH Grant UL1 TR002345 and Siteman Comprehen- sive Cancer Center and NCI Cancer Center Support NIH Grant P30 CA091842. The content is solely the responsibility of the authors and does not necessarily represent the ofﬁcial views of the funders. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Washington University School of Medicine (approval # 202109160). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. In the case of minor subjects, parents provided written consent and minor subjects provided verbal assent. Data Availability Statement: Individual subject data is available via the Open Science Foundation page for the project in ref. [12]. Data Availability Statement: Individual subject data is available via the Open Science Foundation page for the project in ref. [12]. Acknowledgments: The authors gratefully acknowledge help from Drs. Morera Maiquez and Jackson regarding study design and methods, and from Jonathan M. Koller for programming related to text messaging. A previous version of this manuscript appeared in OSF Preprints [30]. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.1. Limitations As mentioned above, our study is limited by a relatively small sample size. In addition, there were some participants for whom device usage and survey completion were limited, and it is possible that these participants would have had a more negative view of the device. Thus, while we were able to draw interesting conclusions about the utility of such a device, we were less able to deﬁnitively determine results for some of our secondary analyses such as the length of stimulation improvement and which participant characteristics predicted response to stimulation. In addition, our study relied on participant self-reporting of tic frequency and intensity rather than more objective measures such as videotaping and clinician ratings. Although participant results were generally consistent between the EMA responses during the study and the end-of-study surveys, participants’ perception of their own tics may differ from what objective measurements of tics would show. j Finally, although participants were trained on the use of the device at the beginning of the study period, we have no independent way of conﬁrming that the participants used the device as recommended after that point. Repeating this study with a larger sample size would provide more support regarding our primary ﬁndings and more power to detect differences in our secondary analyses. In addition, a newer, less cumbersome device could be tested with a similar study design. If there is interest in further characterizing how long improvement lasts after stimulation, having participants ﬁll out surveys at multiple timepoints after stimulation would provide signiﬁcantly more data. J. Clin. Med. 2023, 12, 2545 17 of 19 17 of 19 4.2. Conclusions Overall, the results of this study are in line with the previously published RCT results from the University of Nottingham [8]. In that open, laboratory-based study, 10 Hz rhythmic MNS stimulation produced notable improvements in tic frequency and intensity in 6 one-minute stimulation blocks. The present results support the real-world utility and acceptability of MNS to patients. Generally, these results regarding efﬁcacy are in line with the results from the RCT in the same participants, but this open-label study identiﬁed additional barriers to daily use of the device in practice. 4.3. Clinical Signiﬁcance The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. 1. Cuenca, J.; Glazebrook, C.; Kendall, T.; Hedderly, T.; Heyman, I.; Jackson, G.; Murphy, T.; Rickards, H.; Robertson, M.; Stern, J.; et al. 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https://openalex.org/W3151174963	https://www.journalpsychiatry.com/jour/article/download/590/492	Russian	null	Endogenous Manic-Paraphrenic States	Psihiatriâ	2,021	cc-by	8,308	Endogenous Manic-Paraphrenic States REVIEW Sizov S.V., Oleichik I.V., Baranov P.A. FSBSI “Mental Health Research Centre”, Moscow, Russian Federation Sizov S.V., Oleichik I.V., Baranov P.A. FSBSI “Mental Health Research Centre”, Moscow, Russian Federation tic and foreign researchers on the clinical and bi ork of endogenous paroxysmal states psychoses. M nic”, “manic-paraphrenic state”, publications avai ebofscience, eLibrary. Conclusion: the following ic states at the present time; 2) evolution of rese the study of the issue; 3) a modern point of view assiﬁ cations of mental disorders; 4) topical issue lusional states; typology; paroxysmal schizop nic-Paraphrenic States. Psychiatry (Moscow) (Psikh 0-101 спространенностью [1–4]. Кроме того, Эндогенные маниакально-парафренные состояния Сизов С.В., Олейчик И.В., Баранов П.А. ФГБНУ «Научный центр психического здоровья», Москва, Российская Федерация Psychiatry (Moscow) 19(1)'2021'90-101 https://doi.org/10.30629/2618-6667-2021-19-1-90-101 УДК 616.89-02, 616.893, 616.895, 616.89-08 Psychiatry (Moscow) 19(1)'20 Abstract Purpose of the work: analysis of changes in the views of domestic and foreign researchers on the clinical and biological features of manic-paraphrenic states developing within the framework of endogenous paroxysmal states psychoses. Material and method: by the keywords “mania, paraphrenia, manic, paraphrenic”, “manic-paraphrenic state”, publications available to authors were selected in the databases MEDLINE/PubMed, Scopus, Webofscience, eLibrary. Conclusion: the following areas of research have been identiﬁ ed: 1) the prevalence of manic-paraphrenic states at the present time; 2) evolution of researchers’ opinions regarding these psychoses throughout the entire period of the study of the issue; 3) a modern point of view on the nosology of manic-paraphrenic states and their place in current classiﬁ cations of mental disorders; 4) topical issues of the pathogenesis of such disorders. Keywords: paraphrenia; manic-paraphrenic states; manic-delusional states; typology; paroxysmal schizophrenia; schizoaffective disorder; bipolar disorder. For citation: Sizov S.V., Oleichik I.V., Baranov P.A. Endogenous Manic-Paraphrenic States. Psychiatry (Moscow) (Psikhiatriya). 2021;19(1):90–101. https://doi.org/10.30629/2618-6667-2021-19-1-90-101 There is no conﬂ ict of interest Резюме Резюме Цель работы: анализ изменения взглядов отечественных и зарубежных исследователей на клинические и биологиче- ские особенности маниакально-парафренных состояний, развивающихся в рамках эндогенных приступообразных психозов. Материал и метод: по ключевым словам «мания, парафрения, manic, paraphrenic», «маниакально-парафренное состояние» отобраны доступные авторам публикации в базах MEDLINE/PubMed, Scopus, Webofscience, eLibrary. Заключение: выделены следующие направления исследований: 1) распространенность маниакально-парафренных состояний в настоящее время; 2) эволюция представлений исследователей в отношении данных психозов на протяжении всего периода изучения вопро- са; 3) современная точка зрения на нозологическую принадлежность маниакально-парафренных состояний и их место в классификациях психических расстройств, используемых в настоящее время; 4) актуальные вопросы патогенеза подобных расстройств. р р Ключевые слова: парафрения; маниакально-парафренные состояния; маниакально-бредовые состояния; типология; приступообразная шизофрения; шизоаффективное расстройство; биполярное аффективное рас- стройство. Для цитирования: Сизов С.В., Олейчик И.В. Баранов П.А. Эндогенные маниакально-парафренные состояния. Психиатрия. 2021;19(1):90–101. https://doi.org/10.30629/2618-6667-2021-19-1-90-101 Конфликт интересов отсутствует КЛИНИКО-ПСИХОПАТОЛОГИЧЕСКАЯ ОЦЕНКА МАНИАКАЛЬНО-ПАРАФРЕННЫХ СОСТОЯНИЙ, ПРОБЛЕМА ДЕФИНИЦИИ И НОЗОЛОГИЧЕСКОЙ КВАЛИФИКАЦИИ «острой паранойи» характер аффекта определяет фа- булу бредовых построений. В то же время комплекс- ный анализ взаимовлияния аффективной и бредовой составляющей маниакально-бредового синдрома ав- тором не проводился. Первые упоминания о маниакальных психотических состояниях появились еще в древней Греции. Описа- ния Аретея Каппадокийского соответствовали в со- временном понимании психозам аффективного круга и острым бредовым психозам с помрачением созна- ния. На рубеже XVII–XVIII веков сам термин «мания» в некоторых исследованиях и вовсе использовался для обозначения большинства бредовых психозов и объе- динялся с другими расстройствами в группу паранойи. С середины XVIII века сформировалось несколько ос- новных направлений в изучении проблемы маниакаль- но-бредовых психозов в рамках различных научных школ, что послужило значительным толчком к после- дующим многочисленным исследованиям в этом на- правлении. В это время ряд исследователей (Pinel P., 1797; Vogel Z., 1799) сделали акцент в своих работах на критериях прогноза и динамике маниакально-бре- довых состояний, при этом отошли от статичного под- хода, характерного для французской психиатрической школы. Было установлено, что мании имеют опреде- ленную характерную последовательность своего раз- вития (Heinroth J., 1818; Giuislain G., 1838) и маниа- кально-бредовые состояния являются одной из стадий последнего. H. Dagonet (1872) выделил интермитти- рующий вариант течения мегаломанического бреда, при котором кроме периодически появляющихся идей величия также наблюдался выраженный маниакаль- ный аффект. Более детально проблема аффективной формы «острой паранойи» изучалась W. Griesinger (1881) и P. Pol (1889). Этими авторами был введен термин «горделивое помешательство», под которым подра- зумевались маниакальные состояния с «переоценкой собственного достоинства», а также возбуждением, сопровождаемым «горделивыми представлениями». Таким образом, впервые была описана клиническая картина маниакальных состояний с идеями величия и завышенной самооценки, которая не потеряла своей актуальности до настоящего времени. 91 Развивая учение W. Griesinger, R. Kraft-Ebing (1889) выделил маниакальные состояния, значительно отли- чающиеся от типичных маний: резонирующую манию, манию с «болезненной извращенностью нравствен- ности» и манию с раздражительностью. В работах W. Weygandt (1889, 1901), ученика R. Kraft-Ebing, была предложена оригинальная типология маниакальных состояний, а также впервые проанализирована вза- имосвязь их психопатологической структуры с пре- морбидными особенностями личности пациентов; при этом оценка нозологической принадлежности данных психозов не проводилась. Полемизируя с L. Kirn (1878), который считал, что течение циркулярного психоза не- избежно влечет за собой изменения личности и разви- тие слабоумия, W. Weygandt высказывал мнение, что мании могут чередоваться с «чистыми», свободными от болезненных проявлений интервалами и не приводить к деменции. Эта дискуссия фактически отражала раз- личие существовавших в то время точек зрения в от- ношении течения и прогноза циркулярных психозов. ВВЕДЕНИЕ распространенностью [1–4]. Кроме того, особая значимость исследования клинико-психопато- логической структуры данных вариантов эндо- генных психозов обусловлена необходимостью их своевременной и правильной диагностики, а также определения прогноза и адекватной те- рапевтической тактики [2, 3]. Исследование эндогенных психозов, проте- кающих с картиной маниакально-парафренных состояний в приступах, остается одной из наи- более актуальных задач клинической психиа- трии, что, прежде всего, связано с их высокой МИА’ ISSN1683-8319 (print)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 Psychiatry (Moscow) 19(1)'2021'90-101 до наследственной отягощенности и прогностических предикторов. ближе всего к маниакально-парафренным состояниям были экспансивная и фантастическая формы. Одним из характерных признаков парафрении E. Kraepelin считал измененность восприятия своего психическо- го «Я». В последующих работах он допускал развитие в рамках маниакально-депрессивного психоза (МДП) исключительно экспансивной парафрении. р р В конце XIX века и в российской психиатрии стали появляться работы, посвященные изучению маниа- кальных состояний. При этом большое внимание уде- лялось тщательному изучению психопатологической структуры данных состояний, рассматривавшихся в рамках синдромального подхода. Особого внимания заслуживает работа С.С. Корсакова (1893), который при описании так называемых маниакальных везаний ука- зывал на то, что с типичными симптомами мании могут сочетаться бредовые идеи величия, преследования, эротический бред и признаки помрачения сознания. Важное значение имели также работы А.Ф. Эрлицкого (1896) и В.П. Сербского (1905, 1906), отличительной чертой которых был детальный анализ психопатологии психотических маний, а также исследование особен- ностей патокинеза симптомов в течении заболевания и взаимосвязи структуры приступов с характером на- следственной отягощенности. Работы этих авторов свидетельствуют о том, что изучение маниакальных состояний в отечественной психиатрии шло не только в рамках синдромологических или психопатологиче- ских аспектов, но и в направлении их нозологического разграничения. Важную роль в проблеме изучения психопатологии маниакально-парафренных состояний сыграли работы E. Dupre и B. Logre (1911), описавших бред воображе- ния, основанный на интуиции и выдумке, и идеи ве- личия, для которых была характерна отчетливая зави- симость от полюса аффекта, а также течение в форме периодически возникающих приступов. В дальнейшем вопрос определения нозологической принадлежности маниакальных психозов решался не- однозначно. Ряд исследователей в рамках концепции атипичных смешанных психозов допускали возмож- ность сочетания признаков раннего слабоумия и МДП одновременно (Halberstadt G., 1924; Осипов В.П., 1931). Французские авторы по-прежнему отстаивали син- дромологический подход к классификации психозов (Claud H., 1924; Rouar J., 1938), а некоторые психиатры придерживались точки зрения о необходимости выде- ления периодических психозов в отдельную нозологи- ческую форму (Meduna L., 1935). В дальнейшем вопрос определения нозологической принадлежности маниакальных психозов решался не- однозначно. Ряд исследователей в рамках концепции атипичных смешанных психозов допускали возмож- ность сочетания признаков раннего слабоумия и МДП одновременно (Halberstadt G., 1924; Осипов В.П., 1931). Французские авторы по-прежнему отстаивали син- дромологический подход к классификации психозов (Claud H., 1924; Rouar J., 1938), а некоторые психиатры придерживались точки зрения о необходимости выде- ления периодических психозов в отдельную нозологи- ческую форму (Meduna L., 1935). В эти же годы В.М. Бехтерев (1899) изучал па- рафренные состояния в рамках периодической формы острой паранойи. КЛИНИКО-ПСИХОПАТОЛОГИЧЕСКАЯ ОЦЕНКА МАНИАКАЛЬНО-ПАРАФРЕННЫХ СОСТОЯНИЙ, ПРОБЛЕМА ДЕФИНИЦИИ И НОЗОЛОГИЧЕСКОЙ КВАЛИФИКАЦИИ Научные обзоры фф До середины XIX века маниакально-бредовые пси- хозы не имели четкой нозологической квалификации. J. Esquirol (1838), один из основателей клинико-но- зологического подхода в психиатрии, создал первую типологию маниакальных состояний и описал динами- ку их развития, при этом указав, что вслед за маниа- кальной экзальтацией в клинической картине болезни могут возникать бредовые идеи, галлюцинации, а так- же развиваться «неистовое возбуждение». W. Sander (1868) выделил особую форму бредовых расстройств, где бред величия, высокого происхождения, бессмер- тия наблюдался практически одновременно с идеями преследования. Хотя автор подчеркивал в основном длительное и непрерывное течение болезни и исход в слабоумие, он тем не менее отмечал, что в некоторых случаях заболевание может протекать относительно благоприятно. K. Westphal (1878) ввел термин «острая паранойя». Это состояние развивалось в виде отдель- ных приступов, сменявшихся интервалами времени, свободными от какой-либо симптоматики либо содер- жавшими ее «остаточные» проявления. Различали про- стую, галлюцинаторную и аффективную форму острой паранойи. Указывалось на явную взаимосвязь аффек- тивной составляющей психоза с бредом и галлюцина- циями. K. Westphal считал, что при аффективной форме р ц р у р На рубеже XIX–ХХ веков активное изучение маниа- кальных состояний проводилось последователями обе- их школ. Однако в немецкой школе (Shule H., 1880) ис- следование психозов осуществлялось преимуществен- но с применением синдромологического метода, в то время как во французской — клинического (Magnan V., 1878). H. Shule в противоположность R. Krafft-Ebing заявлял о необходимости разграничения циркуляр- ного и периодического помешательства, он выступал против нозологической оценки психозов в качестве мании и меланхолии, отводя данным определениям лишь синдромальную роль. Кроме того, автор, раз- вивая учение W. Westfall, выдвинул дифференциаль- но-диагностические критерии острой и хронической паранойи, а также высказал мнение о взаимосвязи пси- хопатологической структуры синдрома с характером аффекта, особенностями течения заболевания и ва- риантами его исхода. V. Magnan, являясь сторонником динамического подхода, в противоположность H. Shule призывал не ограничиваться лишь синдромологиче- ским изучением маний, а рассматривать весь спектр проявлений заболевания: от этиологических факторов MIA’ ISSN2618-6667 (online)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 Psychiatry (Moscow) 19(1)'2021'90-101 Среди них, по описанию, МИА’ ISSN1683-8319 (print)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 Важным этапом развития учения о парафрениях стало окончательное разделение парафренного син- дрома на хроническую и острую формы. Системати- ческая парафрения E. Kraepelin выделялась в картине непрерывно текущей параноидной шизофрении [5, 8], а острая парафрения рассматривалась в рамках при- ступообразной (циркулярной онейроидно-кататониче- ской) формы эндогенного процесса (Снежневский А.В., 1969; Вербальская Л.М., 1964). В.Н. Фаворина (1956) и А.С. Тиганов (1965) впервые описали острый па- рафренный синдром в рамках рекуррентной формы течения шизофрении. В то же время Г.И. Зальцман (1967), Б.В. Соколова (1972), К.А. Адильханова (1971), В.А. Концевой (1973) рассматривали острый парафрен- ный синдром в рамках приступообразно-прогредиент- ного эндогенного процесса. аффекта расстройствами — бредом воздействия и психическими автоматизмами. Отдельной точки зрения придерживался W. Mayer-Gross (1932), кото- рый не признавал существование атипичных психо- зов и квалифицировал маниакально-парафренные состояния как вариант шизофрении, который в своей клинической картине сочетал проявления аффек- тивной и собственно процессуальной симптоматики. В последующем W. Mayer-Gross (1960), выделяя пер- вичные бредовые переживания, считал важными бре- дообразующими факторами парафренного синдрома осознание больными особого значения собственной измененности и бредовое отношение к оценке своего психического «Я». Большинством исследователей середины ХХ века парафрения расценивалась как синдром в структуре острой либо хронической паранойи, шизофрении, МДП, а также шизоаффективных состояний [5–8]. Н.Г. Шумский (1958), в своей работе «К клинике па- рафренной (фантастически-параноидной) шизофре- нии» наряду с хроническими формами этого заболе- вания описал и его острую форму — периодическую бредовую шизофрению. Автор отмечал, что характер- ной особенностью заболевания, помимо периодически возникающего фантастического бреда, следует счи- тать наличие выраженных аффективных расстройств. В картине приступов описывается образный бред фантастического содержания, а также преобладание феномена ложных узнаваний и проявлений бреда интерметаморфозы. В работах Н.Г. Шумского (1959), В.Ф. Землич (1962) и Э.С. Петровой (1967) приведены подробные описания фантастическо-параноидной, конфабуляторной и галлюцинаторной форм парафрен- ного синдрома. Н.Г. Шумский (1958), в своей работе «К клинике па- рафренной (фантастически-параноидной) шизофре- нии» наряду с хроническими формами этого заболе- вания описал и его острую форму — периодическую бредовую шизофрению. Автор отмечал, что характер- ной особенностью заболевания, помимо периодически возникающего фантастического бреда, следует счи- тать наличие выраженных аффективных расстройств. В картине приступов описывается образный бред фантастического содержания, а также преобладание феномена ложных узнаваний и проявлений бреда интерметаморфозы. В работах Н.Г. Шумского (1959), В.Ф. Землич (1962) и Э.С. Петровой (1967) приведены подробные описания фантастическо-параноидной, конфабуляторной и галлюцинаторной форм парафрен- ного синдрома. Анализ проведенных нозологических исследова- ний парафрении позволил авторам создать система- тику различных ее вариантов. Ю.А. Psychiatry (Moscow) 19(1)'2021'90-101 Им были подробно описаны бре- довые идеи превосходства и особых способностей, реформаторства и мирового могущества, которые со- четались с идеями преследования, ущерба и манихей- ским бредом. Изучением бреда величия при маниях в рамках острой паранойи занимался и П.Б. Ганнушкин (1904), который выделял особую взаимосвязь бреда величия с нарушением восприятия собственного «Я». Размах бреда и искажение образа «Я» могли варьиро- вать от приземленной, бытовой фабулы до фантасти- чески-мегаломанической. Начало и середина XX века характеризовались созданием разнообразных клинических типологий маниакальных состояний: выделялись мании с бре- дом, галлюцинациями, психопатоподобными рас- стройствами (Forster O., 1908; Thalbitzer S., 1910; Rittershaus E., 1923). K. Shreder (1918, 1920, 1926) подробно описал атипичные случаи МДП, в которые включал аффективные приступы с бредом, галлюци- нозом, кататоническим возбуждением и спутанно- стью. N. Skliar (1924) подчеркивал, что обязательным условием развития парафрении должно быть нали- чие в клинической картине приступа аффективных расстройств: мании или депрессии; при этом данное состояние может сопровождаться кататонией и не- глубокой деменцией. K. Kolle (1931), напротив, стре- мился связать структуру парафрений с возрастом, считая, что в пубертатном периоде они протекают, сочетаясь с кататонической симптоматикой, а в позд- нем — наиболее часто возникают в «чистом» виде. Некоторые авторы заявляли о том, что парафрения протекает преимущественно в рамках МДП с атипич- ным течением (Bowman K., Raumond A., 1931 и др.). В то же время O. Bumke (1924) считал, что острый ва- риант парафрении характерен для симптоматических психозов, преимущественно органического генеза. A. Bostroem (1920) выделил «острый экспансивный аутопсихоз» с внезапным появлением бредовых идей величия, переустройства мира, могущества, спо- собностей к экстрасенсорике, которые развивались на фоне маниакального аффекта. Автором было ука- зано на случаи сочетания экспансивных идей с «не- специфичными», неконгруентными для маниакального Таким образом, аффективные психозы и, в част- ности, маниакальные состояния, расценивались в исследованиях начала XX века в качестве «острой паранойи», «аменции» и недифференцированных «острых психотических состояний». В ряде работ этого времени поднимался вопрос о необходимости установления взаимосвязи аффективной и бредо- вой составляющей синдрома, однако из-за крайней неоднородности в описании и квалификации выше- описанных психозов данная проблема оставалась далекой от разрешения. Наиболее четкую в то время позицию по вопро- су дифференциации различных форм периодических психозов выразил E. Kraepelin (1906). Он впервые применил такие термины, как «периодическое поме- шательство» и «бредовое помешательство». В 1912 г. E. Kraepelin выделил парафрению в качестве самосто- ятельной нозологической формы и описал четыре ее варианта: систематическую, экспансивную, конфабу- ляторную и фантастическую. СОВРЕМЕННОЕ ОСВЕЩЕНИЕ ПРОБЛЕМЫ МАНИАКАЛЬНО- ПАРАФРЕННЫХ СОСТОЯНИЙ приступообразно-прогредиентной шизофрении. Ав- тор выделила пять типов парафренных синдромов, среди которых наиболее близка к маниакально-па- рафренному состоянию была экспансивная парафре- ния. В данной работе подробно описана динамика развития парафренного синдрома, а также проведен анализ особенностей бреда, характера расстройств сознания и структуры маниакального аффекта. В.А. Концевой (1973) выделял полиморфные и моно- морфные типы острых парафренных приступов. Пер- вые сочетали в себе как галлюцинаторно-бредовые, так и аффективные расстройства. При вторых, напро- тив, доминировали бредовые расстройства и отсут- ствовала аффективная составляющая. В 1999 г. группой исследователей (P. Appelbaum с соавт.) были изучены различные типы бреда на ма- териале 1136 больных. У значительного количества пациентов был диагностирован бред величия, более распространенным оказался только бред преследова- ния. При этом установлено, что бред величия характе- ризовался практически полным отсутствием критики к состоянию и тем не менее не был связан с развити- ем процессуальных негативных расстройств. Автора- ми был установлен парадокс: несмотря на то что па- циентам с бредом величия присуща высокая степень убежденности в правильности своих суждений, они практически не демонстрировали бредового поведе- ния, в отличие от больных с другими типами бреда. В этой работе впервые упоминалось о необходимости исследований по выявлению биологических марке- ров бреда величия с целью достижения более высо- кого уровня достоверности диагностики. Кроме того, в данной работе было показано, что бред величия чаще возникает в рамках течения биполярного аффектив- ного расстройства (БАР) (59%), чем при шизофрении (49%). Распространенность бреда величия изучалась и J. Junginger с соавт. (1992), которые также устано- вили более высокую частоту его возникновения при аффективных расстройствах, чем при шизофрении. К концу ХХ века наметилась тенденция к сокраще- нию числа как отечественных, так и зарубежных работ, касающихся маниакально-парафренных и маниакаль- но-бредовых состояний. Имели место лишь отдельные, преимущественно отечественные, публикации по дан- ной теме, которые приводятся ниже. р р В частности, Т.П. Платоновой (1984) были описа- ны парафренные состояния с острым чувственным бредом, острые псевдогаллюцинаторные и острые конфабуляторные парафрении, а также парафрении с явлениями психического автоматизма. В.И. Дикая (1985) в рамках разработки типологии синдрома Кандинского–Клерамбо выделила вариант с уча- стием маниакального аффекта и переходом психоза в состояние парафрении. С.Ю. Циркин (1980) провел дифференциацию синдрома Кандинского–Клерам- бо, основываясь на особенностях составляющих его структуру бредовых расстройств. Синдром Кандин- ского, протекающий с острым образным (фантастиче- ски-парафренным) бредом, отличался преобладанием воображения над «бредовым восприятием» (по тер- минологии К. Jaspers, 1923), господством «бредовых представлений» и «бредовых осознанностей». По мнению С.Ю. Psychiatry (Moscow) 19(1)'2021'90-101 Антропов (1964), развивая учение О. Bumke, выделял систематическую парафрению как отдельную нозологическую форму. Сторонником подобного подхода был также Б.Я. Пер- вомайский (1958), который описал самостоятельный тип парафрении — гипофрению. По мнению автора, ей свойственно постепенное начало, развитие малоси- стематизированных идей величия и наличие критики. Одним из минусов работ нозологического направления в изучении маниакально-парафренных состояний яв- ляется тот факт, что в них сравнительно мало внимания уделялось анализу психопатологической структуры синдрома; основной акцент делался на разграничении симптомов непрерывного и приступообразного тече- ния в картине психоза. Научные обзоры А.В. Снежневский (1969) описал сочетание идей величия с психическими автоматизмами на фоне аф- фективной симптоматики. А.С. Тиганов в серии ра- бот (1965, 1968, 1969) выделил три группы острых парафрений: с бредом восприятия K. Jaspers, гал- люцинаторную и конфабуляторную, рассматривая их в качестве бредовой составляющей маниакаль- но-парафренного синдрома. А.С. Тигановым в ходе дальнейших исследований была создана типология психотических маний, к которым автор отнес бредо- вую манию, манию с острым чувственным и острым фантастическим бредом, а также манию с онейро- идно-кататоническими расстройствами. Т.Ф. Папа- допулосом (1975) была разработана оригинальная классификация приступов эндогенных заболеваний, основывающаяся как на механизме бредообразо- вания, так и на характеристике тяжести бредовых расстройств. В рамках этапа одного из вариантов приступов при приступообразно-прогредиентной шизофрении автор выделил парафрению. При ре- куррентной шизофрении он расценивал острую па- рафрению как этап развития онейроидно-кататони- ческого приступа. Особое значение в истории изучения маниакаль- но-парафренных состояний имеют работы школы K. Kleist — K. Leonhard (1964, 1968). Исследова- тели полагали, что кроме шизофрении и МДП су- ществует «третья болезнь» — группа циклоидных психозов, занимающих промежуточное место меж- ду двумя основными эндогенными заболеваниями. Авторы выделили три группы эндогенных психозов (фазные психозы, циклоидные психозы и несистем- ную шизофрению) и предполагали, что фазные и ци- клоидные психозы проходят бесследно, в то время как несистемная шизофрения приводит к дефекту в структуре личности. Среди циклоидных психозов К. Leonhard (1968) выделял психозы страха–счастья (Angst–Gluck–Psychosen), психозы, протекающие со спутанностью (Verwirrtheitspsychosen), и психозы с гиперкинезами или акинетическими состояниями (Motilitatpsychosen). Наиболее близкими к мани- акально-парафренным состояниям по клинической картине оказывались «психозы счастья», которые проявлялись психотическим аффектом, экстазом, иде- ями величия и частым развитием галлюцинаторной симптоматики. Б.В. Соколовой (1971) был проведен под- робный анализ психопатологической структу- ры острых парафренных состояний в рамках Б.В. Соколовой (1971) был проведен под- робный анализ психопатологической структу- ры острых парафренных состояний в рамках MIA’ ISSN2618-6667 (online)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 СОВРЕМЕННОЕ ОСВЕЩЕНИЕ ПРОБЛЕМЫ МАНИАКАЛЬНО- ПАРАФРЕННЫХ СОСТОЯНИЙ При этом подчеркивается, что особенностью данно- го состояния оказывается отсутствие наследствен- ной отягощенности и отчетливого доманифестного периода, какой-либо взаимосвязи с преморбидной стуктурой личности, а также слабая выраженность негативных симптомов и расстройств мышления в со- четании с очень медленным прогрессированием болез- ни [13]. Рядом исследователей было установлено, что «шизофреноформный психоз с очень поздним нача- лом» часто ассоциирован с органическими заболева- ниями, такими как опухоли мозга, острое нарушение мозгового кровообращения и нейродегенеративные процессы [14]. Многие авторы связывают развитие поздней парафрении с наличием нейрофибриллярных клубочков (NFT) в основном в пределах энторинальной коры [10, 15]. состояний до настоящего времени остается неопре- деленной. В последнее время продолжаются дискуссии отно- сительно возможности применения оценочных диагно- стических шкал при маниакальных и маниакально-бре- довых состояниях. Так, ряд зарубежных авторов [21, 22] ссылается на достаточную валидность применения шкалы мании The Bech-Rafaelsen Mania Scale (MAS) на- ряду с популярной в отечественной психиатрии шка- лой мании Янга (YMRS). Авторы указывают на схожую диагностическую ценность шкалы MAS при меньших временных затратах. Тем не менее в настоящее вре- мя данная шкала практически не используется. Еще одна шкала для оценки мании разработана Альтманом в Университете Иллинойса для определения наличия и/или выраженности маниакальных (гипоманиакаль- ных) симптомов (Altman self-rating mania scale). Она состоит из пяти пунктов, основанных на диагностиче- ских критериях мании согласно классификации DSM-IV. Пункты шкалы оценивают психическое состояние боль- ного на основе изменений в настроении, самооценке, потребности в сне, речи и активности. Шкала Альтма- на имеет высокую степень корреляции с оценочными шкалами, основанными на клиническом интервью, и, в частности, шкалой мании Янга. Следует отметить тот факт, что в исследованиях последнего десятилетия в области психиатрии наме- тилась тенденция к замене синдромального клиниче- ского подхода на узкосимптоматический, направлен- ный лишь на оценку отдельных психопатологических дефиниций [23]. В рамках данного направления были разработаны когнитивные модели, обычно ассоцииро- ванные с психопатологическим содержанием психоза, например слуховыми галлюцинациями [23–25] и бре- дом преследования [23, 26]. Также рассматривались общие механизмы развития позитивных симптомов психоза [27, 28]. При этом бред величия остается не- достаточно изученным и с вышеописанных позиций, которые, впрочем, близки скорее к патопсихологиче- ским, нежели психопатологическим. р [ ] При анализе современного состояния проблемы ма- ниакально-парафренных состояний следует отметить, что в зарубежной литературе активно ведется дискус- сия о правомерности применения при психотических маниях диагноза БАР и шизоаффективного расстрой- ства/психоза (ШАП) [16, 17]. СОВРЕМЕННОЕ ОСВЕЩЕНИЕ ПРОБЛЕМЫ МАНИАКАЛЬНО- ПАРАФРЕННЫХ СОСТОЯНИЙ Циркина, образный бред соответствует бреду воображения французских авторов (Dupre E., Logre B., 1911, 1925), а также острому образному бре- ду, выделенному Г.П. Киндрас (1974). В соответствии с наблюдениями автора, на высоте состояния в фабу- ле острого образного бреда идеи воздействия начина- ли переплетаться с бредом величия. М.В. Варавикова (1994) описывала маниакальные состояния с бредом воображения. Среди общих признаков бреда вообра- жения в ее работе были отмечены следующие особен- ности: 1) внезапность возникновения основной фабу- лы, ее изменчивость; 2) преобладание в тематике бре- да идей предвидения, предсказания, особой миссии, любовного притязания; 3) наличие патологических ощущений по типу «прилива энергии»; 4) относитель- ная упорядоченность поведения и удовлетворитель- ная социальная адаптация больных. Е.Ш. Гибадулин (1993), изучая мании юношеского возраста, описывал варианты подобных психозов с бредовыми идеями ве- личия, особого предназначения, мессианства, а также преследования и отравления. фф р р р фр Н.В. Субботская (2005) разработала типологию острых парафренных состояний на основании раз- личия механизмов бредообразования. Путем деталь- ного психопатологического исследования автором были описаны три типологических варианта острой парафрении: с доминированием чувственного бреда, с преобладанием проявлений синдрома Кандинского– Клерамбо и с высоким удельным весом в клинической картине психоза конфабуляторных расстройств. В то же время взаимосвязь особенностей структуры бре- довых расстройств с характеристиками аффективной симптоматики в картине синдрома в работе подробно не исследовалась. А.В. Соколов (2012) как одну из разновидностей маниакально-бредовых состояний с картиной бреда преувеличенной самооценки описал парафренный вариант, который развивался в тесной взаимосвязи с маниакальным аффектом и формиро- вался либо по закономерностям стереотипа разви- тия хронического бреда V. Magnan, либо с участием острого чувственного бреда воображения. На оформ- ление психопатологической картины этих вариантов маниакально-парафренного состояний оказывали влияние особенности маниакального аффекта, кото- рые обнаруживали корреляцию как с характеристи- ками бредообразования, так и с динамикой бредовых расстройств. За последние годы в доступной нам зарубежной научной литературе представлены лишь отдельные публикации, посвященные клинике и психопатологии МИА’ ISSN1683-8319 (print)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 маниакально-бредовых и маниакально-парафренных состояний, их нозологической и прогностической оценке, а также подходам к терапии данных психозов. При этом многие зарубежные исследователи считают, что парафренные психозы могут возникать исключи- тельно в позднем возрасте, и их развитие неотъемлемо связано с когнитивными нарушениями, приводящими к деменции [10]. Некоторые авторы даже полагают, что парафренные состояния являются продромом демен- ции [11, 12]. Кроме того, в зарубежной литературе для обозначения парафрении используется термин «ши- зофреноформный психоз с очень поздним началом». Psychiatry (Moscow) 19(1)'2021'90-101 и нормотимика — в 3,5 раза по сравнению с моноте- рапией нормотимиками [40]. Кроме того, указывается, что антипсихотики являются небезопасными препара- тами, приводящими к увеличению смертности пациен- тов в 1,5–3 раза [42]. Следует отметить и способность нейролептиков индуцировать депрессии, отмеченную еще Г.Я. Авруцким (1988) на примере хлорпромази- на и М.Ю. Поповым (2012) на примере галоперидола. Существует точка зрения, согласно которой депрессо- генное влияние антипсихотиков, в том числе атипич- ных, обусловлено их влиянием на дофаминергическую трансмиссию [43–45]. Не меньшее внимание уделяется и вопросу индуцирования нейролептиками псевдоде- фицитарных симптомов у различных групп пациентов, при этом отмечается значительное сходство клиниче- ской картины негативных расстройств при шизофре- нии и депрессивной симптоматики, вызванной приме- нением антипсихотиков [46]. В то же время существует точка зрения [47], согласно которой антипсихотики второго поколения за счет меньшего ингибирования D2-рецепторов и сродства к некоторым другим ней- ротрансмиттерным системам обладают значительно меньшей способностью индуцировать когнитивные и депрессивные расстройства, а, возможно, могут и ча- стично редуцировать последние. Альтернативным ме- тодом купирования маниакальных состояний является применение нормотимиков. Так, изучение результатов рандомизированного исследования C. Bowden c соавт. (1994) показало, что и литий, и вальпроаты были эф- фективны в лечении психотической мании (более, чем плацебо). Эти данные нашли свое отражение в раз- работке терапевтических схем, например достаточно широко известного «Техасского алгоритма» [48]. время появляется все больше работ, свидетельствую- щих о нарастающем разочаровании в сугубо димен- сиональном подходе [32]. Все большее число исследо- вателей говорит о необходимости возврата к феноме- нологическому методу и категориальной диагностике [33, 34]. Значительный вклад в исследование взаимосвязи между аффективными и другими психопатологически- ми расстройствами в структуре маниакального синдро- ма внесли A. Gonzales-Pinto с соавт. (2003), которые установили три основных симптома маний: гедонизм, гиперактивность и дисфорию с агрессией, раздражи- тельностью и недостатком критики. Авторами была выявлена корреляция этих трех симптомов с расстрой- ствами психотического спектра: галлюцинациями, бре- довой симптоматикой и нарушениями мышления. N. Smith с соавт. (2005) изучали вопрос о том, явля- ется ли бред величия одним из механизмов психологи- ческой защиты от низкой самооценки в преморбидной структуре личности или прямым отражением эмоций и связанных с ними процессов. В своем исследова- нии авторы установили, что бред величия, хотя и ос- новывается на завышенной личностной самооценке у исследуемых, но возникает исключительно на фоне повышенного настроения с эйфорией во время мани- акального психоза. Авторы работы даже полагали, что у людей с бредом величия характерное для них нега- тивное отношение по отношению к окружающим мо- жет способствовать повышению социального статуса. НЕЙРОБИОЛОГИЧЕСКИЕ ОСОБЕННОСТИ МАНИАКАЛЬНО-ПАРАФРЕННЫХ СОСТОЯНИЙ До настоящего времени помимо психопатологиче- ских особенностей маниакально-бредовых и маниа- кально-парафренных состояний остаются недостаточ- но изученными как их нейробиологические (нейрофи- зиологические, иммунологические, биохимические), так и патопсихологические аспекты. Несмотря на на- блюдающийся в последние годы акцент на биологи- ческих исследованиях в отечественной и зарубежной психиатрии [49–54], комплексного изучения нейробио- логических, биохимических, иммунологических и па- топсихологических особенностей маниакально-бредо- вых и маниакально-парафренных состояний до настоя- щего времени практически не проводилось. Psychiatry (Moscow) 19(1)'2021'90-101 Кроме того, по мнению исследователей, в настоящее время актуальным представляется обнаружение опре- деленных личностных предикторов в развитии бреда величия. Существует также точка зрения, что бред величия может играть определенную роль в развитии персекуторного бреда [10, 35–37]. P. Conus и S. Cotton с соавт. (2010) провели анализ исхода психических болезней, протекающих с картиной маниакально-бре- довых психозов. Исследование выявило негативный прогноз заболевания при развитии бредовой мании в манифестной фазе, что согласуется с более ранними работами [38]. СОВРЕМЕННОЕ ОСВЕЩЕНИЕ ПРОБЛЕМЫ МАНИАКАЛЬНО- ПАРАФРЕННЫХ СОСТОЯНИЙ При этом большинство авторов относят маниакально-бредовые и маниакаль- но-парафренные состояния к расстройствам аффек- тивного регистра, квалифицируя их, таким образом, в рамках БАР даже при наличии в клинической картине психоза симптомов первого ранга, по K. Schneider [18]. С другой стороны, W. Maier с соавт. (1992) установили, что пациенты с ШАП, в отличие от больных, страдаю- щих БАР, имеют четкую наследственную отягощенность (у родственников таких больных часто диагностиро- валась шизофрения) [19]. Исследования когнитивного функционирования показали, что у больных ШАП на- рушения интеллектуальных и поведенческих функций, отсутствующие у пациентов с БАР, проявляются уже на доманифестном этапе заболевания. При сравнении пациентов с ШАП и БАР в периоде эутимии [20] у па- циентов, страдающих ШАП, был выявлен когнитивный дефицит, который не отмечался у больных БАР, причем показатели последних были сопоставимы с контроль- ной группой здоровых исследуемых. В то же время при изучении клинических аспектов данных расстройств [19] было установлено, что у пациентов с ШАП не обна- руживалось явных негативных симптомов, свойствен- ных больным шизофренией. Таким образом, нозоло- гическая квалификация маниакально-парафренных Научные обзоры В течение последних десятилетий типологическая дифференциация маниакальных состояний основыва- ется преимущественно на квалификации выявляемых в их структуре бредовых расстройств, оцениваемых в качестве конгруентных либо неконгруентных аф- фекту [29–31]. При этом данное разделение никак не увязывается с нозологической оценкой психоза и его прогнозом. В современных классификациях психи- ческих расстройств (МКБ-10, DSM-5) данные психозы рассматриваются исключительно в рамках БАР. Более того, большинство зарубежных авторов отмечает, что при нозологической оценке маниакально-бредовых психозов имеет место гипердиагностика шизофрении [30]. Следует отметить, что, несмотря на наблюдающийся в конце XX и начала XXI века тренд в психиатрической науке в сторону отказа от традиционного клинико-пси- хопатологического метода исследования, в последнее MIA’ ISSN2618-6667 (online)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 Психиатрия 19(1)'2021'90-101 Помимо вышеизложенного, данные литературы [60] свидетельствуют о повышении у больных шизофренией и аффективным заболеванием риска развития веноз- ных и артериальных тромбозов. Предполагают, что уве- личение такого риска связано с развитием у больных ожирения висцерального типа, метаболического син- дрома, системного воспаления или нейровоспаления [58], а также с длительной терапией как традиционны- ми, так и атипичными антипсихотическими средствами [59, 60]. Кроме того, есть основания предполагать, что увеличение частоты тромбообразования у больных эн- догенными психическими заболеваниями может быть обусловлено повышенной свертываемостью плазмы крови при шизофрении и аффективных психозах. Со- гласно последним данным О.С. Брусова с соавт. (2018, 2019), у больных с эндогенными маниакально-бредо- выми психозами наблюдается повышенная сверты- ваемость плазмы крови (гиперкоагуляция), которая нормализуется на фоне психофармакотерапии. Полу- ченные данные свидетельствуют о том, что у больных шизофрений и в значительно меньше степени у боль- ных аффективным заболеванием в стадии обострения присутствует повышенный риск тромботических со- бытий. Эти патологические процессы могут быть па- тогенетическим звеном формирования у больных эн- догенными психическими заболеваниями когнитивных расстройств после каждого перенесенного приступа. Исследования последних десятилетий обогати- лись гипотезой об участии нейровоспалительных процессов в патогенезе эндогенных психозов. Так, работа F. Dickerson с соавт. (2013) показывает изме- нения ряда иммунных параметров при маниакальных расстройствах (повышение уровней антител к пепти- ду NR2 рецептора NMDA, IgG к глиадину, а также IgM к Toxoplasma gondii). Нейровоспаление может быть вызвано стрессом, ишемией/гипоксией, сопутствую- щим инфекционным процессом или быть результатом генетической предрасположенности, которая спо- собствует поддержанию патологического процесса в мозге и формированию нейроанатомических нару- шений, что в дальнейшем приводит к расстройствам психической деятельности. Было установлено, что ма- ниакально-парафренные состояния, наблюдающиеся в рамках эндогенных приступообразных заболеваний, сопровождаются разным уровнем активации иммунной системы [56]. Кроме того, при этих психозах запуска- ются не только воспалительные, но и аутоиммунные реакции, отражающие тяжесть текущего патологиче- ского процесса в мозге. Как правило, у таких больных повышен уровень противоспалительных цитокинов, белков острой фазы, хемокинов и молекул клеточной адгезии [57]. Высокий уровень антител к нейроантиге- нам служит маркером, свидетельствующим о тяжести патологического процесса [50]. Научные обзоры ВОПРОСЫ ТЕРАПИИ МАНИАКАЛЬНО- ПАРАФРЕННЫХ СОСТОЯНИЙ Вопросы терапии маниакально-парафренных и ма- ниакально-бредовых состояний также остаются пред- метом оживленной дискуссии. Одни авторы предлага- ют купировать данную симптоматику исключительно нормотимиками [29, 39, 40], другие, напротив, рекомен- дуют терапию маниакальных фаз только применением антипсихотиков, при этом утверждается, что эффект лечения наступает быстрее и более выражен, чем при применении солей лития [41]. В то же время имеются данные о том, что назначение антипсихотиков в виде монотерапии при маниях ассоциируется с увеличени- ем частоты несмертельного суицидального поведения в 9,4 раза, а использование комбинации антипсихотика Немногочисленные нейрофизиологические иссле- дования по данной теме свидетельствуют о том, что при острых психотических состояниях, в том числе маниакально-бредовых и маниакально-парафренных, как правило, регистрируется десинхронизированная ЭЭГ со снижением спектральной мощности альфа-диа- пазона и часто с усилением бета-активности, что МИА’ ISSN1683-8319 (print)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 свидетельствует о повышенной активации стволовых структур и значительном дефиците тормозных систем коры головного мозга. В недавнем клинико-нейрофи- зиологическом исследовании [55] было показано, что по данным количественной ЭЭГ у пациентов с маниа- кально-бредовыми состояниями в рамках приступо- образной шизофрении (без более тонкого подразде- ления на синдромально различающиеся группы) бо- лее тяжелое клиническое состояние до начала курса терапии, отражающееся в более высоких значениях общей суммы баллов шкалы PANSS, ассоциируется с повышенной активацией лобных и центральных зон и сниженной активацией височных областей левого полушария коры головного мозга. проявлений психоза, т.е. может служить дополнитель- ным инструментом для мониторинга, оценки качества терапевтической ремиссии и прогноза дальнейшего течения заболевания [50, 56]. ЛИТЕРАТУРА/REFERENCES 1. Точилов ВА. О трудностях в диагностике и лечении атипичных аффективных психозов. Обозрение пси- хиатрии и медицинской психологии имени В.М. Бех- терева. 1992;4:28–36. Tochilov VA. O trudnostiakh v diagnostike i lechenii atipichnykh affektivnykh psikhozov. Obozrenie psikh- iatrii i meditsinskoi psikhologii imeni V.M. Bekhtereva. 1992;4:28–36. (In Russ.). 9. 9. Appelbaum P, Clark Robbins P, Roth Loren H. Di- mensional Approach to Delusions: Comparison Across Types and Diagnoses. The American Journal of psychiatry. 1999;156:1938–1943. DOI: 10.1176/ ajp.156.12.1938 10. Casanova M. The Pathology of Paraphrenia. Current Psychiatry Reports. 2010;12:196–201. DOI: 10.1007/ s11920-010-0108-8 2. Пашковский ВЭ. Клинические особенности бреда мессианства при психических расстройствах шизо- френического спектра. Журнал неврологии и психи- атрии имени С.С. Корсакова. 2006;106(4):15–20. Pashkovskii VE. Klinicheskie osobennosti breda mes- sianstva pri psikhicheskikh rasstroistvakh shizof- renicheskogo spektra. Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova. 2006;106(4):15–20. (In Russ.). 11. Brodaty H, Sachdev P, Koschera A, Monk D, Cul- len B. Long-term outcome of late-onset schizo- phrenia: 5-year follow-up study. Br. J. Psychiatry. 2003;183:213–219. DOI: 10.1192/bjp.183.3.213. PMID: 12948993 12. Girard C. Clinical characterization of late- and very late-onset ﬁ rst psychotic episode in psychi- atric inpatients. The American Journal of Geriatric Psychiatry. 2008:16(6):478–487. DOI: 10.1097/JGP. 0b013e31816c7b3c ( ) ( ) 3. Субботская НВ. Психопатология острого парафрен- ного синдрома и его типологические варианты. Психиатрия. 2005;5:26–31. Subbotskaya NV. Psychopathology of acute para- phrenic syndrome and its typological variants. Psy- chiatry. 2005;5:26–31. (In Russ.). 13. Howard R, Rabins P. Late paraphrenia revisited. Br. J. Psychiatry. 1997;171:406–408. DOI: 10.1192/ bjp.171.5.406. PMID: 9463595 4. Соколов АВ. Психопатологические особенности бреда преувеличенной самооценки в картине эн- догенных маниакально-бредовых состояний. Жур- нал неврологии и психиатрии имени С.С. Корсакова. 2012;112(2):18–24. Sokolov AV. Psychopathological features and delusion of exaggerated self-esteem in endogenous manic-delusion states. Zhurnal nevrologii i psixiatrii imeni S.S. Korsakova. 2012;112(2):18–24. (In Russ.). 4. Соколов АВ. Психопатологические особенности бреда преувеличенной самооценки в картине эн- догенных маниакально-бредовых состояний. Жур- нал неврологии и психиатрии имени С.С. Корсакова. 2012;112(2):18–24. 14. Reeves RR, Brister JC. Psychosis in late life: emerging issues. J. Psychosoc. Nurs. Ment. Health Serv. 2008;46(11):45–52. DOI: 10.3928/02793695- 20081101-09. PMID: 19051578 15. Yamada M. Senile Dementia of the Neuroﬁ brillary Tangle Type (SD-NFT). Brain Nerve. 2018;5(70):533– 541. DOI: 10.11477/mf.1416201035 16. Conus P, Cotton S, Bechdolf A, Nelson B, Chenen A. A preliminary evaluation of the validity of at-risk criteria for bipolar disorders in help-seeking ad- olescents and young adults. Journal of Affective Disorder. 2010;1(127):316–320. DOI: 10.1016/j. jad.2010.06.016 5. Шумский НГ. К клинике парафренной (фантасти- чески-параноидной) шизофрении. ЗАКЛЮЧЕНИЕ На основании приведенных данных можно сделать вывод о необходимости дальнейшего изучения корре- ляций психопатологических особенностей маниакаль- но-бредовых и маниакально-парафренных состояний с нейробиологическими показателями и поиска пре- дикторов развития и биологических маркеров данных психозов в рамках мультидисциплинарных исследова- ний. Интерес представляет дальнейший анализ имму- нологических особенностей разных клинических вари- антов маниакально-парафренных состояний. Результа- ты подобных исследований могли бы способствовать уточнению как диагностики маниакально-парафрен- ных состояний, так и прогноза заболеваний, в течении которых они возникают. Одним из воспалительных маркеров, вовлеченных в патогенез эндогенных приступообразных заболева- ний, является лейкоцитарная эластаза (ЛЭ). ЛЭ — про- теолитический фермент нейтрофилов, участвующий в реакциях проницаемости сосудистой стенки и вы- брасывающийся во внеклеточное пространство при развертывании воспалительных реакций. Повышение активности ЛЭ в острой стадии эндогенного приступо- образного заболевания сопровождается повышением функциональной активности D1-протеиназного инги- битора (D1-ПИ), относящегося к белкам острой фазы воспаления. В качестве аутоиммунных маркеров так- же рассматриваются повышенные уровни аутоантител к нейроантигенам [50, 56]. Кроме того, установлено, что комплексное определение иммунологических по- казателей в динамике заболевания отражает изме- нения состояния пациентов задолго до клинических Таким образом, несмотря на длительную историю исследований маниакально-парафренных и маниа- кально-бредовых состояний, исчерпывающего ана- лиза их психопатологической структуры, патокинеза расстройств в динамике синдрома, особенностей со- отношения маниакальных и бредовых (парафренных) расстройств в их картине проведено не было. Также до настоящего времени не определены нейробиоло- гические маркеры данных состояний, сохраняется не- однозначность их нозологической оценки, отсутствуют MIA’ ISSN2618-6667 (online)’ journalpsychiatry.com DOI 10.30629/2618-6667-2021-19-1-90-101 Psychiatry (Moscow) 19(1)'2021'90-101 Sokolova BV. The clinical picture and psychopathology of acute paraphrenic states in schizophreniа. Zhurnal nevropatologii i psikhiatrii imeni S.S. Korsakova. 1968;5:754–758. 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DOI: 10.17116/ jnevro201711711191-100 Brusov OS, Matveev IA, Kirillov PS, Faktor MI, Kar- pova NS, Vasil’eva EF, Katasonov AB, Zozulya SA, Klyushnik TP. Risk assessment of thrombotic events in patients with schizophrenia and schizoaffective disorder in the acute state: the “ﬁ brinodynamics” technology. Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova. 2017;117(11):91–100. (In Russ.). DOI: 10.17116/jnevro201711711191-100 53. Брусов ОС, Матвеев ИА, Кириллов ПС, Фактор МИ, Карпова НС, Васильева ЕФ, Катасонов АБ, Зозу- ля СА, Клюшник ТП. Оценка риска тромботических нарушений у больных шизофренией и шизоаффек- тивными расстройствами в остром периоде болез- ни с использованием технологии «фибринодина- мика». ЛИТЕРАТУРА/REFERENCES Possibilities of the use of immunological indicators for the as- sessment of the risk of manifestation of endogenous psychoses in patients with nonpsychoticdisorders of the juvenile age. Zhurnal nevrologii i psikhiatrii imeni S.S. 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PMID: 11072939 Сведения об авторах Сизов Степан Владимирович, научный сотрудник, отдел по изучению эндогенных психических расстройств и аффективных состояний, ФГБНУ «Научный центр психического здоровья», Москва, Российская Федерация, https://orcid.org/0000-0002-8213-5122 p // g/ E-mail: sizov.stepan@list.ru E-mail: sizov.stepan@list.ru p @ Олейчик Игорь Валентинович, доктор медицинских наук, отдел по изучению эндогенных психических расстройств и аффективных состояний, ФГБНУ «Научный центр психического здоровья», Москва, Российская Федерация, https://orcid.org/0000-0002-8344-0620 Научные обзоры Information about the authors Information about the authors Stepan V. Sizov, Department of Endogenous Mental Disorders and Affective Conditions, FSBSI “Mental Health Research Centre”, Moscow, Russian Federation, https://orcid.org/0000-0002-8213-5122 E-mail: sizov.stepan@list.ru Igor V. Oleichik, MD, PhD, Dr. of Sci. (Med.), Principal Researcher, Department of Endogenous Mental Disorders and Affective Conditions, Mental Health Research Centre, FSBSI “Mental Health Research Centre”, Moscow, Russian Federation, https://orcid.org/0000-0002-8344-0620 E-mail: i.oleichik@mail.ru Petr A. Baranov, MD, PhD, Cand. of Sci. (Med.), Researcher, Department for the Study of Endogenous Mental Disorders and Affective Conditions, FSBSI “Mental Health Research Centre”, Moscow, Russian Federation, https:// orcid.org/0000-0002-4423-4007 E-mail: pab1960@mail.ru Автор для корреспонденции/Corresponding author Сизов Степан Владимирович/Stepan V. Sizov E-mail: sizov.stepan@list.ru Дата поступления 27.07.2020 Received 27.07.2020 Дата рецензии 27.10.2020 Revised 27.10.2020 Дата принятия 12.11.2020 Accepted for publication 12.11.2020 Stepan V. Sizov, Department of Endogenous Mental Disorders and Affective Conditions, FSBSI “Mental Health Research Centre”, Moscow, Russian Federation, https://orcid.org/0000-0002-8213-5122 E-mail: sizov.stepan@list.ru Igor V. Oleichik, MD, PhD, Dr. of Sci. (Med.), Principal Researcher, Department of Endogenous Mental Disorders and Affective Conditions, Mental Health Research Centre, FSBSI “Mental Health Research Centre”, Moscow, Russian Federation, https://orcid.org/0000-0002-8344-0620 E-mail: i.oleichik@mail.ru Petr A. Baranov, MD, PhD, Cand. of Sci. (Med.), Researcher, Department for the Study of Endogenous Mental Disorders and Affective Conditions, FSBSI “Mental Health Research Centre”, Moscow, Russian Federation, https:// orcid.org/0000-0002-4423-4007 E-mail: pab1960@mail.ru E-mail: pab1960@mail.ru Автор для корреспонденции/Corresponding author Сизов Степан Владимирович/Stepan V. Sizov E-mail: sizov.stepan@list.ru
https://openalex.org/W4386301421	https://zenodo.org/records/8299700/files/D3.3%20PIACERE%20Abstractions%20DOML%20and%20DOML-E%20v3-v1.0.pdf	English	null	D3.3 PIACERE Abstractions DOML and DOML-E v3-v1.0	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	21,567	D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Deliverable D3.3 PIACERE Abstractions, DOML and DOML-E – v3 Editor(s): Sergio Canzoneri, Elisabetta Di Nitto Responsible Partner: Politecnico di Milano/ PoliMi Status-Version: Final - v1.0 Date: 31.05.2023 Distribution level (CO, PU): Public DRAFT Deliverable D3.3 PIACERE Abstractions, DOML and DOML-E – v3 Editor(s): Sergio Canzoneri, Elisabetta Di Nitto Responsible Partner: Politecnico di Milano/ PoliMi Status-Version: Final - v1.0 Date: 31.05.2023 Distribution level (CO, PU): Public DRAFT Editor(s): Sergio Canzoneri, Elisabetta Di Nitto Responsible Partner: Politecnico di Milano/ PoliMi Status-Version: Final - v1.0 Date: 31.05.2023 Distribution level (CO, PU): Public D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 Project Number: 101000162 Project Title: PIACERE Title of Deliverable: PIACERE Abstractions, DOML and DOML-E – v3 Due Date of Delivery to the EC 31.05.2023 Workpackage responsible for the Deliverable: WP3 - Plan and create Infrastructure as Code Editor(s): Politecnico di Milano/PoliMi Contributor(s): Go4it, HPE, Prodevelop, Tecnalia Reviewer(s): Ismael Torres (Prodevelop) Approved by: All Partners Recommended/mandatory readers: WP4, WP5, WP6, WP7 Abstract: This deliverable is the output of tasks 3.1, 3.2 and 3.3. It presents the final version of the DOML (v3.0). DOML is a domain-specific language designed for modelling the cloud applications and the infrastructural resources, hiding the specificities and technicalities of the current IaC solutions and increases the productivity of these teams. DOML is complemented by DOML-E (KR4), which is the set of extension mechanisms defined for the language. They allow new infrastructural components, e.g., for software execution, network communication, cloud services, or data storage, to be incorporated in the DOML language. This deliverable presents the DOML metamodel and syntax and its extension mechanisms, DOML-E. Moreover, it includes an overview of the changes in the language since the previous deliverable D3.2 and discusses about the level of accomplishment of the requirements formulated within the PIACERE project. A preliminary evaluation of DOML is presented together with some examples of DOML usage. Finally, the deliverable includes an overview about the lessons learnt and future directions for the extension of the language beyond the scope of the PIACERE project. Deliverable D3.3 PIACERE Abstractions, DOML and DOML-E – v3 Editor(s): Sergio Canzoneri, Elisabetta Di Nitto Responsible Partner: Politecnico di Milano/ PoliMi Status-Version: Final - v1.0 Date: 31.05.2023 Distribution level (CO, PU): Public DRAFT Keyword List: Model-driven engineering, metamodels, modelling abstractions, Infrastructure as Code Licensing information: This work is licensed under Creative Commons Attribution-ShareAlike 3.0 Unported (CC BY-SA 3.0) http://creativecommons.org/licenses/by-sa/3.0/ Disclaimer This document reflects only the author’s views and neither Agency nor the Commission are responsible for DRAFT Project Number: 101000162 Project Title: PIACERE Title of Deliverable: PIACERE Abstractions, DOML and DOML-E – v3 Due Date of Delivery to the EC 31.05.2023 Workpackage responsible for the Deliverable: WP3 - Plan and create Infrastructure as Code Editor(s): Politecnico di Milano/PoliMi Contributor(s): Go4it, HPE, Prodevelop, Tecnalia Reviewer(s): Ismael Torres (Prodevelop) Approved by: All Partners Recommended/mandatory readers: WP4, WP5, WP6, WP7 T This deliverable is the output of tasks 3.1, 3.2 and 3.3. It presents the final version of the DOML (v3.0). DOML is a domain-specific language designed for modelling the cloud applications and the infrastructural resources, hiding the specificities and technicalities of the current IaC solutions and increases the productivity of these teams. DOML is complemented by DOML-E (KR4), which is the set of extension mechanisms defined for the language. They allow new infrastructural components, e.g., for software execution, network communication, cloud services, or data storage, to be incorporated in the DOML language. This deliverable presents the DOML metamodel and syntax and its extension mechanisms, DOML-E. Moreover, it includes an overview of the changes in the language since the previous deliverable D3.2 and discusses about the level of accomplishment of the requirements formulated within the PIACERE project. A preliminary evaluation of DOML is presented together with some examples of DOML usage. Finally, the deliverable includes an overview about the lessons learnt and future directions for the extension of the language beyond the scope of the PIACERE project. RAFT Page 2 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Document Description Version Date Modifications Introduced Modification Reason Modified by v0.1 16.03.2023 Table of contents defined PoliMi v0.2 06.04.2023 Material collected ALL partners v0.3 15.05.2023 Complete draft version ready for review PoliMi v.0.4 19.05.2023 Reviewed version Prodevelop v0.5 29.05.2023 Final version after review. PoliMi v1.0 30.05.2023 Final quality check. Ready for submission TECNALIA DRAFT Document Description Version Date Modifications Introduced Modification Reason Modified by v0.1 16.03.2023 Table of contents defined PoliMi v0.2 06.04.2023 Material collected ALL partners v0.3 15.05.2023 Complete draft version ready for review PoliMi v.0.4 19.05.2023 Reviewed version Prodevelop v0.5 29.05.2023 Final version after review. Deliverable D3.3 PIACERE Abstractions, DOML and DOML-E – v3 Editor(s): Sergio Canzoneri, Elisabetta Di Nitto Responsible Partner: Politecnico di Milano/ PoliMi Status-Version: Final - v1.0 Date: 31.05.2023 Distribution level (CO, PU): Public DRAFT PoliMi v1.0 30.05.2023 Final quality check. Ready for submission TECNALIA T Document Description TECNALIA T © PIACERE Consortium Contract No. GA 101000162 Page 3 of 51 www.piacere-project.eu v1.0 30.05.2023 submission TECNALIA DRAFT Contract No. GA 101000162 Page 3 of 51 © PIACERE Consortium www.piacere-project.eu D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 © PIACERE C ti C t t N GA 101000162 P 4 f 51 Table of contents Terms and abbreviations ............................................................................................................... 6 Executive Summary ....................................................................................................................... 7 1 Introduction .......................................................................................................................... 8 1.1 About this Deliverable ................................................................................................... 8 1.2 Document Structure ...................................................................................................... 9 2 DOML (KR1) and DOML-E (KR4) .......................................................................................... 10 2.1 Overview ..................................................................................................................... 10 2.1.1 DOML main characteristics ................................................................................. 11 2.1.2 DOML metamodel ............................................................................................... 12 2.1.3 DOML syntax ....................................................................................................... 14 2.1.4 DOML-E ............................................................................................................... 15 2.1.5 DOML supporting components ........................................................................... 17 2.2 Changes in the latest version ...................................................................................... 18 2.3 Functional description and requirements coverage ................................................... 19 2.3.1 Requirements coverage ...................................................................................... 19 2.3.2 Scenarios ............................................................................................................. 22 2.4 Main innovations ......................................................................................................... 25 3 Overview of preliminary experiments................................................................................. 27 3.1 Empirical evaluation .................................................................................................... 27 3.2 Reference test cases example ..................................................................................... 28 3.2.1 Introduction and comparative table ................................................................... 28 3.2.2 WordPress Website ............................................................................................. 29 3.2.3 NginX ................................................................................................................... 34 4 Lessons Learnt and Plan for Future Development .............................................................. 38 5 Conclusions ......................................................................................................................... 40 6 References ........................................................................................................................... 41 APPENDIX: Further details about the DOML ............................................................................... 42 1 Simplified version of the Ericsson case ............................................................................... 42 1.1 Case short description ................................................................................................. 42 1.2 DOML model ............................................................................................................... 42 2 User manual to extend DOML ............................................................................................. 50 2.1 Required tools ............................................................................................................. 50 2.2 Metamodel update ..................................................................................................... 50 2.3 Syntax update .............................................................................................................. 50 2.4 Testing applied changes and updating the ICG ........................................................... 51 DRAFT Table of contents D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 List of tables TABLE 1. REQUIREMENTS ON THE GENERAL CHARACTERISTICS OF DOML. ................................................. 19 TABLE 2. REQUIREMENTS ON THE SPECIFIC ELEMENTS TO BE MODELLED IN DOML. .................................... 20 TABLE 3. COMPARISON BETWEEN DOML AND OTHER IAC APPROACHES. .................................................. 27 TABLE 4. COMPARATIVE TABLE FOR TEST CASES EXAMPLES ...................................................................... 29 List of figures FIGURE 1. DOML POSITIONING IN THE PIACERE APPROACH. ................................................................. 10 FIGURE 2. Deliverable D3.3 PIACERE Abstractions, DOML and DOML-E – v3 Editor(s): Sergio Canzoneri, Elisabetta Di Nitto Responsible Partner: Politecnico di Milano/ PoliMi Status-Version: Final - v1.0 Date: 31.05.2023 Distribution level (CO, PU): Public DRAFT COMMONS LAYER DIAGRAM ............................................................................................... 12 FIGURE 3. APPLICATION LAYER DIAGRAM ............................................................................................. 13 FIGURE 4. INFRASTRUCTURE LAYER DIAGRAM ....................................................................................... 13 FIGURE 5. CONCRETE LAYER DIAGRAM ................................................................................................ 14 FIGURE 6. OPTIMIZATION LAYER DIAGRAM .......................................................................................... 14 DRAFT List of tables TABLE 1. REQUIREMENTS ON THE GENERAL CHARACTERISTICS OF DOML. ................................................. 19 TABLE 2. REQUIREMENTS ON THE SPECIFIC ELEMENTS TO BE MODELLED IN DOML. .................................... 20 TABLE 3. COMPARISON BETWEEN DOML AND OTHER IAC APPROACHES. .................................................. 27 TABLE 4. COMPARATIVE TABLE FOR TEST CASES EXAMPLES ...................................................................... 29 List of figures FIGURE 1. DOML POSITIONING IN THE PIACERE APPROACH. ................................................................. 10 FIGURE 2. COMMONS LAYER DIAGRAM ............................................................................................... 12 FIGURE 3. APPLICATION LAYER DIAGRAM ............................................................................................. 13 FIGURE 4. INFRASTRUCTURE LAYER DIAGRAM ....................................................................................... 13 FIGURE 5. CONCRETE LAYER DIAGRAM ................................................................................................ 14 FIGURE 6. OPTIMIZATION LAYER DIAGRAM .......................................................................................... 14 DRAFT DRAFT Contract No. GA 101000162 © PIACERE Consortium www.piacere-project.eu Page 5 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Terms and abbreviations CMS Content Management System CSP Cloud Service Provider DevOps Development and Operation DoA Description of Action EC European Commission EDMM Essential Deployment Metamodel FaaS Function as a Service GA Grant Agreement of the project IaC Infrastructure as Code ICG IaC Code Generation ICMP Internet Control Message Protocol IEP IaC Execution Platform IOP IaC Optimization KPI Key Performance Indicator MC Model Checker NFR Non-Functional Requirement SW Software TBCG Template-Based Code Generation VM Virtual Machine AWS Amazon Web Services DRAFT Terms and abbreviations CMS Content Management System CSP Cloud Service Provider DevOps Development and Operation DoA Description of Action EC European Commission EDMM Essential Deployment Metamodel FaaS Function as a Service GA Grant Agreement of the project IaC Infrastructure as Code ICG IaC Code Generation ICMP Internet Control Message Protocol IEP IaC Execution Platform IOP IaC Optimization KPI Key Performance Indicator MC Model Checker NFR Non-Functional Requirement SW Software TBCG Template-Based Code Generation VM Virtual Machine AWS Amazon Web Services AFT DRAF Contract No. GA 101000162 © PIACERE Consortium www.piacere-project.eu Page 6 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 1 Introduction This deliverable presents the DOML language and the mechanisms that have been developed to support its extension. It is an update of the previous deliverable D3.2 [1] but it is written to be self-contained. Through the PIACERE project development, multiple versions of the DOML language have been released, starting from DOML 1.0 presented in deliverable D3.1 [4], to DOML 3.0 which is the focus of the current deliverable. DOML is characterised by a metamodel, encoded in ECore [5]-- a metamodeling approach offered by Eclipse -- and by a syntax. Thanks to the usage of Xtext [6], the DOML editor guides the user through the DOML syntax and allows him/her to define syntactically correct models. The DOML-DOMLX conversion is a supporting service that, given a DOML model can generate a serialized representation of its data structures, the DOMLX, which is the DOML PIACERE internal representation. By the end of the project, the DOML-DOMLX conversion service will support also the backward translation from DOMLX into the DOML syntax. Thanks to DOMLX, the PIACERE tools can maintain their independency from the specific syntax adopted for the DOML. Moreover, the backward translation that will be offered by the DOML-DOMLX conversion service will allow even the transformation of DOML models written according to a previous version of the language into the latest version, thus greatly contributing to the backward compatibility of the language. FT Executive Summary This document extends deliverable D3.2 [1], including the progress of the DOML development to fulfil the requirements during the evolution of the project. DOML (PIACERE KR1) is a domain-specific language designed for modelling cloud applications and infrastructural resources, hiding the specificities and technicalities of current IaC solutions and increasing productivity of these teams. DOML models are created using the PIACERE IDE (PIACERE KR2), which provides users with guidance and it also integrates all other design-time PIACERE tools. Then, the DOML models are translated through the Infrastructural Code Generator (ICG, PIACERE KR3), into the target IaC languages for complex applications. The DOML is complemented by DOML-E (KR4), which is a set of extension mechanisms defined for the language. They allow new infrastructural components, e.g., for software execution, network communication, cloud services, or data storage, to be incorporated in the DOML language. This deliverable provides an overview of DOML and DOML-e. Furthermore, it highlights the changes that have been introduced in DOML compared to what was reported in the previous deliverables and presents the status of requirements fulfilment. Additionally, the document highlights the main innovations introduced by DOML and DOML-e and provides an overview of the experiments developed so far by using the DOML. Finally, it presents the main lessons learnt, an outlook to the future beyond the scope of the PIACERE project, and the conclusions of this deliverable. It includes in the Appendix the DOML details for one of the PIACERE Use Cases and a guide on how to extend DOML. AFT The deliverable is also accompanied by two external annexes, the first one [2] provides a detailed definition of all concepts of the DOML, the second one (Canzoneri, 2023) is a tutorial that allows end users to become proficient with the language. Both are released as separated documents to facilitate their usage and evolution independently of this deliverable. RAF DR © PIACERE Consortium www.piacere-project.eu Page 7 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 1.1 About this Deliverable GA 101000162 Page 8 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 • Elaboration of the examples of use of the DOML language using the PIACERE IDE, their validation through the Model Checker, their optimization through the IOP tool, and the generation of the corresponding IaC code through the ICG. • Elaboration of the examples of use of the DOML language using the PIACERE IDE, their validation through the Model Checker, their optimization through the IOP tool, and the generation of the corresponding IaC code through the ICG. As discussed in further details in this deliverable, of the 18 requirements defined in PIACERE and associated to the DOML, 16 have been achieved completely and 2 partially. 1.1 About this Deliverable The purpose of this deliverable is to provide a general presentation of the DOML and its extension mechanisms, DOML-E. RAF As mentioned above, the main objective of the DOML has been to reduce the effort needed to automate the deployment and operation of an application in combination with its underlying infrastructure. This has resulted in the development of a high-level modeling language that is then translated, through the ICG, into de-facto IaC standard languages supporting the Ops phases of the software lifecycle. DRA The DOML has been developed taking as a reference the resources and configurability options made available by the main cloud providers, considering the main characteristics of the IaC languages used as a reference, and taking into account the needs of the PIACERE case studies. DR The purpose of this deliverable is to report on the work done, to show in practice how DOML works and how it can be used in concrete examples, and to provide a summary of lessons learnt and the plan for future development beyond the end of the PIACERE project. D Being this the final version of a series of three deliverables, it has been chosen to make it self- contained. As such, part of its content is a repetition and revision of what has been presented in previous deliverables. The main innovations introduced since the previous version of this deliverable D3.2 [1], consist in the following aspects: The main innovations introduced since the previous version of this deliverable D3.2 [1], consist in the following aspects: • Consolidation and clean-up of the DOML modelling language which has now reached version 3.0. Through the PIACERE project the following main versions of the language have been released: 1.0 at the end of the first project year, 2.0 and 2.1 during the second project year, 2.2.1 and 2.2.2 in the third year. • Consolidation and clean-up of the DOML modelling language which has now reached version 3.0. Through the PIACERE project the following main versions of the language have been released: 1.0 at the end of the first project year, 2.0 and 2.1 during the second project year, 2.2.1 and 2.2.2 in the third year. • Consolidation of the extension mechanisms (DOML-E). Consolidation of the extension mechanisms (DOML-E). • Consolidation of the extension mechanisms (DOML-E). Contract No. 1.2 Document Structure The document is organized as follows: • Section 2 provides a general overview of the DOML and DOML-e, of their changes compared to the version reported in D3.2, and of their main innovations. • Section 2 provides a general overview of the DOML and DOML-e, of their changes compared to the version reported in D3.2, and of their main innovations. • Section 3 provides an overview of the examples of usage of the DOML and DOML-e. p p g • Section 4 summarizes the lessons learnt and the plan for future development. • Section 4 summarizes the lessons learnt and the plan for future development. • Section 5 concludes the deliverable. The deliverable is accompanied by an Appendix providing further details on a DOML model example that has been used as a reference to extend the language and on how to extend the DOML language by introducing new concepts in the DOML metamodel. Finally, the deliverable includes also Annex 1 [2] which presents the detailed specification of the DOML concepts and Annex 2 (Canzoneri, 2023) which provides a tutorial of the language usage. FT © PIACERE Consortium Contract No. GA 101000162 Page 9 of 51 www.piacere-project.eu DRAFT DRAFT Contract No. GA 101000162 © PIACERE Consortium www.piacere-project.eu Page 9 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 2.1 Overview The DevOps Modelling Language (DOML) aims at offering a high-level declarative approach to the definition of an application and its infrastructure. DOML models are mainly structured in three layers. Specifically, software components (e.g., web servers, databases, etc.) are described in the application layer, abstracting away from the infrastructure on which they are supposed to run. Infrastructure components are specified in the abstract infrastructure layer, and then linked to the applications they are supposed to host. This layer models infrastructural facilities, such as virtual machines, networks, containers, etc., without referring to their actual concretization in specific technologies (e.g., AWS or OpenStack VMs, Docker containers). This last aspect is tackled by the concrete infrastructure layer, where the user specifies the infrastructure components offered by the Cloud Service Provider (CSP). The adoption of this three layers approach allows us to overcome a limitation of the currently available Infrastructure as Code (IaC) languages that tend to focus on specific aspects, either the configuration of the specific infrastructural elements or the installation and configuration of software elements, thus making difficult for DevOps teams to have a complete overview of their whole system [7]. FT As it happens for typical programming languages, DOML offers some extension mechanisms, DOML-E, that keep the language open to the addition of new resources, both at the abstract and concrete layers. AFT Figure 1. DOML positioning in the PIACERE approach. DRAF Figure 1. DOML positioning in the PIACERE approach. Figure 1 shows the positioning of the DOML in the PIACERE ecosystem. The numbers associated to the arrows are described in detail in [8] and represent the steps of the workflow activated when a user wants to create a DOML model. More specifically, the user exploits the IDE to design the model. The DOML ecore [5] and Xtext [6] representations allow the IDE to support the user by providing suggestions about the syntax to use. Through the IDE, the user can exploit all other tools in the figure. More specifically, through the Infrastructural Elements Catalogues he/she can obtain information about the resources that are known to PIACERE. Through the Model Checker it is possible to verify the correctness of the model in terms of internal consistency and fulfilment of explicitly defined requirements. Through the IOP it is possible to obtain a mapping of the abstract infrastructure layer into a concrete infrastructure, through the IGC the Contract No. 2.1 Overview GA 101000162 Page 10 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 generation of IaC and, finally, through the IaC Scan Runner the verification, from the security perspective, of the final code. The DOML ecore and Xtext implementations are available in the PIACERE public repository together with a tutorial presenting the usage of the DOML1. In the following of this section, the main characteristics of the DOML are presented together with the metamodel that regulates its usage and the language syntax to be used by external users. Furthermore, DOML extension mechanisms are explained, and finally, the DOML supporting components are presented. These include: i) a web service that translates DOML specifications in an internal format used by some of the other PIACERE tools, the DOMLX, and vice versa; ii) a component called DOMLIZER that is in charge of receiving resource descriptions available in the catalogue and transforming them into DOML fragments. 2.1.1 DOML main characteristics This will be the subject of future work beyond the scope of the PIACERE project. DRAFT • Facilitate the instantiation of an abstract infrastructure on top of different resource providers: one of the issues that is often faced by DevOps teams is the need to deploy a specific application on different cloud providers. This is due to multiple reasons, ranging from the opportunities deriving from special deals offered by different providers in different cases to the need to avoid so-called vendor locking, that is, the case when it becomes very expensive and difficult for the team to move to a different provider. DR • Facilitate the instantiation of an abstract infrastructure on top of different resource providers: one of the issues that is often faced by DevOps teams is the need to deploy a specific application on different cloud providers. This is due to multiple reasons, ranging from the opportunities deriving from special deals offered by different providers in different cases to the need to avoid so-called vendor locking, that is, the case when it becomes very expensive and difficult for the team to move to a different provider. DR • Facilitate the instantiation of an abstract infrastructure on top of different resource providers: one of the issues that is often faced by DevOps teams is the need to deploy a specific application on different cloud providers. This is due to multiple reasons, ranging from the opportunities deriving from special deals offered by different providers in different cases to the need to avoid so-called vendor locking, that is, the case when it becomes very expensive and difficult for the team to move to a different provider. The multi-layered approach offered by the DOML has the potential to reduce the effort of the team in exploiting resources from different providers. In fact, the application and the abstract infrastructure layers of a DOML model can be reused when moving from DR The multi-layered approach offered by the DOML has the potential to reduce the effort of the team in exploiting resources from different providers. In fact, the application and the abstract infrastructure layers of a DOML model can be reused when moving from one provider to the other, while associations to multiple providers’ resources can coexist in the same model. • Keep the external representation of a DOML model separate from the internal one. 2.1.1 DOML main characteristics The DOML provides the following main characteristics. The DOML provides the following main characteristics. • Reduce the need for polyglotism in DevOps teams: as it has been mentioned in the previous section, typical IaC approaches are focused on specific aspects of the system life cycle. For instance, Terraform is focused on resource provisioning, Ansible on the configuration and execution of software layers on top of existing resources, container technologies such as Docker on the creation of proper execution environments for software components, orchestrators such as Kubernetes on the management of containers and their possibility to scale in/out. This implies the need to have a polyglot DevOps team able to show a reasonable level of proficiency on multiple IaC languages and approaches. DOML, in combination with the translation features offered by ICG [9], allows its users to define a single DOML model that, at the time of writing, can be used for resource provisioning and software configuration and execution. While the other aspects that have been enumerated are not covered yet, they can be potentially addressed through the development of ICG templates developed to translate the DOML model into proper target IaC languages. This will be the subject of future work beyond the scope of the PIACERE project. DRAFT • Reduce the need for polyglotism in DevOps teams: as it has been mentioned in the previous section, typical IaC approaches are focused on specific aspects of the system life cycle. For instance, Terraform is focused on resource provisioning, Ansible on the configuration and execution of software layers on top of existing resources, container technologies such as Docker on the creation of proper execution environments for software components, orchestrators such as Kubernetes on the management of containers and their possibility to scale in/out. This implies the need to have a polyglot DevOps team able to show a reasonable level of proficiency on multiple IaC languages and approaches. DOML, in combination with the translation features offered by ICG [9], allows its users to define a single DOML model that, at the time of writing, can be used for resource provisioning and software configuration and execution. While the other aspects that have been enumerated are not covered yet, they can be potentially addressed through the development of ICG templates developed to translate the DOML model into proper target IaC languages. 2.1.1 DOML main characteristics As it has been mentioned before, the DOML internal representation is separated from the external one. Thanks to this design choice, different external representations can be associated to the same DOML model. This can be useful to accommodate preferences of different users. While the first external representation developed is a textual one, as part of the IDE development, the usage of Eclipse-based frameworks that support the • Keep the external representation of a DOML model separate from the internal one. As it has been mentioned before, the DOML internal representation is separated from the external one. Thanks to this design choice, different external representations can be associated to the same DOML model. This can be useful to accommodate preferences of different users. While the first external representation developed is a textual one, as part of the IDE development, the usage of Eclipse-based frameworks that support the © PIACERE Consortium Contract No. GA 101000162 Page 11 of 51 www.piacere-project.eu 1 https://git.code.tecnalia.com/piacere/public/the-platform/doml © PIACERE Consortium Contract No. GA 101000162 Page 11 of 51 www.piacere-project.eu 1 https://git.code.tecnalia.com/piacere/public/the-platform/doml Page 11 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 creation of a graphical representation has been experimented. A proof of concept of this is under development and will be made available by the end of PIACERE project. 2.1.2 DOML metamodel The DOML metamodel consists of several “layers”, which incrementally enrich the description of the cloud-based applications that will be managed inside PIACERE. Each layer provides a unique point of view of the applications; yet all the layers are built up for a comprehensive application description. This approach allows developers to describe how cloud applications are structured in an abstract manner, mapping the different software components to the concrete infrastructure elements, enabling the usage of different concretizations to match one particular deployment. 2.1.2.1 Commons Layer The Commons Layer contains the main abstract application agnostic concepts that are shared among different layers (see Figure 2). The DOML extension mechanisms (DOML-E) are also addressed in this layer by setting up the basic elements that will allow creating new concepts and properties in the top layers. T Figure 2. Commons Layer diagram DRAFT Figure 2. Commons Layer diagram DR 2.1.2.2 Application Layer D The Application Layer (see Figure 3) contains the information to describe the components and building blocks that compose the applications, as well as the functional requirements of each of them in terms of software interfaces and APIs. Finally, this layer describes how the application is deployed into the different infrastructure components. D Contract No. GA 101000162 Contract No. GA 101000162 Contract No. GA 101000162 Page 12 of 51 © PIACERE Consortium www.piacere-project.eu © PIACERE Consortium www.piacere-project.eu D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 Figure 3. Application Layer diagram T Figure 3. Application Layer diagram T © PIACERE Consortium www.piacere-project.eu 2.1.2.3 Infrastructure Layer The Infrastructure Layer (see Figure 4 for an overview) defines the abstract infrastructure elements that will be used to deploy the application components. The readability of the figure could not be improved for lack of space. A detailed description of all its elements is available in the Annex [2]. Concepts in this layer will include information that is relevant to meet the requirements of the applications. However, most of the concepts in this layer will require a concretization, or in other words, a more concrete instance they will be mapped on. For example, a virtual machine (VM) in this layer must be mapped to a concrete virtual machine instance, either a VM from AWS or a specific VM deployed by the user. RAFT Figure 4. Infrastructure Layer diagram DRA Figure 4. Infrastructure Layer diagram 2.1.2.4 Concrete Layer The Concrete Layer (see Figure 5) provides the tools to concretize the infrastructure elements in the Infrastructure Layer and map them onto specific infrastructure instances either provided by cloud runtime providers, such as AWS or Google Cloud, or provided by the users. Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 Figure 5. Concrete Layer diagram Figure 5. Concrete Layer diagram 2.1.2.5 Optimization Layer The Optimization Layer (see Figure 6) defines all the information required for the optimizers to locate the best configurations for cloud applications described with the DOML, such as optimization objectives and non-functional requirements, as well as means to capture the optimization solutions. T Figure 6. Optimization Layer diagram DRAFT Figure 6. Optimization Layer diagram DR 2.1.3 DOML syntax D 2.1.3 DOML syntax D In order to offer to end users a tool to define a DOML model, a syntax has been developed by means of the Eclipse Xtext™ [6] framework. Most approaches make the text syntax directly derive from the metamodel (e.g., using tools such as the wizard provided by Xtext); however, this kind of approaches leads to a one-to-one mapping of each element in the metamodel. To achieve a high degree of flexibility for the language and improve its readability and ease-of-use, it has been decided to use a different approach, developing an ad hoc syntax, yet carefully aligned with the metamodel. D Diagrams showing the fully detailed syntax are available in [2] and a tutorial for beginners to get familiar with the syntax can be found in [3]. The syntax structure follows the layered, incremental approach used for the metamodel. In a DOML model, an application can be described in four layers: application layer, abstract / concrete infrastructure layer and optimization layer. In a declarative manner, layers are described in sequence, allowing the user to completely describe an application from all the different viewpoints. Contract No. GA 101000162 Page 14 of 51 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Most of the elements can be introduced by means of a keyword and a unique identifier: all the aspects related to such elements can be described through the usage of key-value pairs defined within curly braces. A set of attributes is defined for each kind of element, covering all the main features. Some code examples can be found in Section 4. 2.1.4 DOML-E In order to meet the needs of the continuously evolving cloud markets, DOML includes extension mechanisms that allow the users to create new concepts from the existing ones. These extensions mechanisms are referred to as DOML-E. The DOML is currently extended in the following ways: • Creation of new concepts. The new concepts will require the definition of a metaclassName. Extension elements exist in all the DOML layers, e.g., the Application Layer includes the class ExtApplicationComponent that incorporates into the Application Layer a new type of ApplicationComponent. Further details on the definition of these extension classes are provided in the Annex. T • Definition of new properties. The set of properties and attributes associated to one particular DOML concept can be extended to further increase its expressiveness. T • Definition of new properties. The set of properties and attributes associated to one particular DOML concept can be extended to further increase its expressiveness. T • Usage of the GenericResource concept. FT • Usage of the GenericResource concept. • Usage of the GenericResource concept. FT In the following subsections, the detailed steps for extending DOML are illustrated. More details on the implementation-level steps for extending metamodel are presented in Section 2 of the Appendix “Further details”. AFT In the following subsections, the detailed steps for extending DOML are illustrated. More details on the implementation-level steps for extending metamodel are presented in Section 2 of the Appendix “Further details”. AFT 2.1.4.1 Creation of New Concepts Suppose that a new service concept and a new docker service concept would need to be introduced in the infrastructure layer. RA To this end, the metamodel will be firstly modified by creating in the Commons package a metaclass named Service which extends the abstract class DOMLElement and includes the necessary attributes and references. For the sake of simplicity and clarity, the following example Service has been created: DRA class Service extends DOMLElement { attr Integer port; attr String [] constraints; } D In the above example, a Service is a DOMLElement which has a port attribute and several possible constraints expressed with String (for simplicity). ow considering that the container service is simultaneously a computing node and a service, ultiple inheritance is used to model this concept: lass Container extends ComputingNode, commons.Service { ref ContainerImage #generatedContainers generatedFrom; val ContainerConfig [] configs; In above example, the existing container has been extended by another superclass. Note that the references inside the class are associated to its ComputingNode characteristic. Contract No. GA 101000162 Page 15 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: Now the corresponding concrete syntax is ready to be created for them. For the service, it cou be used a grammar fragment, which could be used for any other service-related concept, show Now the corresponding concrete syntax is ready to be created for them. For the service, it could be used a grammar fragment, which could be used for any other service-related concept, shown as follows: fragment Service returns commons::Service: ( Now the corresponding concrete syntax is ready to be created for them. For the service, it could be used a grammar fragment, which could be used for any other service-related concept, shown as follows: fragment Service returns commons::Service: ( ('port' port=INT)? & ('constraints' '[' constraints+=STRING (',' constraints+=STRING)* ']' )? ) ; Now the corresponding concrete syntax is ready to be created for them. For the service, it could be used a grammar fragment, which could be used for any other service-related concept, shown as follows: fragment Service returns commons::Service: ( ('port' port=INT)? & ('constraints' '[' constraints+=STRING (',' constraints+=STRING)* ']' )? 2.1.4.1 Creation of New Concepts ) ; Since the Container concept can be reused, it has been updated by adding the characteristics as a service: Container returns infra::Container: 'container' name=ID (('service' '{' DOMLElement Service) \| '{' DOMLElement) configs+=ContainerConfig* ('disabled_monitorings' disabledMonitorings+=STRING (','disabledMonitorings+=STRING))? '}' ; Having introduced the above modifications in the metamodel and in the syntax, the new container service construct can be used. The fragment below is a very simple example of DOML script defined by the above model for a container service: container service dns_server { port 53 constraints [ ‘C1’, ‘C2’ ] host vm1 { … } } 2.1.4.2 Definition of New Properties In current DOML, most concepts contain properties that can be expressed by key-value pairs. This is implemented by adding the property attribute in the superclass DOMLElement, since almost all DOML concepts extend it. The detailed implementation is as follows: abstract class DOMLElement { ... val Property [] annotations; } abstract class Property { attr String key; ref DOMLElement reference; op Object getValue(); } class IProperty extends Property { attr Integer value; } The above fragment of DOML metamodel shows an example of integer property definition. DRAFT Since the Container concept can be reused, it has been updated by adding the characteristics as a service: Container returns infra::Container: 'container' name=ID (('service' '{' DOMLElement Service) \| '{' DOMLElement) configs+=ContainerConfig* ('disabled_monitorings' disabledMonitorings+=STRING (','disabledMonitorings+=STRING))? '}' ; FT Since the Container concept can be reused, it has been updated by adding the characteristics as a service: Container returns infra::Container: 'container' name=ID (('service' '{' DOMLElement Service) \| '{' DOMLElement) configs+=ContainerConfig ('disabled_monitorings' disabledMonitorings+=STRING (','disabledMonitorings+=STRING))? '}' ; FT Since the Container concept can be reused, it has been updated by adding the characteristics as a service: Container returns infra::Container: 'container' name=ID (('service' '{' DOMLElement Service) \| '{' DOMLElement) configs+=ContainerConfig ('disabled_monitorings' disabledMonitorings+=STRING (','disabledMonitorings+=STRING))? '}' ; FT Having introduced the above modifications in the metamodel and in the syntax, the ne container service construct can be used. The fragment below is a very simple example of DOM script defined by the above model for a container service: RAFT 2.1.4.2 Definition of New Properties DR In current DOML, most concepts contain properties that can be expressed by key-value pairs. This is implemented by adding the property attribute in the superclass DOMLElement, since almost all DOML concepts extend it. The detailed implementation is as follows: D In current DOML, most concepts contain properties that can be expressed by key-value pairs. In the above example, different types of properties are defined for a concrete FaaS componen of an application. generic_resource vsphere_cl { preexisting true refs_to vsphere_dc type “compute_cluster” gname “cl1” } Examples of what generic resources could model are the following: data centers, clusters, pools, etc. 2.1.5 DOML supporting components DRAF The DOML definition of a generic resource is as follows: AF This class is a specialization of the ConcreteElement class and, as such, it inherits the “preexisting” attribute and the “refs_to” association to another ConcreteElement object: this is particularly relevant, since this feature could be used to model already existing infrastructural resources. DR This class is a specialization of the ConcreteElement class and, as such, it inherits the “preexisting” attribute and the “refs_to” association to another ConcreteElement object: this is particularly relevant, since this feature could be used to model already existing infrastructural resources. DR generic_resource vsphere_cl { preexisting true refs_to vsphere_dc type “compute_cluster” gname “cl1” } Examples of what generic resources could model are the following: data centers, clusters, pools, etc. In the above example, different types of properties are defined for a concrete FaaS componen of an application. In the above example, different types of properties are defined for a concrete FaaS component of an application. In the above example, different types of properties are defined for a concrete FaaS component of an application. When defining new properties, the Infrastructural Code Generator should be updated accordingly. In the User Manual in Deliverable D3.6 [9] an explanation on how to do it can be found. T © PIACERE Consortium Contract No. GA 101000162 Page 17 of 51 www.piacere-project.eu When defining new properties, the Infrastructural Code Generator should be updated accordingly. In the User Manual in Deliverable D3.6 [9] an explanation on how to do it can be found. 2.1.4.3 Usage of the GenericResource concept The GenericResource class has been introduced in DOML 2.2 to support some concrete generic resources that are specialized in different IaC languages. For any generic resource, the current version of DOML allows to specify its type and name. The DOML definition of a generic resource is as follows: generic_resource vsphere_dc { type “datacenter” gname “dc1” } This class is a specialization of the ConcreteElement class and, as such, it inherits the “preexisting” attribute and the “refs_to” association to another ConcreteElement object: this is particularly relevant, since this feature could be used to model already existing infrastructural resources. generic_resource vsphere_cl { preexisting true refs_to vsphere_dc type “compute_cluster” gname “cl1” } Examples of what generic resources could model are the following: data centers, clusters, pools, etc. 2.1.5 DOML supporting components 2.1.5.1 DOMLIZER The objective of the DOMLIZER is to simplify the access of new DOML users to existing resources, like, for example, existing virtual machine descriptions, resources made available by runtime DRAFT 2.1.4.3 Usage of the GenericResource concept T The GenericResource class has been introduced in DOML 2.2 to support some concrete generic resources that are specialized in different IaC languages. FT For any generic resource, the current version of DOML allows to specify its type and name. AFT The DOML definition of a generic resource is as follows: generic_resource vsphere_dc { type “datacenter” gname “dc1” } This class is a specialization of the ConcreteElement class and, as such, it inherits the “preexisting” attribute and the “refs_to” association to another ConcreteElement object: this is particularly relevant, since this feature could be used to model already existing infrastructural resources. 2.1.4.1 Creation of New Concepts This is implemented by adding the property attribute in the superclass DOMLElement, since almost all DOML concepts extend it. The detailed implementation is as follows: D abstract class DOMLElement { ... val Property [] annotations; } abstract class Property { attr String key; ref DOMLElement reference; op Object getValue(); } class IProperty extends Property { attr Integer value; } © PIACERE Consortium Contract No. GA 101000162 Page 16 of 51 www.piacere-project.eu The above fragment of DOML metamodel shows an example of integer property definition. Other properties like string, float, etc. are defined analogously. he above fragment of DOML metamodel shows an example of integer property definition. ther properties like string, float, etc. are defined analogously. D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 A doml script example is as follows: faas concrete_f { properties { lambda_role_name = "DemoLambdaRole" lambda_runtime = "python3.8" lambda_handler = "image_resize.lambda_handler" lambda_timeout = 5 lambda_memory = 128 } maps f } A doml script example is as follows: faas concrete_f { properties { lambda_role_name = "DemoLambdaRole" lambda_runtime = "python3.8" lambda_handler = "image_resize.lambda_handler" lambda_timeout = 5 lambda_memory = 128 } maps f } A doml script example is as follows: A doml script example is as follows: 2.1.5.1 DOMLIZER The objective of the DOMLIZER is to simplify the access of new DOML users to existing resources, like, for example, existing virtual machine descriptions, resources made available by runtime © PIACERE Consortium Contract No. GA 101000162 Page 17 of 51 www.piacere-project.eu The objective of the DOMLIZER is to simplify the access of new DOML users to existing resources, like, for example, existing virtual machine descriptions, resources made available by runtime Page 17 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 providers, etc. The PIACERE framework includes an element called the Infrastructure Elements Catalogue-IEC (hereinafter the Catalogue), which stores definitions of such existing resources. The information stored in the Catalogue, however, is not in DOML format, as it includes specific data that is relevant for some of the tools in the PIACERE framework, but not to the user (e.g., monitoring and IOP). The DOMLIZER tool provides the required API to link the elements in the Catalogue to DOML models, performing a translation and including only the information relevant to the user in the model. During the last year DOMLIZER has been improved to provide conversion support for the new types of resources that have been introduced into the Catalogue: • Existing resource element. This element describes existing virtual machines, databases and storages. • Image element. This element describes existing virtual machine images. The DOMLIZER API allows the tools to request a Catalogue element that is returned according to the DOML syntax. Among the other tools, the IDE has been equipped with a shortcut to the DOMLIZER API to easily include Catalogue elements into the current DOML model with just one click. T The DOMLIZER API allows the tools to request a Catalogue element that is returned according to the DOML syntax. Among the other tools, the IDE has been equipped with a shortcut to the DOMLIZER API to easily include Catalogue elements into the current DOML model with just one click. T 2.1.5.2 DOML-DOMLX conversion service The IDE has integrated in the last year a REST service to convert DOML models from the textual DOML files to the XML-based DOMLX format. This tool supports the rest of the IDE tools to easily access the DOML format that best suits them to implement the functionality they are providing. AFT The implementation of the tool has been done using Xtext and Eclipse Modelling Framework. At the moment, the development of the backward conversion from DOMLX to DOML is under development. To ensure that files converted back from DOMLX are maintainable and readable by users, the service is being implemented using a DOML specific formatter that keeps the DOML files tidy and clean. DRAF The service has been deployed in: https://d2x.ci.piacere.digital.tecnalia.dev/ and it uses the text of the DOML/DOMLX file as input for the conversion. DR 2.2 Changes in the latest version D Compared to DOML v2.1, some modifications have been applied to both the metamodel an the syntax in DOML v3.0. D Major changes consist in the introduction of some new concepts in DOML. A few concepts (e.g., “DeployableElement”, “Node”) have been introduced to improve the formal representation of some components, while some other concepts have been added to express new concepts needed for the use case scenarios (e.g., “Source”) and to extend the variety of aspects covered in DOML in a proper manner (e.g., “MonitoringRule”, “GenericResource”). This has led to achieve a higher degree of completeness and expressiveness of the language. Furthermore, some minor changes have been applied to fix inconsistencies between the metamodel and the syntax and to solve some technical issues arisen during the development of use cases. Finally, a feasibility study has been conducted to improve the uniformity and conciseness of the syntax, which was consequently slightly modified to be easier to use. © PIACERE Consortium Contract No. GA 101000162 Page 18 of 51 www.piacere-project.eu Further details on such changes can be found in the Annex [2]. Further details on such changes can be found in the Annex [2]. Contract No. GA 101000162 Page 18 of 51 Page 18 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 2.3 Functional description and requirements coverage The development of the DOML has been guided by the requirements that have been defined with the collaboration of all PIACERE partners as part of Deliverables D2.1 [10] and D2.2_v1.1 [8] (this last one provides the latest version of such requirements and of the PIACERE architecture). Additionally, the work done has been based on the definition of specific scenarios that have guided the development of the new version of DOML from the beginning of the second year of the project. In this section, it has been provided a summary of the current accomplishment of the requirements relevant to the DOML and a final version of the specific scenarios fulfilled by the language. 2.3.1 Requirements coverage For the sake of clarity, the requirements are split in two tables, one focusing on the general characteristics of the DOML (Table 2) and another concerning the elements of applications and infrastructures the DOML should represent (Table 3). For each requirement, an explanation of the level of achievement is provided together with an explanation. Requirements have been also reordered to have the most general ones at the beginning of the Tables 2-3 followed by more specific ones. For the sake of traceability, the requirement identifiers defined as part of WP2 have been kept. T Table 1. Requirements on the general characteristics of DOML. Req ID Description Level of achievement and justification REQ63 DOML must be unambiguous. Achieved: DOML is formally defined in terms of its translation into the corresponding IaC code fragments. As such, it is not ambiguous by definition. REQ62 DOML must support different views. Achieved: DOML allows models to be defined on a per-layer basis. Layers represent different viewpoints on the system: 1) in the application layer, the definition of the application components and the dependencies between them. 2) in the abstract infrastructure layer, an abstract definition of the needed infrastructure, represented in terms of categories of elements and their mapping with the application-level components they are in charge of executing. 3) in the concrete infrastructure layer, a definition of the proper configuration information for the concrete infrastructure elements to be used and their association to the corresponding abstract elements. 4) In the optimization layer, a definition of multi-objective optimization problem of infrastructure resource provisioning. REQ70 The DOML should allow users to state correctness properties in a suitable sub- language (possibly Formal Logic). Achieved: This requirement is addressed in the DOML in two different ways: 1) The main correctness relationships among elements in the specification are directly defined as part of the language semantics and are verified by the Model Checker [11]. 2) It is also possible to express in the DOML some generic constraints that, once again, are verified by the Model Checker. REQ76 DOML should allow the user to model Achieved: DOML models include the information relevant to the listed phases. DRAFT © PIACERE C ti C t t N GA 101000162 P 19 f 51 Table 1. Requirements on the general characteristics of DOML. Req ID Description Level of achievement and justification REQ63 DOML must be unambiguous. 2.3.1 Requirements coverage Achieved: DOML is formally defined in terms of its translation into the corresponding IaC code fragments. As such, it is not ambiguous by definition. REQ62 DOML must support different views. Achieved: DOML allows models to be defined on a per-layer basis. Layers represent different viewpoints on the system: 1) in the application layer, the definition of the application components and the dependencies between them. 2) in the abstract infrastructure layer, an abstract definition of the needed infrastructure, represented in terms of categories of elements and their mapping with the application-level components they are in charge of executing. 3) in the concrete infrastructure layer, a definition of the proper configuration information for the concrete infrastructure elements to be used and their association to the corresponding abstract elements. 4) In the optimization layer, a definition of multi-objective optimization problem of infrastructure resource provisioning. REQ70 The DOML should allow users to state correctness properties in a suitable sub- language (possibly Formal Logic). Achieved: This requirement is addressed in the DOML in two different ways: 1) The main correctness relationships among elements in the specification are directly defined as part of the language semantics and are verified by the Model Checker [11]. 2) It is also possible to express in the DOML some generic constraints that, once again, are verified by the Model Checker. REQ76 DOML should allow the user to model Achieved: DOML models include the information relevant to the listed phases. DRAFT Table 1. Requirements on the general characteristics of DOML. FT Table 1. Requirements on the general characteristics of DOML. FT 1) in the application layer, the definition of the application components and the dependencies between them. DR 2) in the abstract infrastructure layer, an abstract definition of the needed infrastructure, represented in terms of categories of elements and their mapping with the application-level components they are in charge of executing. DR 3) in the concrete infrastructure layer, a definition of the proper configuration information for the concrete infrastructure elements to be used and their association to the corresponding abstract elements. Contract No. 2.3.1 Requirements coverage GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 information needed for each of the four considered DevOps activities (Provisioning, Configuration, Deployment, Orchestration) REQ57 It is desirable to enable both forward and backward translations from DOML to IaC and vice versa Partially achieved: DOML currently supports to the forward translation to different IaC, e.g., Terraform and Ansible. Enabling backward translations could open up the possibility to incorporate existing IaC definitions into the DOML, thus increasing reuse and the potential impact of the DOML itself. For the above reason, we considered this as an interesting requirement. However, it could not be addressed in the timeframe of the project, given the complexity of the forward translation that had to be studied and considered in several different cases. T Table 2. Requirements on the specific elements to be modelled in DOML. Req ID Description Level of achievement and justification REQ01 The DOML must be able to model infrastructural elements. Achieved: This requirement is addressed by the DOML by offering primitives to represent the most relevant infrastructural elements: containers, virtual machines, network elements, security groups, etc. Clearly, the exhaustive definition of infrastructural elements as base types in the DOML is not possible. For this, the DOML will offer the possibility to define new elements through the extension mechanisms (DOML-E). REQ25 DOML should support the modelling of security rules (e.g., by type tcp/udp..., and ingress/egress port definition) Achieved: This requirement is fulfilled by the new concept of security group, which contains both ingress and egress security rules. It is also possible to specify the communication protocol. REQ26 DOML should support the modelling of security groups (containers for security rules) Achieved: This requirement is addressed by a specific construct in the language. REQ27 DOML should support the modelling, provisioning, configuration, and usage of container engine execution technologies (e.g., docker-host) Achieved: The DOML addresses this requirement by offering constructs to define a container, a container image, and a container file. A container can then be mapped on multiple hosts and ports. REQ28 DOML should support the modelling of containerized application deployment (e.g., pull/run/restart/stop docker containers) Achieved: As stated for REQ27, the DOML offers the possibility to model containers and its constituents. As stated for REQ76, the DOML does not support the explicit modelling of workflows to which the pull/run/restart/stop activities belong to. 2.3.1 Requirements coverage DOML, however, supports the possibility to link elements to script files of various kinds. This opens up the possibility to define specific low-level operations on containers as part of these script files. DRAFT Achieved: This requirement is addressed by the DOML by offering primitives to represent the most relevant infrastructural elements: containers, virtual machines, network elements, security groups, etc. Clearly, the exhaustive definition of infrastructural elements as base types in the DOML is not possible. For this, the DOML will offer the possibility to define new elements through the extension mechanisms (DOML-E). RAFT Achieved: This requirement is fulfilled by the new concept of security group, which contains both ingress and egress security rules. It is also possible to specify the communication protocol. R Contract No. GA 101000162 Contract No. GA 101000162 Page 20 of 51 © PIACERE Consortium www.piacere-project.eu D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 REQ29 DOML should support the modelling of VM provisioning for different platforms such as (OpenStack, AWS) for canary and production environments Achieved: This requirement is fulfilled with the possibility to support different platforms for VM provisioning. REQ30 DOML should enable support for policy definition constraints for QoS/NFR requirements Achieved: DOML supports the definition on QoS/NFR requirements (see REQ61) as well as the definition of monitoring rules. Further needs for the definition of additional policies did not emerge so far. REQ58 DOML should offer the modelling abstractions to define the outcomes of the IoP Achieved: This requirement has been fulfilled by introducing the optimization solution concept which is composed of the results of the optimization together with the decision variables. REQ59 The DOML should allow users to define rules and constraints for redeployment, reconfiguration, and other mitigation actions Achieved: The DOML addresses it by supporting the definition of the requirements and constraints that should be considered while performing mitigation actions. These concern, for instance: • the structural characteristics of the infrastructural elements to be used (if the user states that a VM with 16 GB of RAM should be used for executing a certain application component, any change of VM should ensure that this requirement is still fulfilled) or • the definition of non-functional requirements predicating on response time, availability, or other characteristics of application components. 2.3.1 Requirements coverage Moreover, the DOML language supports the definition of monitoring rules, in terms of monitoring conditions that can trigger the execution of other monitoring and reconfiguration strategies and the configuration of such strategies. REQ60 DOML should support the modelling of security metrics both at the level of infrastructure and application Achieved: DOML now includes a specific syntax to describe security metrics and monitoring rules associated to them. Now the DOML supports the definition of strategies based on Ansible playbook to enforce some security strategies. REQ61 DOML must support the modelling of NFRs and of SLOs Achieved: NFRs and SLOs definition is supported in DOML and used to describe the constraints for the IOP (infrastructure optimization). REQ36 DOML to enable writing infrastructure tests. Partially achieved: Infrastructure testing typically focus on injecting faults in specific points of the infrastructure and then observing the reaction of the system. Chaos engineering is the discipline that focuses on this aspect. A study on the tools adopted in chaos engineering has been conducted as part of a thesis work [12]. Some of the available tools can be configured with the definition of the infrastructure and application to be tested. In this respect, a DOML model describing an application and the underlying infrastructure can potentially be used as an input for such tools. This aspect will be explored as future work beyond the end of the project. DRAFT Achieved: This requirement is fulfilled with the possibility to support different platforms for VM provisioning. • the structural characteristics of the infrastructural elements to be used (if the user states that a VM with 16 GB of RAM should be used for executing a certain application component, any change of VM should ensure that this requirement is still fulfilled) or AFT • the definition of non-functional requirements predicating on response time, availability, or other characteristics of application components. AFT Page 21 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 REQ111 The user could have the possibility to add external custom own IaC Achieved: This requirement has been introduced in the last project year to enable reuse of pre-existing IaC artifacts. This scenario has been implemented as-is. © PIACERE Consortium Contract No. GA 101000162 Page 22 of 51 www.piacere-project.eu 2.3.2.2 Scenario 2: insert a new DOML element in a model with the guidance of the IDE helpers Scenario: Insert a new DOML element in a DOML model Given An empty DOML model When user starts typing the keyword software_component or infrastructure or ... And continues with an identifier for the element to be added And adds needed details (properties or attributes defined for the specific element type) Then the new element is created 2.3.2.1 Scenario 1: create an empty DOML model D Scenario: Create a new empty DOML model Given An installed PIACERE IDE When user starts a new PIACERE DevOps project Then a new DOML file is created 2.3.1 Requirements coverage The DOML has been modified to allow the user to specify that a software component can be managed through a pre-existing source file interpreted by an engine that can be specified as part of a DOML model. More details on this point are described in Section 2.3.2.4. Achieved: This requirement has been introduced in the last project year to enable reuse of pre-existing IaC artifacts. The DOML has been modified to allow the user to specify that a software component can be managed through a pre-existing source file interpreted by an engine that can be specified as part of a DOML model. More details on this point are described in Section 2.3.2.4. 2.3.2 Scenarios This section presents the scenarios of DOML usage that have been specifically analysed and addressed starting from the second project year. The structure of these scenarios is aligned with the guidelines associated to agile development using Gherkin syntax (https://cucumber.io/docs/gherkin/). They concern the following aspects: T • Create an empty DOML model and insert elements in a guided way in the model. T • Define a container and associate a software component to it. FT • Associate a software component to specific IaC code. FT • Define functional and non-functional requirements. AFT • Extend the DOML with new resources/providers. AF The main feature to focus on is the following. RA The main feature to focus on is the following. RA Feature: Creation of a new DOML for a specific software application RA Feature: Creation of a new DOML for a specific software application RA Feature: Creation of a new DOML for a specific software application As a PIACERE user I want to create a new DOML model to automate the provisioning of the corresponding resources and the deployment of the whole software stack and its configuration DRA As a PIACERE user I want to create a new DOML model to automate the provisioning of the corresponding resources and the deployment of the whole software stack and its configuration DRA The following scenarios defined have been all realized. While the purpose and the main characteristic of each scenario remained the same through the development of the DOML, the adopted DOML syntax has been improved. The one shown in the following subsections is the last one aligned with DOML 3.0. DR 2.3.2.2 Scenario 2: insert a new DOML element in a model with the guidance of the IDE helpers Scenario: Insert a new DOML element in a DOML model Given An empty DOML model p y When user starts typing the keyword software_component or infrastructure or And continues with an identifier for the element to be added And adds needed details (properties or attributes defined for the specific element type) Then the new element is created Page 22 of 51 Page 22 of 51 Contract No. GA 101000162 © PIACERE Consortium www.piacere-project.eu D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 This scenario has been implemented as-is. © PIACERE Consortium Contract No. GA 101000162 Page 23 of 51 www.piacere-project.eu Scenario: Associate a software component to specific IaC code Given a DOML model When a user digits something like the following software_component nio3_git { 2.3.2.5 Scenario 6: create an autoscaling group 3 5 Sce a o 6 c eate a autosca g g oup Scenario: Create an autoscaling group Given a DOML model When a user digits: autoscale_group ag { vm vm_template { cpu_count 2 mem_mb 1024.0 iface i1 { belongs_to net1 } credentials ssh_pass } min 1 max 2 } Then autoscaling group is created, And it contains a template for creating a VM instance with the specific requirements on CPU, memory, etc. And the scale is specified by the minimum and maximum number of VMs RAFT Scenario: Create an autoscaling group Given a DOML model Given a DOML model When a user digits: autoscale_group ag { When a user digits: When a user digits: autoscale_group ag { autoscale_group ag { vm vm_template { cpu_count 2 mem_mb 1024.0 credentials ssh_pass credentials ssh_pass } min 1 max 2 } min 1 max 2 Then autoscaling group is created, And it contains a template for creating a VM instance with the specific requirements on CPU, memory, etc. And the scale is specified by the minimum and maximum number of VMs RA This scenario hasn’t changed with regards to the previous version presented in D3.2 [1], since there were not modifications related to autoscaling groups in the DOML syntax from v2.1 to v3.0. DR 2.3.2.3 Scenario 3 and 4: define a container and associate a component to i The code is meant to be executed starting from the specified entry, with the specified backend (Ansible in the example). source s1 { entry "ansible/provision.yml" backend "ansible" } properties { nexus_docker_registry_user = "*"; nexus docker registry password = ""; } The software_component nio3_git relevant code is found in a folder in the local workspace. The code is meant to be executed starting from the specified entry, with the specified backend (Ansible in the example). Compared to its previous version in D3.2 [1], this scenario has been slightly changed, since it has been decided to have the relevant code for the necessary software components located in a folder in the project workspace instead of supporting the usage of external URIs in DOML v3.0. 2.3.2.3 Scenario 3 and 4: define a container and associate a component to i Scenario: Define the container as part of the infrastructure and associate a component to it Given a DOML model When user digits something like: When user digits something like: When user digits something like: software_component c1 { source s1 { // e.g., ansible_code.yml entry "..." // e.g., ansible backend "..." container co1 { host vm1 { ... } } cont_image co1_img { generates co1 image "docker.hub.io/myhub/co1:1.0" } vm vm1 { os "CentOS-7-2111" cpu_count 2 mem_mb 8192.0 iface i1 { //belongs_to net1 } } ... deployment infra_config { c1 -> co1 } Then software_component c1 is meant to be deployed within the container co1, which is created from the specified image and is mapped into vm vm1. DRAFT container co1 { host vm1 { cont_image co1_img { generates co1 image "docker.hub.io/myhub/co1:1.0" } A Then software_component c1 is meant to be deployed within the container co1, which is created from the specified image and is mapped into vm vm1. D In D3.2 [1] this scenario was divided in two different ones. Here they have been grouped together as the container details are not anymore defined within the definition of a component as it was in the initial idea. In DOML 3.0 the container instantiation is handled through the usage of a specific syntax to describe from which image has been generated. The association between a container and a VM is managed as a host configuration within the container definition, instead of being specified in the deployment section. The credentials-related part is not necessary anymore, since the access to the docker repository, where the container image resides, is managed by the PIACERE IEM component [13]. 2.3.2.4 Scenario 5: associate a software component to a specific IaC code © PIACERE Consortium Contract No. GA 101000162 Page 23 of 51 www.piacere-project.eu 2.3.2.4 Scenario 5: associate a software component to a specific IaC code Scenario: Associate a software component to specific IaC code Given a DOML model When a user digits something like the following software_component nio3_git { Page 23 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 Version 1.0 – Final Date: 31.05.2023 source s1 { entry "ansible/provision.yml" backend "ansible" } properties { nexus_docker_registry_user = ""; nexus_docker_registry_password = "*"; } } The software_component nio3_git relevant code is found in a folder in the local workspace. 2.3.2.7 Scenario 8: extend the DOML with new resources T Scenario: Extending the DOML with new resources Given the existing DOML metamodel When a user creates a new class extending a specific DOML metaclass for a new resource And adding the needed attributes and references to other elements in the class And creating the desired concrete syntax in the grammar definition AFT Scenario: Extending the DOML with new resources Given the existing DOML metamodel When a user creates a new class extending a specific DOML metaclass for a new resource A d ddi th d d tt ib t d f t th l t i th l FT When a user creates a new class extending a specific DOML metaclass for a new resource FT And adding the needed attributes and references to other elements in the class And creating the desired concrete syntax in the grammar definition Then a new DOML supporting a specific new resource is created AFT This scenario has been implemented as-is, as discussed in Section 2.1.4. RAF and vm has attribute infrastructure.ComputingNode->os Os1 "Virtual Machine {vm} has no iface" } Then functional requirements are created (which can be some external requirements in external DSL like the example) } Then functional requirements are created (which can be some external requirements in external DSL like the example) q p ) And they are dedicated to the verification tools When a user digits: q p And they are dedicated to the verification tools When a user digits: g nonfunctional_requirements { _ q req1 "Cost <= 70.0" max 70.0 => "cost"; _ req1 "Cost <= 70.0" max 70.0 => "cost" req1 "Cost <= 70.0" max 70.0 => "cost"; q req2 "Availability >= 66.5%" min 66.5 => "availability"; } Then the nonfunctional requirements are created (which can be some numerical constraints like the example) And they are dedicated to the Optimization tools This scenario has been implemented as-is. 2.3.2.6 Scenario 7: define functional and non-functional requirements D © PIACERE Consortium Contract No. GA 101000162 Page 24 of 51 www.piacere-project.eu Scenario: Define functional and non-functional requirements Given a DOML model When a user digits: functional_requirements { req_ext ``` > "example requirement to test" # Expr to parse not ( vm is class infrastructure.VirtualMachine and vm is not class infrastructure.Storage or vm is not class infrastructure.Storage implies vm is class infrastructure.Storage ) iff not exists iface, apple ( forall orange ( vm has association infrastructure.ComputingNode->ifaces iface or vm has association infrastructure.ComputingNode->ifaces iface D Scenario: Define functional and non-functional requirements Given a DOML model D When a user digits: When a user digits: functional_requirements { functional_requirements { © PIACERE Consortium Contract No. GA 101000162 Page 24 of 51 www.piacere-project.eu req_ext > "example requirement to test" # Expr to parse not ( vm is class infrastructure.VirtualMachine and vm is not class infrastructure.Storage or vm is not class infrastructure.Storage implies vm is class infrastructure.Storage ) iff not exists iface, apple ( forall orange ( vm has association infrastructure.ComputingNode->ifaces iface or vm has association infrastructure.ComputingNode->ifaces iface > "example requirement to test" p vm is class infrastructure.Storage ) Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final Date: 31.05.2023 2.4 Main innovations R As discussed in Section 2.1.1, one of the main innovations introduced by the DOML concerns the possibility to define an infrastructure at an abstract level, using provider and technology- independent concepts (e.g., virtual machine, container, network, …), and to instantiate it in multiple concrete infrastructures. Such concrete infrastructure definitions can coexist in the same DOML model. Moreover, the selection of the concrete infrastructures to adopt can be delegated to the IOP component that will select the optimal one or more based on criteria that are defined as part of the DOML model itself, thus reducing the effort required to DevOps teams. DRA To evaluate the effectiveness of the DOML from the simplicity-to-use perspective, in [7], DOML models have been compared with Terraform and TOSCA/Cloudify-based specifications. The analysis highlights that Terraform and TOSCA/Cloudify IaC is heavily dependent on the selected provider, where each provider brings in a custom set of resources showing different parameters and configuration possibilities that must be mastered by the user willing to create an infrastructure. Another issue is the difficulty of acquiring from a Terraform code fragment an overview of the entire system to be run as the attention is exclusively focused on the infrastructural aspects. D The possibility offered by DOML to define in a single model both the application and the underlying abstract and concrete infrastructure opens up to the possibility to verify the correctness of the model and, therefore, the instantiation of the application on top of the infrastructure and, in turn, the mapping of the abstract infrastructure on a concrete one. This verification is achieved through the model checker that is integrated within the PIACERE IDE and fully compatible with the DOML. Contract No. GA 101000162 Page 25 of 51 Version 1.0 – Final Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Another important innovation concerns the clear distinction that has been introduced between the external DOML representation and the corresponding internal one (also called DOMLX). The DOML-DOMLX conversion service (see Section 2.1.5) is the new component in charge of managing the DOMLX and any needed translation. Thanks to its availability, the compatibility between different DOML versions can be managed by translating an external DOML representation written in one version of the language into DOMLX and then, from this, back into a different version of the external language representation. 2.4 Main innovations R Finally, other innovations critical to the consolidation of the whole PIACERE project consist in the following aspects: • Consolidation and clean-up of the DOML modelling language which has now reached version 3.0. Through the PIACERE project, the following main versions of the language have been released: 1.0 at the end of the first project year, 2.0 and 2.1 during the second project year, 2.2.1 and 2.2.2 in the third year. • Consolidation and clean-up of the DOML modelling language which has now reached version 3.0. Through the PIACERE project, the following main versions of the language have been released: 1.0 at the end of the first project year, 2.0 and 2.1 during the second project year, 2.2.1 and 2.2.2 in the third year. • Consolidation of the extension mechanisms (DOML-E). • Consolidation of the extension mechanisms (DOML-E). • Transformation of the resource descriptions in the PIACERE catalogue into DOML fragments to support users in selecting resources and incorporating them in a DOML model. This is done through the supporting component called DOMLIZER. T • Elaboration of the examples of use of the DOML language using the PIACERE IDE, their validation through the Model Checker, their optimization through the IOP tool, and the generation of the corresponding IaC code through the ICG. FT • Elaboration of the examples of use of the DOML language using the PIACERE IDE, their validation through the Model Checker, their optimization through the IOP tool, and the generation of the corresponding IaC code through the ICG. FT DRAFT Contract No. GA 101000162 Page 26 of 51 © PIACERE Consortium www.piacere-project.eu D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 3 Overview of preliminary experiments The DOML is currently being used and experimented in the PIACERE case studies and it is expected to have a complete evaluation, which will include also DOML-E, at the end of the project. Until now, the following actions have been performed: Until now, the following actions have been performed: • defined multiple examples that can be called reference test cases and that can be used to check whether the DOML fulfils the expectation and whether the other tools of the PIACERE toolchain work as expected; • conducted a first rigorous empirical evaluation by comparing the performance of the DOML with the one of two well-known existing IaC languages, that is, Terraform and Cloudify. In Section 3.1 , a short summary of the empirical evaluation is presented while in Section 3.2 the reference test case examples are discussed. T In Section 3.1 , a short summary of the empirical evaluation is presented while in Section 3.2 the reference test case examples are discussed. T 2 The reader should note that, as described in Section 2.3.2.4, a DOML model can be linked to external scripts. Such possibility allows users to integrate legacy IaC into a DOML model, but this is not mandatory for supporting the provisioning and configuration operations. 3.2 Reference test cases example 3.2 Reference test cases example 3.2 Reference test cases example 3.1 Empirical evaluation In [7] it has been conducted a first evaluation of the DOML by addressing two specific research questions: FT • RQ1: Can a DOML model represent the information required to generate executable IaC tackling both provisioning and configuration? Is a DOML model more readable and easier to use than the state-of-the-art approaches? AFT • RQ1: Can a DOML model represent the information required to generate executable IaC tackling both provisioning and configuration? Is a DOML model more readable and easier to use than the state-of-the-art approaches? AFT • RQ2: Is a DOML model able to target multiple execution platforms? RAF The first research question has been addressed by comparing the IaC code defined by independent researchers in [14] using two different languages, Terraform and Cloudify with an equivalent DOML model. The objective results obtained in this experiment are summarized in Table 3 where, for each of the three considered approaches, the number of lines of code (#LOC), the number of files (#File) composing the specifications, and the number of used languages (#Languages) are listed. DRA Table 3. Comparison between DOML and other IaC approaches. D Table 3. Comparison between DOML and other IaC approaches. Approach #LOC #Files #Languages DOML 103 1 1 Terraform 305 3 2 Cloudify 506 9 2 D Essentially, the table shows that the DOML model taken as example is more concise compared to its counterparts as it requires less lines of code and does not require the usage of additional external languages that are instead required by Terraform and Cloudify2. 2 The reader should note that, as described in Section 2.3.2.4, a DOML model can be linked to external scripts. Such possibility allows users to integrate legacy IaC into a DOML model, but this is not mandatory for supporting the provisioning and configuration operations. The reader should note that, as described in Section 2.3.2.4, a DOML model can be linked to external ripts. Such possibility allows users to integrate legacy IaC into a DOML model, but this is not mandatory r supporting the provisioning and configuration operations. Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 As for the second research question, it has been shown that a DOML model can target multiple providers by specifying their resources within different concretizations but keeping the abstract infrastructure layer untouched. 3.2.1 Introduction and comparative table During the development of the DOML, four “test case examples” have been developed aiming at exercising the specific elements of the language in different contexts. These examples are the following: • a web application using NginX as web server and a DBMS; • a WordPress application; • an application exploiting the FaaS (Function as a Service) paradigm; • a simplified version of the case study developed by Ericsson for PIACERE and focusing on the configuration of a relatively complex network infrastructure. T • a web application using NginX as web server and a DBMS; • a WordPress application; • a simplified version of the case study developed by Ericsson for PIACERE and focusing on the configuration of a relatively complex network infrastructure. T The NginX and the Wordpress applications are presented in this section while, for the sake of brevity, the Ericsson case is available in the Appendix (Section 7) and the FaaS example, which resulted not to be of interest of the PIACERE partners, is available on the DOML public repository3. The selection of these specific examples aims at ensuring a significant coverage of the language elements, as summarized in Table 4, where, for each DOML layer, the concepts used by each example are listed. AFT © PIACERE Consortium Contract No. GA 101000162 Page 28 of 51 www.piacere-project.eu Example Application Layer Infrastructure Layer Concrete Layer Optimization Layer NginX • DBMS • Software component • VM • Network with Internet gateway • Interconnected subnets • Key pair credentials • Security group • Concrete VM • Concrete Network • 2 Optimization Objectives • NFR: Region requirement • NFR: Provider requirement • NFR: Elements to • be deployed requirement WordPress • DBMS • Software component • Alternative example with SaaS DBMS • VM • Network with subnet • Autoscaling group • Key pair credentials • Security group • VM image • Container • Concrete VM • Concrete Network • Concrete Autoscaling group • Concrete pre- existing VM image with a given image name / 3 https://git.code.tecnalia.com/piacere/public/the-platform/doml DRAF D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. 3.2.1 Introduction and comparative table Date: 31.05.2023 • Container image • Concrete pre- existing container image • Example of two different concrete infrastructures using different providers FaaS • Software component • Source code for a given software component • SaaS • Network • Container • Container image • Key pair credentials • FaaS • Storage • Autoscaling group • Security group • Concrete Network • Concrete Autoscaling group • Concrete FaaS • Concrete Storage • Concrete pre- existing VM image • Concrete pre- existing container image / Simplified Ericsson • Software component • SaaS • Example of provided software interface at a given URL • VM with more than one network interface • Several networks with different interconnected subnets • Networks with Internet gateways • Key pair credentials • Security group • Concrete VM • Concrete Network • 3 Optimization Objectives (all the available ones) • NFR: Cost requirement • NFR: Performance requirement • NFR: Provider requirement • NFR: Elements to be deployed requirement Table 4. Comparative table for test cases examples 3.2.2 WordPress Website WordPress is a popular open-source Content Management System (CMS) that can be used to easily develop blogs and other kinds of websites. WordPress is written in PHP programming DRAFT 3.2.2.1 Application Layer Next, the application layer is defined. application app { dbms postgres { properties { identifier = "education" name = "wp_db" } provides { SQL_interface } } software_component wordPressServer { consumes { SQL_interface } } } DRAFT The application layer consists of two components: the postgres database and the WordPress server. Here the relationships between these components are declared: the database provides a SQL interface, which is consumed by the WordPress server. Additionally, here the properties of the DBMS are defined. They are agnostic with respect to the DBMS implementation in the infrastructure layer, examples are its name and identifier. The database credentials are not handled directly in the DOML code, since having them unencrypted would result in a bad practice, leading to potential security issues. Instead, they are managed by other components in the PIACERE framework. DR 3.2.2 WordPress Website 3.2.2 WordPress Website WordPress is a popular open-source Content Management System (CMS) that can be used to easily develop blogs and other kinds of websites. WordPress is written in PHP programming © PIACERE Consortium Contract No. GA 101000162 Page 29 of 51 www.piacere-project.eu Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 language, so it needs to be run on a server with the appropriate runtime environment properly configured. It also needs a SQL database as a backend for storing website data. language, so it needs to be run on a server with the appropriate runtime environment properly configured. It also needs a SQL database as a backend for storing website data. The structure of the WordPress application is that: a WordPress is running in a container which is hosted in a VM provisioned by a provider, e.g., AWS. VM is defined in an autoscaling group where the size is defined as two. WordPress is connecting to a database through network. The code of the example is reported below, divided into several code snippets, which are explained separately. In practice, all such code snippets are merged in a single DOML file. oml wordpress In the first line of the file, the name of the DOML model is declared. the first line of the file, the name of the DOML model is declared. In the first line of the file, the name of the DOML model is declared. 3.2.2.1 Application Layer Next, the application layer is defined. application app { dbms postgres { properties { identifier = "education" name = "wp_db" } provides { SQL_interface } } software_component wordPressServer { consumes { SQL_interface } } } The application layer consists of two components: the postgres database and the WordPress server. Here the relationships between these components are declared: the database provides a SQL interface, which is consumed by the WordPress server. Additionally, here the properties of the DBMS are defined. They are agnostic with respect to the DBMS implementation in the infrastructure layer, examples are its name and identifier. The database credentials are not handled directly in the DOML code, since having them unencrypted would result in a bad practice, leading to potential security issues. Instead, they are managed by other components in the PIACERE framework. DRAFT 3.2.2.2 Abstract Infrastructure Layer 3.2.2.2 Abstract Infrastructure Layer The abstract infrastructure layer is shown below: infrastructure infra { net net1 { cidr "10.0.1.0/24" protocol "TCP/IP" subnet subnet1 { cidr "10.0.1.0/24" } } The abstract infrastructure layer is shown below: infrastructure infra { net net1 { cidr "10.0.1.0/24" protocol "TCP/IP" subnet subnet1 { cidr "10.0.1.0/24" } © PIACERE Consortium Contract No. GA 101000162 Page 30 of 51 www.piacere-project.eu D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 vm dbms_vm { os "ubuntu" size "micro" iface dbms_iface { belongs_to subnet1 address "10.0.1.2" } credentials ssh_key } autoscale_group ag { vm wp_vm { os "ubuntu" size "micro" cpu_count 2 mem_mb 1024.0 iface i1 { belongs_to subnet1 address "10.0.1.1" } credentials ssh_key } // count = 2 min 2 max 2 } vm_image vm_img { generates wp_vm } key_pair ssh_key { user "user" keyfile "ssh key" } container container1 { properties { WP_DB_HOST = "dbms_vm" WP_DB_USER = "username" WP_DB_PASSWORD = "password" WP_DB_NAME = "database.name" } host wp_vm { container_port 80 vm_port 8080 iface i1 } } cont_image container_image { generates container1 image "docker.hub.io/wordpress/wordpress:5.8.0" } security_group sg { egress icmp { from_port -1 to_port -1 protocol "ICMP" cidr ["0.0.0.0/0"] } DRAFT © PIACERE Consortium Contract No. 3.2.2.2 Abstract Infrastructure Layer GA 101000162 Page 31 of 51 www.piacere-project.eu _ mem_mb 1024.0 iface i1 { belongs_to subnet1 address "10.0.1.1" } credentials ssh_key } // count = 2 min 2 max 2 } vm_image vm_img { generates wp_vm } key_pair ssh_key { user "user" keyfile "ssh key" } container container1 { properties { WP_DB_HOST = "dbms_vm" WP_DB_USER = "username" WP_DB_PASSWORD = "password" WP_DB_NAME = "database.name" } host wp_vm { container_port 80 vm_port 8080 iface i1 } } cont_image container_image { generates container1 image "docker.hub.io/wordpress/wordpress:5.8.0" } security_group sg { egress icmp { from_port -1 to_port -1 protocol "ICMP" cidr ["0.0.0.0/0"] } ingress http { from_port 80 to_port 80 protocol "TCP" cidr ["0.0.0.0/0"] } DRAFT } credentials ssh_key } // count = 2 min 2 max 2 } vm_image vm_img { generates wp_vm } key_pair ssh_key { user "user" keyfile "ssh key" } container container1 { properties { WP_DB_HOST = "dbms_vm" WP_DB_USER = "username" WP_DB_PASSWORD = "password" WP_DB_NAME = "database.name" } host wp_vm { container_port 80 vm_port 8080 iface i1 } } DRAFT } cont_image container_image { generates container1 image "docker.hub.io/wordpress/wordpress:5.8.0" } cont_image container_image { generates container1 image "docker.hub.io/wordpress/wordpress:5.8.0" } cont_image container_image { generates container1 image "docker.hub.io/wordpress/wordpress:5.8.0" } © PIACERE Consortium Contract No. GA 101000162 Page 31 of 51 www.piacere-project.eu D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 ingress https { from_port 443 to_port 443 protocol "TCP" cidr ["0.0.0.0/0"] } ingress ssh { from_port 22 to_port 22 protocol "TCP" cidr ["0.0.0.0/0"] } ifaces i1, dbms_iface } } First, the network is defined, with the specification of the CIDR and protocol and the definition of a subnet. The database VM is defined by specifying a set of attributes, which will determine the VM characteristics. The autoscaling group is defined by providing the template of virtual machine “wp_vm” and the minimum and maximum number of VMs supported. A VM image is used to generate such virtual machine. The key-pair credentials for the virtual machines are then defined. In the next fragment, “container1” is to be hosted on the virtual machine “wp_vm” with the port mapping “8080:80” binding with network interface “i1”. The container is generated from a Docker container image, as specified in the following code fragment. Finally, the security group defines several egress rules (w.r.t. ICMP) and ingress rules (w.r.t. HTTP, HTTPS and SSH) for the network. 3.2.2.2 Abstract Infrastructure Layer AFT Note that no VM image for generating the database VM is provided explicitly. Thus, the ICG will provide one automatically during deployment, by inferring its requirements from the properties of the database component at the application layer, and its being linked with “dbms_vm” and the concrete VM mapping it in the concrete layer. RAF Each component from the application layer is linked to the abstract-infrastructure component that implements it in the following deployment configuration: DRA deployment config1 { wordPressServer => container1, postgres => dbms_vm } active deployment config1 DR The last line states that the deployment configuration above is currently active. In principle, multiple deployment configurations could be defined and switched. 3.2.2.3 Concrete Infrastructure Layer 3.2.2.3 Concrete Infrastructure Layer The abstract infrastructure can be concretized in two ways: con_infra1 uses AWS as a clou provider, while con_infra2 uses OpenStack. The model fragment is shown below: © PIACERE Consortium Contract No. GA 101000162 Page 32 of 51 www.piacere-project.eu concretizations { concrete_infrastructure con_infra1 { provider aws { vm_image concrete_vm_img { preexisting true image_name "ami-012e16cfb2f9e8b0a" maps vm_img } autoscale_group concrete_ag { concretizations { concrete_infrastructure con_infra1 { provider aws { vm_image concrete_vm_img { preexisting true image_name "ami-012e16cfb2f9e8b0a" maps vm_img } autoscale_group concrete_ag { concretizations { concrete_infrastructure con_infra1 { provider aws { vm_image concrete_vm_img { preexisting true image_name "ami-012e16cfb2f9e8b0a" maps vm_img } autoscale_group concrete_ag { Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 maps ag } vm concrete_dbms_vm { maps dbms_vm } net concrete_net { maps net1 } cont_image concrete_wp_image { preexisting true maps container_image } } } concrete_infrastructure con_infra2 { provider openstack { vm_image concrete_vm_img { preexisting true image_name "mantic-20230508" maps vm_img } autoscale_group concrete_ag { maps ag } vm concrete_dbms_vm { maps dbms_vm } net concrete_net { maps net1 } cont_image concrete_wp_image { preexisting true maps container_image } } } active con_infra1 } In both concretizations, a concrete VM for the “dbms_vm” abstract VM and a concrete autoscaling group for the “ag” abstract autoscaling group are defined and linked through the “maps” statement Images and network need to be concretized too Please note that the DRAFT concrete_infrastructure con_infra2 { provider openstack { vm_image concrete_vm_img { preexisting true image_name "mantic-20230508" maps vm_img } autoscale_group concrete_ag { maps ag } vm concrete_dbms_vm { maps dbms_vm } net concrete_net { maps net1 } cont_image concrete_wp_image { preexisting true maps container_image } } } active con_infra1 } DRAFT In both concretizations, a concrete VM for the “dbms_vm” abstract VM and a concrete autoscaling group for the “ag” abstract autoscaling group are defined and linked through the “maps” statement. Images and network need to be concretized, too. Please note that the “wp_vm” VM must not be concretized, since it is defined as a template for the autoscaling group. The VM image name, which is provider specific, is specified in the concrete layer. Any other relevant provider specific property could also be specified here through the usage of properties. 3.2.2.3 Concrete Infrastructure Layer Since the VM image is selected from the provider catalogue and the container image is an existing Docker image, they are both set as preexisting. Finally, the “active” statement sets con_infra1 as the concrete infrastructure to be used fo deployment. Finally, the “active” statement sets con_infra1 as the concrete infrastructure to be used for deployment. © PIACERE Consortium Contract No. GA 101000162 Page 33 of 51 www.piacere-project.eu © PIACERE Consortium Contract No. GA 101000162 Page 33 of 51 www.piacere-project.eu Contract No. GA 101000162 Page 33 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 3.2.2.4 Alternative version with SaaS Database An alternative second version of the WordPress example uses a SaaS database instead of a custom VM deployment. We describe it by reporting only differences compared to the version presented in this section. The application layer changes by replacing the “dbms” component with the following: The application layer changes by replacing the dbms component with the following: saas_dbms postgres { properties { identifier = "education" } provides { SQL_interface } } Thus, the only difference is that its component type is now “saas_dbms”. Thus, the only difference is that its component type is now “saas_dbms”. hus, the only difference is that its component type is now “saas_dbms”. With respect to other layers, the only difference in the Infrastructure Layer is the absence of the “dbms_vm” virtual machine and of its binding with the “database” application in the “deployment” section, and, consequently, the removal of its corresponding “concrete_dbms_vm” concrete instance from both the two concrete infrastructures described in the Concrete Layer. FT With respect to other layers, the only difference in the Infrastructure Layer is the absence of the “dbms_vm” virtual machine and of its binding with the “database” application in the “deployment” section, and, consequently, the removal of its corresponding “concrete_dbms_vm” concrete instance from both the two concrete infrastructures described in the Concrete Layer. FT 3.2.3 NginX This example was developed ad hoc to provide non-expert users with a simple, readab example, showing the syntax and the basic elements to build a DOML model. AF Here a web application, which accesses a database is described. Such an application consists of an NginX web server and a MySQL DBMS, which both run on virtual machines provided by the OpenStack cloud provider. Such virtual machines, which should be both located in Europe, are connected to a common network, which also guarantees access to Internet, and each of them has an associated subnet. Finally, these two subnets are connected to each other. Moreover, the availability and performance of the infrastructure must be maximized, and OpenStack must be used as a cloud provider for the infrastructure. DRA 3.2.3.1 Application Layer D The Application Layer is shown below: D The Application Layer is shown below: D The Application Layer is shown below: doml doml_example1 application app_example1 { dbms mysql { provides { sql_interface } } software_component nginx { consumes { sql_interface } } } D The Application Layer consists of two components: the NginX web server and the MySQL DBMS. The latter one provides a SQL interface to handle data, which is consumed by the web server. © PIACERE Consortium Contract No. GA 101000162 Page 34 of 51 www.piacere-project.eu Page 34 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 3.2.3.2 Abstract Infrastructure Layer The Infrastructure Layer is shown below: infrastructure infra_example1 { vm nginx_vm { arch "x86-64" os "Ubuntu-22.04.2-LTS" mem_mb 1024.0 sto "16" cpu_count 1 size "small" loc { region "00EU" } iface nginx_iface { belongs_to nginx_subnet } credentials nginx_vm_credentials } vm mysql_vm { arch "x86-64" os "Ubuntu-22.04.2-LTS" mem_mb 1024.0 sto "16" cpu_count 1 size "small" loc { region "00EU" } iface mysql_iface { belongs_to mysql_subnet } credentials mysql_vm_credentials } net common_network { subnet nginx_subnet { connections { mysql_subnet } cidr "10.0.144.0/25" } subnet mysql_subnet { connections { nginx_subnet } cidr "10.0.144.128/25" } protocol "TCP/IP" cidr "10.0.144.0/24" gateway igw1 { address "10.0.144.22" } } key_pair nginx_vm_credentials { user "nginx_user" keyfile "ssh key" algorithm "RSA" bits 4096 } key_pair mysql_vm_credentials { user "mysql user" DRAFT 3.2.3.2 Abstract Infrastructure Layer The Infrastructure Layer is shown below: The Infrastructure Layer is shown below: © PIACERE Consortium Contract No. GA 101000162 Page 35 of 51 www.piacere-project.eu mem_mb 1024.0 sto "16" cpu_count 1 size "small" loc { region "00EU" } iface nginx_iface { belongs_to nginx_subnet } credentials nginx_vm_credentials } vm mysql_vm { arch "x86-64" os "Ubuntu-22.04.2-LTS" mem_mb 1024.0 sto "16" cpu_count 1 size "small" loc { region "00EU" } iface mysql_iface { belongs_to mysql_subnet } credentials mysql_vm_credentials } net common_network { subnet nginx_subnet { connections { mysql_subnet } cidr "10.0.144.0/25" } subnet mysql_subnet { connections { nginx_subnet } cidr "10.0.144.128/25" } protocol "TCP/IP" cidr "10.0.144.0/24" gateway igw1 { address "10.0.144.22" } } key_pair nginx_vm_credentials { user "nginx_user" keyfile "ssh key" algorithm "RSA" bits 4096 } key_pair mysql_vm_credentials { user "mysql_user" keyfile "ssh key" algorithm "RSA" bits 4096 } security_group sec_group { egress icmp { DRAFT } vm mysql_vm { arch "x86-64" os "Ubuntu-22.04.2-LTS" mem_mb 1024.0 sto "16" cpu_count 1 size "small" loc { region "00EU" } iface mysql_iface { belongs_to mysql_subnet } credentials mysql_vm_credentials } net common_network { subnet nginx_subnet { connections { mysql_subnet } cidr "10.0.144.0/25" } subnet mysql_subnet { connections { nginx_subnet } id "10 0 144 128/25" DRAFT Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 protocol "ICMP" from_port -1 to_port -1 cidr ["0.0.0.0/0"] } ingress ssh { protocol "TCP" from_port 22 to_port 22 cidr ["0.0.0.0/0"] } ingress http { protocol "TCP" from_port 80 to_port 80 cidr ["0.0.0.0/0"] } ingress https { protocol "TCP" from_port 443 to_port 443 cidr ["0.0.0.0/0"] } ifaces nginx_iface, mysql_iface } } In this layer, the two VMs hosting the web server and the DBMS are described together with the common network to which they need to be connected to. The VMs configuration is set through attributes. For each VM, a network interface belonging to the corresponding subnet is defined together with the credentials to be used to access the VMs. The network is configured to have two different subnets connected to each other and a gateway to guarantee Internet access. Finally, a security group is defined to secure access to the network. Next, the deployment configuration for our application is defined and set as active. The Infrastructure Layer is shown below: deployment config_example1 { mysql => mysql_vm, nginx => nginx_vm } active deployment config_example1 3.2.3.3 Concrete Infrastructure Layer The Concrete Layer is shown below: DRAFT protocol "ICMP" from_port -1 to_port -1 cidr ["0.0.0.0/0"] } ingress ssh { protocol "TCP" from_port 22 to_port 22 cidr ["0.0.0.0/0"] } ingress http { protocol "TCP" from_port 80 to_port 80 cidr ["0.0.0.0/0"] } ingress https { protocol "TCP" from_port 443 to_port 443 cidr ["0.0.0.0/0"] } ifaces nginx_iface, mysql_iface } } In this layer, the two VMs hosting the web server and the DBMS are described together with the common network to which they need to be connected to. AFT © PIACERE Consortium Contract No. GA 101000162 Page 36 of 51 www.piacere-project.eu cidr ["0.0.0.0/0"] } ingress https { protocol "TCP" from_port 443 to_port 443 cidr ["0.0.0.0/0"] } ifaces nginx_iface, mysql_iface } } In this layer, the two VMs hosting the web server and the DBMS are described together with the common network to which they need to be connected to. The VMs configuration is set through attributes. For each VM, a network interface belonging to the corresponding subnet is defined together with the credentials to be used to access the VMs. The network is configured to have two different subnets connected to each other and a gateway to guarantee Internet access. Finally, a security group is defined to secure access to the network. Next, the deployment configuration for our application is defined and set as active. deployment config_example1 { mysql => mysql_vm, nginx => nginx_vm } active deployment config_example1 3.2.3.3 Concrete Infrastructure Layer The Concrete Layer is shown below: concretizations { concrete_infrastructure concrete_example1 { provider openstack { vm concrete_nginx_vm { maps nginx_vm } vm concrete_mysql_vm { maps mysql_vm } net concrete_common_network { maps common_network } } } active concrete_example1 } DRAFT In this layer, the two VMs hosting the web server and the DBMS are described together with the common network to which they need to be connected to. AFT In this layer, the two VMs hosting the web server and the DBMS are described together with the common network to which they need to be connected to. AFT The VMs configuration is set through attributes. For each VM, a network interface belonging to the corresponding subnet is defined together with the credentials to be used to access the VMs. The Infrastructure Layer is shown below: The network is configured to have two different subnets connected to each other and a gateway to guarantee Internet access. Finally, a security group is defined to secure access to the network. Next, the deployment configuration for our application is defined and set as active. DRAF The VMs configuration is set through attributes. For each VM, a network interface belonging to the corresponding subnet is defined together with the credentials to be used to access the VMs. The network is configured to have two different subnets connected to each other and a gateway to guarantee Internet access. Finally, a security group is defined to secure access to the network. Next the deployment configuration for our application is defined and set as active DRAF Next, the deployment configuration for our application is defined and set as active. DRA 3.2.3.3 Concrete Infrastructure Layer D 3.2.3.3 Concrete Infrastructure Layer D The Concrete Layer is shown below: The Concrete Layer is shown below: The Concrete Layer is shown below: © PIACERE Consortium Contract No. GA 101000162 Page 36 of 51 www.piacere-project.eu Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 In this layer, the concrete instances of the corresponding abstract infrastructural elements described in the Infrastructure Layer can be found. The chosen provider is OpenStack, as required for this application. Lastly, the concrete infrastructure is set as active. In this layer, the concrete instances of the corresponding abstract infrastructural elements described in the Infrastructure Layer can be found. The chosen provider is OpenStack, as required for this application. Lastly, the concrete infrastructure is set as active. 3.2.3.4 Optimization Layer 3.2.3.4 Optimization Layer The Optimization Layer is shown below: optimization opt { objectives { "availability" => max "performance" => max } nonfunctional_requirements { Req1 "Region" values "00EU" => "region" Req2 "Provider" values "openstack" => "provider" Req3 "elements" => "VM, VM" } } T 3.2.3.4 Optimization Layer The Optimization Layer is shown below: optimization opt { objectives { "availability" => max "performance" => max } nonfunctional_requirements { Req1 "Region" values "00EU" => "region" Req2 "Provider" values "openstack" => "provider" Req3 "elements" => "VM, VM" } } T The Optimization Layer is shown below: The Optimization Layer is shown below: In the Optimization Layer, it is possible to specify some optimization objectives and non- functional requirements. 4 Lessons Learnt and Plan for Future Development The DOML language represents an attempt to develop a high-level modelling approach for Infrastructure as Code that maps on multiple concrete IaC languages. The experiments carried out so far appear to confirm that the multi-layered approach adopted in DOML is useful as it enables separation of concerns between applications and the underlying infrastructure, but, at the same time, enables an explicit and clear definition of the way applications are mapped on their infrastructures. The possibility to keep the abstract infrastructure definition separated from the concrete one allows users to reuse DOML fragments and map them into different concrete infrastructures. Keeping the DOML internal representation separated from the external one enables the possibility to conceive different external languages both textual and graphical. While the consolidated version of the DOML is a textual notation, experiments on a graphical Domain Specific Language are being conducted. The objective is to derive the graphical representation from the internal DOML representation by exploiting the libraries and tools made available by the Eclipse platform. Some of these experiments have been reported in Deliverable D3.9 [15, 1, 1] and some others are ongoing work that will be reported before the end of the PIACERE project. FT Another important lesson learnt as a result of the latest experiments is that confining crosscutting aspects in a specific layer of the DOML specification is counterproductive. More specifically, at the beginning of the project, for the purpose of parallelizing the research and development work, a simple optimization language has been developed and included in the DOML optimization layer. This particular layer was the only part of the DOML meant to be used by the IOP component to return concrete infrastructural resources fulfilling the optimization objectives and constraints. RAFT In the development of the latest more sophisticated DOML model examples, it has been realized that this approach becomes cumbersome when there are multiple abstract resources, and the user wants to express specific optimization objectives or constraints for each individual resource. In this case, it would have been better to incorporate the optimization aspects within the definition of the resources in the abstract infrastructure layer and to have the IOP to interpret this whole layer to take its decisions. Due to time constraints, at the moment this problem is being addressed in a simplified way by slightly extending the optimization layer language. Improving this part is certainly an important future work. The Infrastructure Layer is shown below: As required for our application, the maximization of both performance and availability are set as optimization objectives. FT Furthermore, as stated in the description of the example, both VMs should be located in the "00EU" region. In order to specify this constraint, a non-functional requirement is defined. Finally, the required provider and the elements we want to deploy as non-functional requirements for the PIACERE Optimizer are defined. AFT DRA Contract No. GA 101000162 Page 37 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 4 Lessons Learnt and Plan for Future Development Another critical point is that a DOML specification typically corresponds to IaC code fragments written in different languages. This implies that a single fragment would result in an incomplete DOML model. For the above reasons, it has been decided to limit the backward transformation on moving from the internal DOML representation, serialized in the so called DOMLX, into the DOML external one. Such internal transformation is at the moment under development and will be released at the end of the project with the objective to used it to support transformations between different versions of the DOML. • REQ57 – “It is desirable to enable both forward and backward translations from DOML to IaC and vice versa”, was considered, since the beginning of the project, as a very challenging, nice to have, and low priority requirement for the part concerning the backward translation due to two main reasons: the need to have a fully stable DOML language before addressing it and the intrinsic difficulty of deriving a DOML model from a lower level specification. Another critical point is that a DOML specification typically corresponds to IaC code fragments written in different languages. This implies that a single fragment would result in an incomplete DOML model. For the above reasons, it has been decided to limit the backward transformation on moving from the internal DOML representation, serialized in the so called DOMLX, into the DOML external one. Such internal transformation is at the moment under development and will be released at the end of the project with the objective to used it to support transformations between different versions of the DOML. In addition to the future developments that have been identified above, an important future development will concern the analysis of the role of DOML models with respect to the usage of AI to automatically derive IaC. Recently, projects aiming at generating infrastructural code from natural language are under development [16]. Clearly, such approaches have a significant potential in terms of reducing the time and effort needed to develop IaC. The problem though is the fact that such systems, at least at the moment, are able to deal with specific tasks, such as, “create a Terraform code for provisioning a VM on AWS”, but not with the more general task to create all it is needed to support the operation of a complex application. 4 Lessons Learnt and Plan for Future Development DRA The extensibility of the DOML is depending on the DOML-E mechanisms defined (see Section 2.1.4). The effectiveness of these messages has to be studied in close connection with the possibility to define in the ICG new templates and modify the existing ones as otherwise any addition into the DOML would not have an impact on the generated IaC code. Given the fact that through the PIACERE project so far, the main focus was the development of the DOML and of ICG, it has not been possible to run extensive experiments in this context. This will be the subject of future work from now till the end of the project and beyond. As it has been discussed in Section 2.3, all requirements defined for the DOML have been addressed except two that have been partially addressed: • REQ36 – “DOML to enable writing infrastructure tests” is aiming at using the DOML to instrument with proper test cases the canary environment or any other testing environment. An analysis of existing tools in the context has suggested us the possibility to adopt the DOML as input language for the setup of chaos engineering tools, that is, • REQ36 – “DOML to enable writing infrastructure tests” is aiming at using the DOML to instrument with proper test cases the canary environment or any other testing environment. An analysis of existing tools in the context has suggested us the possibility to adopt the DOML as input language for the setup of chaos engineering tools, that is, © PIACERE Consortium Contract No. GA 101000162 Page 38 of 51 www.piacere-project.eu Contract No. GA 101000162 Page 38 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 those tools that introduce failures in a distributed system to test its resilience. Further investigation on this aspect is required and will be the subject for future work. • REQ57 – “It is desirable to enable both forward and backward translations from DOML to IaC and vice versa”, was considered, since the beginning of the project, as a very challenging, nice to have, and low priority requirement for the part concerning the backward translation due to two main reasons: the need to have a fully stable DOML language before addressing it and the intrinsic difficulty of deriving a DOML model from a lower level specification. 4 Lessons Learnt and Plan for Future Development AFT In this context, the availability of DOML models providing a precise description of the application and infrastructural elements, together with their expected mapping, could potentially help in ensuring that the low level code generated by the AI is correct and complete. RAF DRA Contract No. GA 101000162 Page 39 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 5 Conclusions This deliverable presents the latest version of DOML (DOML 3.0). Compared to the previous version, it consolidates and cleans up the language and adds specific constructs that were considered critical to address the needs of PIACERE case studies. The deliverable includes a discussion on the fulfilment of requirements, examples of DOML models that demonstrate the modelling details under the new version of DOML, a preliminary analysis of the DOML effectiveness in terms of a comparison with well-known infrastructure languages, and some reflections on the DOML status and on possible future work. DRAFT Contract No. GA 101000162 Page 40 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 6 References [1] B. Xiang, E. Di Nitto and G. Novakova Nedeltcheva, “Deliverable 3.2: PIACERE Abstractions, DOML and DOML-E - v2,” PIACERE Consortium, 2022. [2] PIACERE team, "PIACERE DOML Specification v 3.0.," https://www.piacere- doml.deib.polimi.it/, October 2022. [3] S. Canzoneri, “DOML v3.0 tutorial,” 2023. https://git.code.tecnalia.com/piacere/public/the-platform/doml/-/tree/main/tutorial [4] E. Di Nitto ed., “Deliverable D3.1: PIACERE Abstractions, DOML and DOML-E - v1,” PIACERE Consortium, Dec. 2021. [5] Eclipse Foundation, “Eclipse Modeling Framework,” [Online]. Available: https://www.eclipse.org/modeling/emf/. [6] Eclipse Foundation, Inc., “Eclipse Xtext™,” [Online]. Available: https://www.eclipse.org/Xtext/. T [7] M. Chiari, B. Xiang, G. Novakova Nedeltcheva, E. Di Nitto, L. Blasi, D. Benedetto and L. Niculut, “DOML: A New Modelling Approach To Infrastructure-as-Code,” in CAISE 2023, Zaragoza, 2023. AFT [8] E. Morganti, “Deliverable 2.2 v 1.1 PIACERE DevSecOps Framework Requirements specification, architecture and integration strategy - v2,” PIACERE Consortium, 2023. RAF [9] D. Benedetto and L. Nicolut, “Deliverable D3.6: Infrastructural code generation - v3,” PIACERE Consortium, 2023. RA [10] Morganti, Emanuele, “Deliverable 2.1: PIACERE DevSecOps Framework Requirements specification, architecture and integration strategy - v1,” PIACERE Consortium, Dec. 2021. DR [11] A. Franchini and M. Pradella, “Deliverable D4.3: Verify the Trustworthiness of Infrastructure as Code Task 4.1 Infrastructural Model Verification – v3,” PIACERE Consortium, 2023. D [12] D. E. Iero, “Chaos Engineering: an analysis of processes and tools,” Master Thesis - Politecnico di Milano, 2021. [13] J. Díaz de Arcaya, “Deliverable 5.3: IaC execution platform prototype - v3,” PIACERE Consortium, 2023. [14] L. Reboucas de Carvalho y A. Favacho de Araujo, «Performance comparison of Terraform and Cloudify as multicloud orchestrators,» de CCGRID’20, 2020. [15] E. Villanueva, “Deliverable 3.7: PIACERE IDE - v3,” PIACERE Consortium, 2023. [16] Firefly, “Artificial Intelligence Infrastructure-as-Code Generator,” Firefly, 2023. [Online]. Available: https://aiac.dev/. Contract No. GA 101000162 Page 41 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 1 Simplified version of the Ericsson case Being one of the PIACERE use case validation scenarios, the Ericsson case concerns IoT networks in the context of Public Safety applications. The DOML model presented in this section was built as a simplified version of the required infrastructure for the Ericsson case, to be used for validation purposes. The example, per se, does not introduce new DOML concepts compared to the ones exemplified through the case studies in Section 3, but it is more complex than the others as it includes three different networks in the infrastructure layer. 1.1 Case short description A Public Safety use case network is composed and organized so that dedicated networks can segregate the traffic that flows between the virtual machines to provide an environment that cannot be easily compromised. Therefore, the public safety network includes three different networks for the application components to communicate, and a separate network for operation and management. T In this example, three different software components are deployed on three different virtual machines, provisioned by the OpenStack cloud provider. Besides such software components, the application needs to access two different Software-as-a-Service components providing APIs, accessible through specific URL. AFT Finally, the infrastructure should be configured in such a way to guarantee maximum availability and performance while minimizing the cost. There are two non-functional requirements: the maximum cost should be 300.0, while the minimum performance measure should be 7.0%. RAF © PIACERE Consortium Contract No. GA 101000162 Page 42 of 51 www.piacere-project.eu maximum cost should be 300.0, while the minimum performance measure should be 7.0%. 1.2 DOML model doml uc3_openstack application app { software_component iwg { provides { net_info } } software_component osint { provides { osint_info } consumes { net_info, get_twitter, ewcf_rest_interface } } software_component ewcf { provides { ewcf_rest_interface } consumes { get_firebase } } saas external_twitter { provides { get_twitter @ "https://twitter_api/get" } } DRA p 1.2 DOML model doml uc3_openstack application app { software_component iwg { provides { net_info } } f i { DRA 1.2 DOML model doml uc3_openstack D software_component ewcf { provides { ewcf_rest_interface } consumes { get_firebase } D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 saas external_firebase { saas external_firebase { provides { get_firebase @ "https://firebase_api/get" } infrastructure infra { // VMs region // This VM as a network interface belonging to the OAM network // one belonging to Net1 to communicate with the osint V // This VM as a network interface belonging to the OAM network, // one belonging to Net1 to communicate with the osint VM // and one belonging to Net2 to access the 5G network // This VM as a network interface belonging to the OAM network, // one belonging to Net1 to communicate with the osint VM // and one belonging to Net2 to access the 5G network // g g , // one belonging to Net1 to communicate with the osint VM // d b l i t N t2 t th 5G t k g g // one belonging to Net1 to communicate with the osint V // and one belonging to Net2 to access the 5G network vm igw_vm { vm igw_vm { os "Ubuntu-Focal-20.04-Daily-2022-04-19" os "Ubuntu-Focal-20.04-Daily-2022-04-19" os "Ubuntu-Focal-20.04-Daily-2022-04-19" size "small" iface igw_vm_oam { belongs_to subnet_oam_igw } iface igw_vm_net1 { belongs_to subnet_net1_igw } iface igw_vm_net2 { belongs_to subnet_net2_igw } credentials ssh_key } // This VM has a network interface belonging to the OAM network, // one belonging to Net1 to communicate with the other VMs // and one belonging to Net3 to the access the Internet vm osint vm { DRAFT size "small" iface igw_vm_oam { iface igw_vm_oam { belongs_to subnet_oam_igw iface igw_vm_net1 { iface igw_vm_net1 { belongs_to subnet_net1_igw A iface igw_vm_net2 { R iface igw_vm_net2 { R // This VM has a network interface belonging to the OAM network, // one belonging to Net1 to communicate with the other VMs // and one belonging to Net3 to the access the Internet D vm osint_vm { vm osint_vm { os "Ubuntu-Focal-20.04-Daily-2022-04-19" os "Ubuntu-Focal-20.04-Daily-2022-04-19" size "small" iface osint_vm_oam { iface osint_vm_oam { belongs_to subnet_oam_osint belongs_to subnet_oam_osint iface osint_vm_net1 { iface osint_vm_net1 { belongs_to subnet_net1_osint belongs_to subnet_net1_osint iface osint_vm_net3 { belongs_to subnet_net3_osint Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Ve D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Date: 31.05.2023 credentials ssh_key // This VM has a network interface belonging to the OAM network, // b l i t N t1 t i t ith th i t VM // This VM has a network interface belonging to the OAM network, // one belonging to Net1 to communicate with the osint VM // and one belonging to Net3 to access the Internet © PIACERE Consortium Contract No. GA 101000162 Page 44 of www.piacere-project.eu // This VM has a network interface belonging to the OAM networ // one belonging to Net1 to communicate with the osint // and one belonging to Net3 to access the Internet vm ewcf_vm { os "Ubuntu-Focal-20.04-Daily-2022-04-19" size "small" iface ewcf_vm_oam { belongs_to subnet_oam_ewcf } iface ewcf_vm_net1 { belongs_to subnet_net1_ewcf } iface ewcf_vm_net3 { belongs_to subnet_net3_ewcf } credentials ssh_key } // Operation and management network, to which all the VMs are connected net oam { protocol "TCP/IP" cidr "16.0.0.0/24" subnet subnet_oam_igw { protocol "TCP/IP" cidr "16.0.1.0/26" } subnet subnet_oam_osint { protocol "TCP/IP" cidr "16.0.1.64/26" } subnet subnet_oam_ewcf { protocol "TCP/IP" cidr "16.0.1.128/26" } DRAFT // This VM has a network interface belonging to the OAM network, // one belonging to Net1 to communicate with the osint VM // and one belonging to Net3 to access the Internet g g // one belonging to Net1 to communicate with the osint V // and one belonging to Net3 to access the Internet // one belonging to Net1 to communicate with the osint VM // and one belonging to Net3 to access the Internet // and one belonging to Net3 to access the Internet // and one belonging to Net3 to access the Internet vm ewcf_vm { vm ewcf_vm { vm ewcf_vm { os "Ubuntu-Focal-20.04-Daily-2022-04-19" size "small" iface ewcf_vm_oam { belongs_to subnet_oam_ewcf } iface ewcf_vm_net1 { belongs_to subnet_net1_ewcf } iface ewcf_vm_net3 { belongs_to subnet_net3_ewcf } credentials ssh_key } // Operation and management network, to which all the VMs are connected DRAFT iface ewcf_vm_net1 { iface ewcf_vm_net1 { iface ewcf_vm_net1 { belongs_to subnet_net1_ewcf iface ewcf_vm_net3 { belongs_to subnet_net3_ewcf A Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Date: 31.05.2023 } // This is an internal network and therefore has no Internet gateway net net1 { protocol "TCP/IP" cidr "16.0.1.0/24" // Subnets definition } protocol "TCP/IP" cidr "16.0.1.0/25" } subnet subnet_net1_osint { connections { subnet_net1_igw, subnet_net1_ewcf } protocol "TCP/IP" cidr "16.0.1.64/26" } subnet subnet_net1_ewcf { connections { subnet_net1_osint DRAFT subnet subnet_net1_osint { A subnet subnet_net1_osint { A connections { protocol "TCP/IP" cidr "16.0.1.128/26" Page 45 of 51 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 } gateway net2_igw { address "16.0.2.22" } // Network connecting osint and ewcf to Internet net net3 { protocol "TCP/IP" cidr "16.0.3.0/24" cidr "16.0.3.0/24" subnet subnet_net3_osint { protocol "TCP/IP" cidr "16.0.3.0/25" } subnet subnet_net3_ewcf { protocol "TCP/IP" cidr "16.0.3.128/25" } gateway net3_igw { address "16.0.3.22" } } //Credentials region // h d i l d h DRAFT subnet subnet_net3_osint { subnet subnet_net3_osint { cidr "16.0.3.0/25" subnet subnet_net3_ewcf { protocol "TCP/IP" cidr "16.0.3.128/25" R // These credentials are used to access the VMs // These credentials are used to access the VMs key_pair ssh_key { user "ubuntu" // key to be inserted here keyfile "…" algorithm "RSA" bits 4096 // Security region // This security group is composed of standard security rules // and is associated with all the interfaces © PIACERE Consortium Contract No. GA 101000162 Page 46 of 51 www.piacere-project.eu Page 46 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 to_port 80 cidr ["0.0.0.0/0"] } ingress https { protocol "TCP" from_port 443 to_port 443 cidr ["0.0.0.0/0"] } ingress ssh { protocol "TCP" from_port 22 to_port 22 cidr ["0.0.0.0/0"] } DRAFT to_port 80 cidr ["0.0.0.0/0"] } ingress https { protocol "TCP" from_port 443 to_port 443 cidr ["0.0.0.0/0"] } ingress ssh { protocol "TCP" from_port 22 to_port 22 cidr ["0.0.0.0/0"] DRAFT ingress ssh { D ifaces igw_vm_oam, igw_vm_net1, igw_vm_net2, osint_vm_oam, osint_vm_net1, osint_vm_net3, ewcf_vm_oam, ewcf_vm_net1, ewcf_vm_net3 ifaces igw_vm_oam, igw_vm_net1, igw_vm_net2, osint_vm_oam, osint_vm_net1, osint_vm_net3, ewcf_vm_oam, ewcf_vm_net1, ewcf_vm_net3 deployment config1 { active deployment config1 Contract No. GA 101000162 Page 47 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. 2 User manual to extend DOML This brief manual has the purpose to help users in the process of applying modifications to the DOML language to extend it. D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Date: 31.05.2023 concretizations { concrete_infrastructure con_infra { provider openstack { // Concrete computing nodes region vm concrete_osint_vm { maps osint_vm } vm concrete_igw_vm { maps igw_vm } vm concrete_ewcf_vm { maps ewcf_vm } // Concrete networks region net concrete_oam { maps oam } net concrete_net1 { maps net1 } net concrete_net2 { maps net2 } net concrete_net3 { maps net3 } } } active con_infra } optimization opt { DRAFT vm concrete_ewcf_vm { maps ewcf_vm } // Concrete networks region net concrete_oam { maps oam } net concrete_net1 { maps net1 } net concrete_net2 { maps net2 } net concrete_net3 { maps net3 DRAFT Contract No. GA 101000162 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 } nonfunctional_requirements { req1 "Cost <= 300" max 300.0 => "cost" req2 "Performance >= 7%" min 7.0 => "performance" req3 "Provider" values "openstack" => "provider" req4 "elements" => "VM, VM, VM" DRAFT Contract No. GA 101000162 Page 49 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 Version 1.0 – Final. Date: 31.05.2023 2.2 Metamodel update DR Steps to update the metamodel: D 1. Open the implementation folder as workspace inside the Eclipse IDE. D 2. Open the “eu.piacere.doml” project. D 2. Open the “eu.piacere.doml” project. D 3. Inside the “model” folder, open the “doml.emf” file and apply the needed changes. 4. After saving the file, right click on it in the Project Explorer and in the “Eugenia” sub- menu, select “Generate EMF Editor”. It is strongly recommend installing the Emfatic plugin to make changes to the DOML metamode FT In order to generate the Ecore from the Emfatic code and to being able to generate graphical models from it, the Eugenia tool, which is part of the Eclipse Epsilon plugin, is required. The metamodel diagrams shown in the D3.3 document have been generated using Eugenia. AFT The DOML syntax has been generated by using Eclipse Xtext, which is a framework for development of programming languages and domain-specific languages, as reported in the official website [6]. RA Users will, therefore, need to install the Xtext plugin to apply changes to the syntax. DR 2.1 Required tools First, it must be pointed out that the PIACERE framework and DOML rely on the open-source Eclipse IDE. The Eclipse IDE version used to build DOML v3.0 is “Eclipse 2023-03”: in particular, it is used the Eclipse Modeling Tools package, which can be selected directly from the installer. The Eclipse IDE version used to build DOML v3.0 is “Eclipse 2023-03”: in particular, it is used the Eclipse Modeling Tools package, which can be selected directly from the installer. Such a package already includes Eclipse Modeling Framework (EMF) installed with it. As reported in the official website [5], the EMF project is a modeling framework and code generation facility for building tools and other applications based on a structured data model. This framework has been used to build the DOML Ecore, the formalization of its metamodel. To make the metamodel more readable and easily accessible to programmers, we have made use of Eclipse Emfatic, a textual syntax for EMF Ecore metamodels. The syntax is very intuitive and easy-to-use. FT It is strongly recommend installing the Emfatic plugin to make changes to the DOML metamodel. FT 2.3 Syntax update Steps to update the syntax: 1. Open the implementation folder as workspace inside the Eclipse IDE. 2. Open the “eu.piacere.doml.grammar” project. 2. Open the “eu.piacere.doml.grammar” project. 3. Inside the “src/eu.piacere.doml” folder, open the “Doml.xtext” file and apply the needed changes. 4. After saving the file, right click on it in the Project Explorer and in the “Run as” sub- menu, select “Generate Xtext Artifacts”. © PIACERE Consortium Contract No. GA 101000162 Page 50 of 51 www.piacere-project.eu Contract No. GA 101000162 Page 50 of 51 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 D3.3 – PIACERE Abstractions, DOML and DOML-E – v3 Version 1.0 – Final. Date: 31.05.2023 2.4 Testing applied changes and updating the ICG To test the DOML editor after the modifications have been applied, right click on the “eu.piacere.doml” project in the Project Explorer (Plug-in Development perspective) and in the “Run as” sub-menu, select “Eclipse Application”. At this point, after the application is launched, try creating a new project containing a new file with the “.doml” extension: here the user can test the editor, verifying that the changes to the syntax have been applied correctly. When extending DOML, it is fundamental to remember that not only the metamodel and the syntax must be updated, but also the Infrastructure Code Generator (ICG), which interprets the DOML language and generates the corresponding code in IaC standard languages. To see how to deal with the changes to be applied to the ICG, please refer to the User Manual in the Deliverable [9]. DRAFT Contract No. GA 101000162 Page 51 of 51
https://openalex.org/W2983343307	http://revista.cofen.gov.br/index.php/enfermagem/article/download/1991/566	es		PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA	Enfermagem em Foco	2,019	cc-by	5,221	ARTIGO 21 PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA Tâmara Taynah Medeiros da Silva1, Rodrigo Assis Neves Dantas1, Daniele Vieira Dantas1, Maria Solange Moreira de Lima1, Louise Constância de Melo Alves1, Ilanne Caroline Santos Costa1, Naryllenne Maciel de Araújo1 Objetivo: analisar aspectos epidemiológicos e angiográficos de pacientes submetidos à Angioplastia Transluminal Coronariana eletiva em um hospital de referência em cardiologia do estado do Rio Grande do Norte. Metodologia: trata-se de uma pesquisa exploratória, descritiva, transversal com abordagem quantitativa. A coleta de dados ocorreu de abril/2017 a outubro/2017. Resultados: foram incluídos no estudo 129 pacientes, destes 65,8% eram do sexo masculino. O stent farmacológico foi mais utilizado nos procedimentos com 79 (61,2%). A via femoral predominou entre os dois sexos, com 29 (65,9%) entre mulheres e 54 (63,5%), nos homens.Conclusões:nota-se predomínio de pacientes do sexo masculino, idosos, baixo grau de escolaridade, pardos, casados e aposentados. A análise angiográfica constitui-se de pacientes com predominância de implantação de um (1) stent do tipo farmacológico, Descendente anterior e Coronária direita como coronárias tratadas, sendo a punção de acesso com maior prevalência a por via femoral. Descritores: Intervenção coronária percutânea; Angioplastia; Infarto do miocárdio; Doenças cardiovasculares; Reperfusão miocárdica. PATIENTS SUBMITTED TO CORONARY TRANSLUMINAL ANGIOPLASTY: EPIDEMIOLOGICAL AND ANGIOGRAPHIC ANALYSIS Objective: to analyze epidemiological and angiographic aspects of patients submitted to elective Coronary Transluminal Angioplasty at a referral hospital in cardiology in the state of Rio Grande do Norte. Methodology: This is an exploratory, descriptive, transversal research with a quantitative approach. Data collection occurred from April / 2017 to October / 2017. Results: 129 patients were included in the study, of which 65.8% were male. The pharmacological stent was more used in the procedures with 79 (61.2%). The femoral route predominated between the two sexes, with 29 (65.9%) between women and 54 (63.5%), in men. Conclusions: a predominance of male patients, elderly, low educational level, pardos, married and retired people. Angiographic analysis consists of patients with a predominance of implantation of one (1) stent of the pharmacological type, Anterior descending and Right coronary as treated coronaries, being the access puncture with greater prevalence to the femoral route. Descriptors: Percutaneous coronary intervention, Angioplasty, Myocardial infarction, Cardiovascular diseases, Myocardial reperfusion. PACIENTES SUBMETIDOS A LA ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISIS EPIDEMIOLÓGICA Y ANGIOGRÁFICA Objetivo: analizar aspectos epidemiológicos y angiográficos de pacientes sometidos a la Angioplastia Transluminal Coronariana electiva en un hospital de referencia en cardiología del estado de Rio Grande do Norte. Metodology: setrata de una investigación exploratoria, descriptiva, transversal con abordaje cuantitativo. La recolección de datos ocurrió de abril / 2017 a octubre / 2017. Resultados: fueron incluidos en el estudio 129 pacientes, de estos 65,8% eran del sexo masculino. El stent farmacológico fue más utilizado en los procedimientos con 79 (61,2%). La vía femoral predominó entre los dos sexos, con 29 (65,9%) entre mujeres y 54 (63,5%), en los hombres. Conclusiones: se observa predominio de pacientes del sexo masculino, ancianos, bajo grado de escolaridad, pardos, casados y jubilados. análisis angiográfico consistía en pacientes con despliegue predominante de (1) el tipo stent farmacológico antes de la arteria coronaria descendente y la coronaria derecha tratada como la punción de acceso con la prevalencia más alta a través de la arteria femoral. 1 Universidade Federal do Rio Grande,FURG.Email: Descriptores: Intervención coronaria manuh_souza@msn.com percutânea; Angioplastia; Infarto del miocárdio; Enfermedades cardiovasculares; Reperfusión miocárdica. 1 Universidade Federal do Rio Grande do Norte-UFRN. Autor correspondente: Tâmara Taynah Medeiros da Silva. E-mail: tamaratmds1904@gmail.com Enferm. Foco 2019; 10 (3): 126-133 126 PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA Juliano Teixeira Moraes, Alexandre Ernesto Silva, Tarcísio Laerte Gontijo, Renato Ferreira Ribeiro, Rafaela das Graças Santiago Faria INTRODUÇÃO As Doenças Cardiovasculares (DC)prevalecem como a principal causa de óbitos, correspondendo a 31% de todas as mortes em nível global. No Brasil, estima-se que aproximadamente 350 000 pessoas morreram por DC durante o ano de 2016. Entre 2004 a 2014, mais de um milhão de brasileiros foram acometidos por Doenças Isquêmicas do Coração (DIC), dentre elas, as Síndromes Coronarianas Agudas (SCA), presente no grupo das Doenças Arteriais Coronarianas (DAC)(1-4). As SCA são compreendidas como situações na qual o paciente apresenta evidências de isquemia miocárdica, onde, o tratamento propõe-se a reduzir os danos no tecido miocárdico e restabelecer o fluxo sanguíneo. Entre os métodos de reperfusão temos a Angioplastia Transluminal Coronariana (ATC) que consiste em um procedimento realizado através da inserção de um cateter via arterial que tem por objetivo o remodelamento da placa aterosclerótica para manter a potência do vaso(4-6). A população brasileira vive uma transformação caracterizada pelo aumento percentual da população de idosos gerando um predomínio de doenças crônicas, dentre esses distúrbios, os acometimentos cardiovasculares são os responsáveis por tornar a população idosa como sendo principal público que são frequentemente submetidos à ATC. Os indivíduos prevalentes são do gênero masculino, com faixa etária avançada e com múltiplos fatores de risco associados (7) . Todavia, maior parte das doenças não deve ser julgada como resultado inevitável desse modelo atual, mas como algo que pode ser prevenido junto à atenção primária de saúde(8). Este estudo justifica-se devido a grande quantidade de indivíduos acometidos por doenças cardiovasculares, notadamente os de fatores isquêmicos e relacionados, tornando-se imprescindível conhecer o perfil dos pacientes submetidos ao procedimento de ATC eletiva, de forma que as doenças arteriais coronarianas sejam elucidadas mais adequadamente sistematizando o atendimento dos profissionais de saúde tornando-os capazes de elaborar estratégias de prevenção dos fatores de risco, especialmente os passíveis de modificação e orientar adequadamente o paciente que necessita de ATC eletiva(8). Diante disso, questiona-se: quais aspectos epidemiológicos e angiográficos dos pacientes submetidos à Angioplastia Transluminal Coronariana eletiva em um Hospital de referência em cardiologia do estado do Rio Grande do Norte? Portanto, este artigo tem por objetivo de analisar aspectos epidemiológicos e angiográficos de pacientes submetidos à Angioplastia Transluminal Coronariana eletiva em um hospital de referência em cardiologia do estado do Rio Grande do Norte. ARTIGO 21 METODOLOGIA Tipo de estudo Trata-se de um estudo exploratório, descritivo, transversal, com abordagem quantitativa e dados prospectivos, desenvolvido no Hospital Universitário Onofre Lopes (HUOL) no estado do Rio Grande do Norte. Participantes da pesquisa A amostra contou com 129 pacientes submetidos à ATC eletiva e para definição desta amostragem, utilizou-se uma amostra aleatória simples do tipo probabilística e por conveniência, tendo por base no número de procedimentos realizados em 2016 realizando o cálculo amostral, com erro tolerável de 5%. Os critérios de inclusão foram pacientes maiores de 18 anos, terem sido submetidos à ATC de caráter eletivo e assinatura do Termo de Consentimento Livre Esclarecido (TCLE). Dentre os critérios de exclusão estavam pacientes que foram submetidos á ATC com caráter de urgência e pacientes que foram a óbito. Local do estudo O estudo foi desenvolvido no Hospital Universitário Onofre Lopes (HUOL), estado do Rio Grande do Norte, nos setores da Hemodinâmica, Unidade Cardiovascular e Unidade de Terapia Intensiva (UTI). O local de estudo foi escolhido por ser, atualmente, a única instituição pública que possui laboratório de hemodinâmica sendo responsável pela efetuação de ATC primárias e eletivas. O primeiro atendimento é composto pela administração de terapia adjuvante e seleção do método de reperfusão, desta forma, a instituição cadastra o paciente através do sistema nacional de regulação, onde será realizado o devido agendamento do procedimento, além do HUOL ser o maior hospital de ensino do RN, se disponibilizando a formação de profissionais de saúde (9). Coleta dos dados com explicitação dos instrumentos e procedimentos Os dados foram coletados nos meses de abril a outubro de 2017 através dos prontuários impressos e eletrônicos, sendo registrado em instrumento elaborado previamente. Foram selecionadas para análise as seguintes variáveis: dados demográficos (data do evento, sexo, escolaridade, etnia, estado civil, idade, procedência, profissão, renda); aspectos clínicos e comorbidades (Hipertensão Arterial Sistêmica (HAS), Diabetes Mellitus (DM)), história prévia de infarto, Acidente Vascular Encefálico (AVE) prévio, Cirurgia de Revascularização Miocárdica (CRM) anterior, tabagismo, Enferm. Foco 2019; 10 (3): 126-133 127 ARTIGO 21 PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA Juliano Teixeira Moraes, Alexandre Ernesto Silva, Tarcísio Laerte Gontijo, Renato Ferreira Ribeiro, Rafaela das Graças Santiago Faria sedentarismo, etilismo, obesidade; aspectos do procedimento (local de punção, artérias coronárias tratadas, stents utilizados) e aspectos da evolução (alta para a enfermaria ou alta para a UTI). Variáveis Mulheres (n=44) Idade (anos) (média ± Desvio Padrão) H o m e n s (n=85) 65,4±9,5 62,6±12,0 < 40 anos, n (%) 0 (0,0%) 3 (3,5%) 40 - 49 anos, n (%) 0 (0,0%) 9 (10,6%) Faixa Etária Procedimentos de análise dos dados O software International Buniness Machines Statistical Package for the Social Sciences (IBM SPSS) 21.0 foi utilizado para análise dos dados, atribuindo-se o nível de significância de 5% para todos os testes estatísticos (p < 0.05). Após a tabulação e análise, as informações foram organizadas na forma de tabelas. O perfil epidemiológico por sexo dos pacientes submetidos à ATC em caráter eletivo conduziu-se análise descritiva dos dados, com uso de medidas de tendência central (média e mediana) e dispersão (desvio padrão) para as variáveis contínuas, enquanto as variáveis categóricas foram mensuradas pela frequência de ocorrências. Com o objetivo de avaliar se as variáveis contínuas apresentavam distribuição normal, e consequentemente definir as estatísticas inferenciais que serão conduzidas, foi aplicado o teste Kolmogorov-Sminorv, o qual indicou uma distribuição assimétrica dos dados (p < 0.05). Posto isto, o teste de Mann-Whitney (U) foi utilizado para comparação por sexo das variáveis contínuas. Além disso, para verificar associações entre sexo (homens e mulheres) e às variáveis de aspectos clínicos, comorbidades, fatores de risco, medicamentos em uso, características da ATC e recuperação foi utilizado o teste de Qui-quadrado de Pearson (X2). Adicionalmente, empregou-se o teste Exato de Fisher nos casos de violação dos princípios para uso do Qui-Quadrado de Pearson. Procedimentos éticos O projeto foi apreciado e autorizado pelo Comitê de Ética em Pesquisa (CEP) do HUOL de Natal/RN, sob parecer de n° 1.997.884, certificado de apresentação para apreciação ética: 64183517.0.0000.5292. Além disso, autorização da própria instituição em questão para coleta de dados. A pesquisa teve por base a resolução de n° 466 de 12 de dezembro de 2012, do Conselho Nacional de Saúde. RESULTADOS Dos 129 pacientes abrangidos no estudo, 65,8% são do sexo masculino, enquanto 34,1% são do sexo feminino. As características sociodemográficas da amostra são mostradas na Tabela 1. Tabela 1 - Características demográficas e socioeconômicas dos pacientes submetidos à ATC* em caráter eletivo. Natal, RN, Brasil, 2017 128 Enferm. Foco 2019; 10 (3): 126-133 50 - 59 anos, n (%) 15 (34,1%) 20 (23,5%) 60 - 69 anos, n (%) 13 (29,5%) 30 (35,5%) 70 - 79 anos, n (%) 14 (31,8%) 18 (21,2%) ≥ 80 anos 2 (4,5%) 5 (5,9%) Pardos, n (%) 24 (54,5%) 40 (47,1%) Brancos, n (%) 16 (36,4%) 23 (27,0%) Negros, n (%) 4 (9,1%) 22 (25,9%) Analfabeto, n (%) 9 (20,5%) 26 (30,6%) 1° Grau incompleto, n (%) 20 (45,5%) 25 (29,4%) 1° Grau completo, n (%) 9 (20,5%) 11 (12,9%) 2° Grau incompleto, n (%) 0 (0,0%) 2 (2,4%) 2º Grau completo, n (%) 6 (13,6%) 18 (21,2%) Nível Superior incompleto, n (%) 0 (0,0%) 1 (1,2%) Nível Superior completo, n (%) 0 (0,0%) 2 (2,4%) Com companheiro fixo, n (%) 21 (47,7%) 62 (72,9%) Sem companheiro fixo, n (%) 23 (52,2) 33 (27,0%) < 1 salário mínimo, n (%) 9 (20,5%) 12 (14,1%) 1 - 3 salários mínimos, n (%) 33 (75,0%) 64 (75,3%) > 3 salários mínimos, n (%) 2 (4,5%) 9 (10,6%) Outras ocupações/ mal definidas ‡, n (%) 27 (61,4%) 43 (50,6%) Prestação de Serviços, n (%) 11 (25,0%) 8 (9,4%) Agropecuária e da produção extrativa vegetal e animal, n (%) 2 (4,5%) 8 (9,4%) Comércio e atividades auxiliares, n (%) 2 (4,5%) 7 (8,2%) Técnicas, científicas, artísticas e assemelhadas, n (%) 1 (2,3%) 2 (2,4%) Etnia Escolaridade Estado Civil Renda total por residência (salário mínimo)† Profissão PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA Juliano Teixeira Moraes, Alexandre Ernesto Silva, Tarcísio Laerte Gontijo, Renato Ferreira Ribeiro, Rafaela das Graças Santiago Faria Desempregado, n (%) 1 (2,3%) 0 (0,0%) Histórico familiar, n ARTIGO 21 14 (31,8) 33 (38,8) 0,27b 3 (6,8) 26 (30,6) 0,00 a, b 14 (31,8) 17 (20,0) 0,10b 10 (22,7) 25 (29,4) 0,27b Sedentarismo, n (%) 41 (93,2) 78 (91,8) 0,53c Legenda: () Angioplastia Transluminal Coronariana. (†) R$ 954,00. (‡) Donas Atividade física, n (%) 3 (6,8) 7 (8,2) 0,53c de casa, estudantes, aposentados, pensionistas, autônomos e profissionais do Legenda: () Angioplastia Transluminal Coronariana. Fonte: própria pesquisa. a p sexo. Fonte: própria pesquisa. < 0,05;b p-valor referente ao Qui-quadrado de Pearson; c p-valor referente ao Indústrias de transformação e construção civil, n (%) 0 (0,0%) 7 (8,2%) Transportes e comunicações, n (%) 0 (0,0%) 5 (5,9%) Administrativas, n (%) 0 (0,0%) 4 (4,7%) Defesa nacional e segurança pública, n (%) (%) Etilismo, n (%) Tabagismo atual, n (%) Tabagismo anterior, n (%) 0 (0,0%) 1 (1,2%) teste Exato de Fisher. Na Tabela 1, nota-se a média de idade 65,4 (±9,5) para as mulheres e 62,6 (±12,0) para os homens, com prevalência da cor parda com 49,6% pacientes.Ainda, ressaltam-se os pacientes analfabetos e com primeiro grau incompleto com 27,13% e 34,88%,respectivamente, 64,34% eram casados, 75,20% com renda de um a três salários mínimos e54,26% eram aposentados. A procedência refere-se ao lugar de onde o paciente provém para realizar a ATC eletiva, prevalecendo à origem de hospitais em todo o estado com 43 (33,33%) pacientes, seguido por Residências localizadas na capital, Residências no interior do estado, Unidades de Pronto Atendimento (UPA) com incidência de 38 (29,46%), 30 (23,25%), 18 (13,95%). Os fatores de risco e comorbidades estão expostos na Tabela 2. Observou-se que a comorbidade com maior prevalência foi HAS, presente em 95,5% dos pacientes, seguida de Infarto Agudo do Miocárdio prévio e DM. Todos os pacientes apresentaram pelo menos uma comorbidades, entretanto, dentre as comparações feitas entre os sexos, identificou-se por meio do teste Qui-Quadrado de Pearson, diferenças com significância estatística apenas para a comorbidade de HAS (X2(1) = 3,26; p = 0,05) e o fator de risco etilismo (X2(1) = 9,4; p = 0,00), sugerindo maior frequência de mulheres acometidas por HAS 95,5%, bem como maior etilismo entre os homens 30,6%. Tabela 2 - Comorbidades e fatores de risco entre homens e mulheres submetidos à ATC* em caráter eletivo. Natal, RN, Brasil, 2017 Tabela 3 - Comparação do uso de medicamentos entre homens e mulheres submetidos à ATC eletiva. Natal, RN, Brasil, 2017 Variáveis Antiagregante Mulheres (n=44) Homens (n=85) p-valor Hipertensão Arterial Plaquetário, n 39 (88,6%) 78 (91,8%) 0,38b 39 (88,6%) 74 (87,1%) 0,51b 29 (65,9%) 58 (68,2%) 0,47b 18 (40,2%) 25 (29,4%) 0,13b 9 (20,5%) 16 (18,8%) 0,49b Variáveis Comorbidades Mulheres (n=44) Homens (n=85) p-valor Sistêmica, n (%) Diabetes Mellitus, n (%) Infarto Agudo 42 (95,5) 72 (84,7) 0,05a,b 21 (47,7) 29 (34,1) 0,09b Anti-hiperten- 22 (50,0) 49 (57,6) 0,26b Anticoagulante, n (%) n (%) Hipoglice- Cirurgia de Revascularização miocárdica 2 (4,5) 3 (3,5) 0,55 c Vascular Encefálico, n (%) miante, n (%) Total de medi- - anterior, n (%) Acidente sivos, n (%) Estatina, n (%) do Miocárdio – Prévio, (%) camentos em 3 (6,8) 5 (5,9) 0,55 c uso Fatores de risco Entre Sobrepeso e Obe- dois, n (%) sidade (IMC – Adul- 7 (46,7) 21 (67,7) 0,21c n (%) e De três a cinMais de cinco, Obesidade graus 1, 2 e 3 (IMC – Idosos), um co, n (%) tos), n (%) 11 (37,9) 24 (44,4) 0,90 c 0,00 a, c n (%) Nega uso, n (%) 7 (15,0%) 4 (4,7%) 8 (18,2%) 37 (43,6%) 29 (65,9%) 40 (47,0%) 0 (0,0%) 3 (3,5%) Enferm. Foco 2019; 10 (3): 126-133 129 ARTIGO 21 Não informado, n (%) PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA Juliano Teixeira Moraes, Alexandre Ernesto Silva, Tarcísio Laerte Gontijo, Renato Ferreira Ribeiro, Rafaela das Graças Santiago Faria 0 (0,0%) Radial e femoral, n 1 (1,2%) (%) 0 (0,0%) Legenda: () Angioplastia Transluminal Coronariana. Fonte: própria pesquisa. a p Legenda: () Angioplastia Transluminal Coronariana. Fonte: própria pesquisa. a < 0,05; b p-valor referente ao Qui-quadrado de Pearson; c p-valor referente ao p < 0,05; b p-valor referente ao Qui-quadrado de Pearson; c p-valor referente teste Exato de Fisher. ao teste Exato de Fisher. Entre as classes medicamentosas, nota-se que a mais utilizada são os antiagregantes plaquetários com 91,4%, seguido por anti-hipertensivos com 87,3%, estatinas 67,4%, anticoagulantes com 33,3% e hipoglicemiantes com porcentagem de 19,3%. A média e desvio padrão de medicamentos usados foram de 3,0 ± 1,19 em mulheres e 2,9 ± 1,2 em homens. Em relação aos medicamentos, por meio do teste Exato de Fisher (p=0,00), identificou-se diferença significante no total de medicamentos em uso por homens e mulheres, indicando que as mulheres apresentam maior frequência de uso de três medicações ou mais (84,1% versus80,6%) (Tabela 3). As variáveis: tipo e quantidade de stents, coronária tratada no procedimento de ATC e local da punção para inserção do introdutor vascular está expresso na tabela 4. Da amostra total de pacientes, 61,24% com stent farmacológico implantado, 66,6% utilizou apenas um stent, 36,4% dos pacientes tiveram a artéria coronária descendente anterior como coronária tratada e, como local de punção, a via femoral predominou com 64,3%. As intercorrências durante o procedimento ocorreram em 6,8% das mulheres e 5,9% dos homens. A respeito do local de recuperação pós-intervenção coronária, constatase que, em pacientes do sexo feminino, 81,8% foram para a enfermaria cardiológica e 18,2% para a unidade de terapia intensiva, os homens, 82,4% recuperaram-se na enfermaria e 17,6% na terapia intensiva. Não foram identificadas associações significativas na recuperação (X2(1) =0,006, p = 0,56) e presença de Intercorrências entre homens e mulheres (Exato de Fisher=0,04, p=0,56), visto que, ambos apresentaram percentagens semelhantes de Intercorrências (homens =5,9% versus mulheres=6,8%) e desfecho para unidade de terapia intensiva (mulheres =18,2% vs. homens 17,6%) (Tabela 4). Contudo, relevância significativa foi encontrada entre as percentagens da lesão e o sexo (U=1421,0, p=0,02), indicando que as pacientes do sexo feminino apresentaram lesões mais extensas que os do sexo masculino (91,7% vs 88,2%) (Tabela 4). Tabela 4 - Características angiográficas e recuperação entre homens e mulheres submetidos à ATC eletiva. Natal, RN, Brasil, 2017 Variáveis Mulheres (n=44) Homens (n=85) Porcentagem da le- p-valor são (Média ± Desvio 0,02d 91,7±8,7 88,2±9,7 Padrão) Stents 0,44b Farmacológico, n (%) 24 (54,5%) 55 (69,6%) Convencional, n (%) 18 (40,9%) 25 (29,4%) 2 (4,5%) 5 (5,9%) Convencional e farmacológico, n (%) Número de Stents 0,67c Um, n (%) 27 (61,4%) 59 (69,4%) Dois, n (%) 13 (29,5%) 17 (20,0%) 4 (9,1%) 9 (10,6%) Três ou mais, n (%) Coronária tratada Descendente ante- 0,07c 11 (25,0%) 36 (42,4%) 18 (40,9%) 20 (23,5%) Circunflexa, n (%) 4 (9,1%) 9 (10,6%) Outra, n (%) 2 (4,5%) 10 (11,8%) 9 (20,5%) 10 (11,8%) rior, n (%) Coronária direita, n (%) Mais de uma coronária tratada, n (%) Local da punção 130 1 (2,3%) 0,59c Femoral, n (%) 29 (65,9%) 54 (63,5%) Radial, n (%) 12 (27,3%) 29 (34,1%) Braquial, n (%) 2 (4,6%) 2 (2,4%) Enferm. Foco 2019; 10 (3): 126-133 DISCUSSÃO No presente estudo destaca-se a predominância do sexo masculino com 65,89%, enquanto o sexo feminino corresponde a 34,11% da amostra total, destes, 63,57% apresentam faixa etária maior que 60 anos, o que confirma resultados anteriores, onde revelam o predomínio na realização de ATC em idosos (10-12) do gênero masculino (7, 10-18). Corroborando com os resultados (Tabela 1), um estudo realizado com 624 pacientes submetidos à ATC em um laboratório de Cardiologia da Índia, demonstrou que as mulheres são aproximadamente uma década mais velhas que os homens (67,42 versus 55,26) (10). Estudos brasileiros (11,16,17) ressaltam que as mulheres são protegidas até o período da menopausa em relação ao desenvolvimento de problemas coronarianos, sendo isso justificado pelo fato dos hormônios femininos atuarem beneficamente sobre as coronárias. PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA Juliano Teixeira Moraes, Alexandre Ernesto Silva, Tarcísio Laerte Gontijo, Renato Ferreira Ribeiro, Rafaela das Graças Santiago Faria Pesquisa nacional desenvolvida com 101 pessoas no Hospital do Coração de Sobral-Ceará, evidenciou similarmente etnia parda (60,4%), baixa escolaridade, sendo 44,6% com ensino fundamental e 40,6% informando serem analfabetos, 65,4% recebe menos de três salários mínimos ao mês, 75,2% possuem companheiro fixo e 30,7% são aposentados(16) corroborando com o presente estudo. Sabendo-se que o público-alvo são pacientes de baixo nível de escolaridade, deve-se repensar se as orientações repassadas estão sendo bem compreendidas para melhor atuação na prevenção de futuras ocorrências(16). As comorbidades apresentadas pelos pacientes submetidos à ATC eletiva neste estudo, a HAS está presente em 95,5% das mulheres e 84,7% dos homens. Igualmente, inúmeros estudos apontam a HAS como mais prevalente (7, 10, 16-17). Além disso, em estudo desenvolvido com 1.271 pacientes do Reino Unido apontou que entre homens e mulheres, 54,4% já apresentam infarto do miocárdio anteriormente(19). O estudo realizado(7) com 994 pacientes na região sul do Brasil explicitou o prevalecimento de 32,9% da amostra com DM e 5,8% apresentaram AVE prévio, reforçando nossos resultados que evidencia 38,7% e 6,2%, em DM e AVE prévio, respectivamente. Entre os fatores de risco, destacamos os números significativos de pacientes com índice de massa corpórea elevada (48,8%) e sedentarismo (92,2%). Análise realizada na Alemanha mostra a predominância do sobrepeso nos pacientes estudados (20). Podemos observar, também, que 94,2% do número total dos pacientes são sedentários, validando os dados obtidos, estudos retratam percentuais elevados (91,7% e 71%) de pacientes sedentários (21,22). Ainda entre os fatores de risco, é sabido que, o extabagista é igualmente propenso a desenvolver doenças cardiovasculares em relação ao fumante ativo, já que as substâncias presentes no cigarro geram efeitos deletérios sobre as artérias coronárias (22,23). Na população deste estudo, 24,0% são tabagistas atuais, enquanto 27,1% são ex-fumantes. Diante disso, pesquisa realizado no Sudeste Asiático, dos 2.426 pacientes da pesquisa, 43,4% eram fumantes (12). Ademais, diversos estudos demonstra o tabagismo como um dos fatores de risco presente em grande parte dos pacientes sujeitos a ATC(7,10,16,18). Ainda, 90,7% faziam uso de medicamentos antiagregantes plaquetários e 75,2%, de estatinas. Em análise (12) realizada no Sudeste Asiático com 2.246 pacientes, 99,8% utilizavam antiagregante, enquanto 96,58% usavam estatinas. Juntamente, de 624 indivíduos submetidos àATC em um laboratório de cateterismo cardíaco da Índia, o uso de terapia medicamentosa superou o desta pesquisa, sendo 86,8% para antiagregante plaquetário, 100% para estatinas (10). De acordo ARTIGO 21 com manual de conduta sobre síndromes coronarianas agudas, o uso de antiagregante plaquetário deve ser iniciado em todos os pacientes que apresente síndrome coronariana, independente de qual estratégia de reperfusão deseja-se efetuar (6). O stent farmacológico foi mais utilizado. De modo igual, pesquisa realizada com 161, o stent farmacológico predominou com 61,5%, seguido de convencional, com 28,8%(17). Opostamente, pesquisa brasileira com 489 pacientes submetidos à ATC, 98,6% utilizaram stent convencionais(23). Ainda, pesquisa do Oriente Médio (12) com 2.246 pacientes, o uso de um stent predominou (59,11%), fortalecendo os dados encontrados neste estudo, onde tanto homens como mulheres, recebem apenas um stent. Na Tabela 4, percebe-se que entre as coronárias tratadas, semelhantemente entre homens e mulheres, a mais prevalente foi a descendente anterior(36,4%) seguida da coronária direita e circunflexa. De acordo com pesquisa realizada em são Paulo-Brasil com 161 pacientes submetidos à angioplastia, 35,4% receberam stent na artéria descendente anterior, seguido decoronária direita com 29,8% e 21,7% em circunflexa(18). Por fim, em relação ao local da punção, a via femoral foi mais utilizada no presente estudo (Tabela 4), seguida da radial. Estudo realizado em país asiático (10) demonstra a via femoral como mais utilizada, com porcentagem de 82,6%. Entretanto, pesquisa realizada no Sul brasileiro (11) resultou em 72,3% dos pacientes com via radial de punção mais prevalente. Diversos autores expõem a via radial como mais vantajosa, pois, apresenta menores taxas de complicações, como sangramento e mortalidade por fatores cardíacos (7, 15,18). Este estudo apresenta limitações por ser observacional, unicêntrico e possuir uma amostra pequena. Admitimos a possibilidade de viés visto que foram obtidos através dos registros em prontuários Limitações do estudo Este estudo apresenta limitações por ser observacional, unicêntrico e possuir uma amostra limitada de pacientes. Contribuições do estudo para a prática Acredita-se que os dados desta pesquisa podem contribuir na criação de estratégias para prevenir agravos após procedimento angiográfico, garantindo uma melhor qualidade de vida desses pacientes. Por isso, nota-se a importância de se continuar analisando o perfil clínico e epidemiológico de pacientes submetidos à Intervenção Coronária Percutânea, visando implementar uma linha de cuidado na redução de danos através da capacitação da equipe de saúde. Contribuindo assim, na qualidade da assistência oferecida. Enferm. Foco 2019; 10 (3): 126-133 131 ARTIGO 21 PACIENTES SUBMETIDOS À ANGIOPLASTIA TRANSLUMINAL CORONARIANA: ANÁLISE EPIDEMIOLÓGICA E ANGIOGRÁFICA Juliano Teixeira Moraes, Alexandre Ernesto Silva, Tarcísio Laerte Gontijo, Renato Ferreira Ribeiro, Rafaela das Graças Santiago Faria CONCLUSÃO Conclui-se que o perfil sócio demográfico dos pacientes submetidos à ATC eletiva são pacientes do sexo masculino, idosos com baixo grau de escolaridade, pardos, casados e aposentados. A análise angiográfica constitui-se de pacientes com predominância de implantação de um (1) stent do tipo farmacológico, descendente anterior e coronária direita como coronárias tratadas, sendo a punção de acesso com maior prevalência a por via femoral. Observa-se que alguns indicadores do processo sofrem influência de aspectos estruturais e organizacionais da rede de atenção a Saúde, com isso, acredita-se que a consolidação da qualidade da assistência através da elaboração de políticas públicas de saúde que abranjam a organização da linha de 132 Enferm. Foco 2019; 10 (3): 126-133 cuidado do Estado, sendo indispensável à agregação dos diversos serviços da rede de saúde, visando atenuar eventos futuros e melhorar a efetividade da terapia. Assim, é imprescindível também a adequação dos profissionais, através do conhecimento do público-alvo a adoção de ferramentas metodológicas diversas para o melhor entendimento do paciente submetido à ATC eletiva para que a continuidade do processo do cuidado seja eficaz. Contribuição dos autores Declara-se que, nesta pesquisa, todos os autores participaram da concepção, análise e interpretação dos dados, redação do artigo, revisão crítica e revisão final. 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https://openalex.org/W3199954582	https://europepmc.org/articles/pmc8442309?pdf=render	English	null	The unidirectional phylogeny of Homo sapiens anchors the origin of modern humans in Eurasia	Hereditas	2,021	cc-by	5,646	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Árnason Hereditas (2021) 158:36 https://doi.org/10.1186/s41065-021-00197-7 Open Access Abstract Background: The Out of Africa hypothesis, OOAH, was challenged recently in an extended mtDNA analysis, PPA (Progressive Phylogenetic Analysis), that identified the African human populations as paraphyletic, a finding that contradicted the common OOAH understanding that Hss had originated in Africa and invaded Eurasia from there. The results were consistent with the molecular Out of Eurasia hypothesis, OOEH, and Eurasian palaeontology, a subject that has been largely disregarded in the discussion of OOAH. Results: In the present study the mtDNA tree, a phylogeny based on maternal inheritance, was compared to the nuclear DNA tree of the paternally transmitted Y-chromosome haplotypes, Y-DNAs. The comparison showed full phylogenetic coherence between these two separate sets of data. The results were consistent with potentially four translocations of modern humans from Eurasia into Africa, the earliest taking place ≈ 250,000 years before present, YBP. The results were in accordance with the postulates behind OOEH at the same time as they lent no support to the OOAH. Conclusions: The conformity between the mtDNA and Y-DNA phylogenies of Hss is consistent with the understand- ing that Eurasia was the donor and not the receiver in human evolution. The evolutionary problems related to OOAH became similarly exposed by the mtDNA introgression that took place from Hss into Neanderthals ≈ 500,000 YBP, a circumstance that demonstrated the early coexistence of the two lineages in Eurasia. Keywords: Human evolution, Molecular phylogenetics, Out of Eurasia hypothesis, OOEH, Out of Africa hypothesis, OOAH, mtDNA, nuDNA, Y-DNA with the common OOAH understanding that Hss had originated in Africa and invaded Eurasia from there. The unidirectional phylogeny of Homo sapiens anchors the origin of modern humans in Eurasia Úlfur Árnason1,2* Úlfur Árnason1,2* Background A continuous unidirectional evolution from the root of the tree to the tip of each individual branch is an indis- putable requirement for the validity of any phylogenetic tree. In a recent study [1] the direction of evolution in the tree of modern man, Homo sapiens sapiens, Hss, and man’s closest extinct relatives was determined in accord- ance with this condition. The results were inconsistent In their study [1] the authors introduced a new molecu- lar approach, Progressive Phylogenetic Analysis (PPA) that allowed establishment of the direction of evolution in the Hss tree. The results showed that the African Hss populations constituted a paraphyletic assembly, a find- ing that compromised the foundation of the OOAH. In comparison the established PPA phylogeny was consist- ent with the OOEH and the Eurasian palaeontology of both Hss and Hsn(Hsnn +Hsnd), a topic that has been largely ignored by the adherents of OOAH. Correspondence: ulfur.arnason@gmail.com; ulfur.arnason@med.lu.se 1 Department of Clinical Sciences Lund, Lund University, Lund, Sweden Full list of author information is available at the end of the article Correspondence: ulfur.arnason@gmail.com; ulfur.arnason@med.lu.se 1 Department of Clinical Sciences Lund, Lund University, Lund, Sweden Full list of author information is available at the end of the article Hsn divides into Hsnn, Neanderthals proper, and Hsnd, Denisova, with Hsnn dividing further into SH-Hsnn (Hsnn at Sima de los Huesos) and Hsnn, a branch arising as the result of the mtDNA introgression that took place from Hss into Hsnn ≈ 500,000 YBP [1] Fig. 1 The nuDNA phylogeny leading to Hss, Homo sapiens sapiens. Blue: Eurasian lineages. Red: African lineages. H. erectus has been placed at the root of the tree in accordance with the artefact sequence related to the Eurasian existence of He 2,12 MYBP [15]. The divergence between Hs, H. sapiens, and Ha, H. antecessor, has been dated to ≈ 850,000 YBP [16, 17], that between Hss and Hsn, H. s. neanderthalensis, to ≈ 800,000 YBP and that between Lund and Mbuti/San to ≈ 250,000 YBP. Hsn divides into Hsnn, Neanderthals proper, and Hsnd, Denisova, with Hsnn dividing further into SH-Hsnn (Hsnn at Sima de los Huesos) and Hsnn, a branch arising as the result of the mtDNA introgression that took place from Hss into Hsnn ≈ 500,000 YBP [1] Here the evolution of Hss was addressed in the light of the phylogenies of two separate sets of data, viz. the nuclear DNA, nuDNA, tree of the paternally transmit- ted Y-chromosome haplotypes, Y-DNAs [2–4], and the mtDNA tree, a non-nuclear tree with maternal inherit- ance. The findings supported conclusively the results pre- sented in the initial Y-DNA studies [3, 4] and the more recent mtDNA findings that have challenged OOAH [1, 5, 6]. compared. Thus, the comparison comprised two evo- lutionarily separate sets of data, viz. a paternal nuclear marker, Y-DNA, and a non-nuclear marker, mtDNA, based on maternal inheritance. The comparison yielded results that were mutually consistent with each other and with the OOEH while they lent no support to the out of Africa hypothesis. The Y-DNA evolution of Neanderthals and Deniso- vans was addressed in a recent study [12] that related the Neanderthal mtDNA replacement to an ancient gene flow from an early lineage related to modern humans. The authors [12] referred to two studies [13, 14] in this connection, both acknowledging Hss origin in Africa and OOAH. One of these studies [13] presented a com- prehensive OOAH account that rested upon Hs origin in Africa followed by an African divergence between Hss and Hsn (Neanderthals/Denisovans). © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 5 Árnason Hereditas (2021) 158:36 Fig. 1 The nuDNA phylogeny leading to Hss, Homo sapiens sapiens. Blue: Eurasian lineages. Red: African lineages. H. erectus has been placed at the root of the tree in accordance with the artefact sequence related to the Eurasian existence of He 2,12 MYBP [15]. The divergence between Hs, H. sapiens, and Ha, H. antecessor, has been dated to ≈ 850,000 YBP [16, 17], that between Hss and Hsn, H. s. neanderthalensis, to ≈ 800,000 YBP and that between Lund and Mbuti/San to ≈ 250,000 YBP. Hsn divides into Hsnn, Neanderthals proper, and Hsnd, Denisova, with Hsnn dividing further into SH-Hsnn (Hsnn at Sima de los Huesos) and Hsnn, a branch arising as the result of the mtDNA introgression that took place from Hss into Hsnn ≈ 500,000 YBP [1] Fig. 1 The nuDNA phylogeny leading to Hss, Homo sapiens sapiens. Blue: Eurasian lineages. Red: African lineages. H. erectus has been placed at the root of the tree in accordance with the artefact sequence related to the Eurasian existence of He 2,12 MYBP [15]. The divergence between Hs, H. sapiens, and Ha, H. antecessor, has been dated to ≈ 850,000 YBP [16, 17], that between Hss and Hsn, H. s. neanderthalensis, to ≈ 800,000 YBP and that between Lund and Mbuti/San to ≈ 250,000 YBP. This stage was fol- lowed by an out of Africa exodus of Hsn and a Eurasian divergence of Hsn into Neanderthals and Denisovans and a late out of Africa exodus of Hss. These circumstances have limited support in palaeontology and molecular findings related to Hs(Hss + Hsn) evolution. During the latter part of the 1980s, fragmentary sequence data from mtDNA became introduced in molecular phylogenetics with the aim of establishing the relationships within and among different species. In this era the group of Allan C. Wilson presented results that, according to the authors [7, 8], provided evidence that Hss had originated in Africa and subsequently migrated from there into Eurasia. The hypothesis soon became the norm in the discussions of Hss evolution, although the molecular basis for the understanding was questionable and the palaeontological support for it was lacking. Fur- thermore, examination of the same data in other molecu- lar studies [9–11] did not favour the trees upon which the OOAH postulate was proposed. The extensive studies of human Y chromosome haplo- types referred to above [3, 4] have yielded comprehen- sive phylogenetic results that are highly relevant to the discussion of OOAH. Contrary to the common OOAH understanding these studies identified a basal divergence between the non-African and African Hss populations, a finding that was incompatible with the OOAH position of a late Hss exodus out of Africa. The out of Eurasia phylogeny Figure 1 outlines basal Hs relationships in accordance with recent advances in Eurasian palaeontology and phy- logenetics [1]. The Eurasian-derived parts of the figure are marked blue and the African contribution red. Homo erectus, He, has been placed at the root of the tree consist- ent with He entering Eurasia from Africa > 2 million years ago [15] in agreement with the Eurasian palaeontology of Here the topologies of the Y-DNA tree [2, 4] and the PPA phylogeny of the mtDNA tree of Hss [1] were Page 3 of 5 Árnason Hereditas (2021) 158:36 Fig. 2 The mtDNA relationships of Hs demonstrating the paraphyly of the African Hss populations as resolved by PPA. Blue: non-African taxa; red: African taxa. Hsnn: Hsnn other than SH-Hsnn. The arrowheads signify the mtDNA introgression that gave rise to Hsnn. The limitation of Hsnn to Eurasia places the mtDNA introgression in this continent, reversing the direction of Hss evolution behind OOAH. The Hss part of the tree underlines the phylogenetic continuity among non-African populations and the paraphyly of the African populations including the two Yoruba [1]. AuAb: Australian aborigines; PNG: Papua New Guinean; Han: Chinese; Lund: The first described non-chimaeric human mtDNA molecule [19]; French: A European, as representing previous genomic findings [20] Fig. 2 The mtDNA relationships of Hs demonstrating the paraphyly of the African Hss populations as resolved by PPA. Blue: non-African taxa; red: African taxa. Hsnn: Hsnn other than SH-Hsnn. The arrowheads signify the mtDNA introgression that gave rise to Hsnn. The limitation of Hsnn to Eurasia places the mtDNA introgression in this continent, reversing the direction of Hss evolution behind OOAH. The Hss part of the tree underlines the phylogenetic continuity among non-African populations and the paraphyly of the African populations including the two Yoruba [1]. AuAb: Australian aborigines; PNG: Papua New Guinean; Han: Chinese; Lund: The first described non-chimaeric human mtDNA molecule [19]; French: A European, as representing previous genomic findings [20] the contemporary coexistence of both Hss and Hsnn in Eurasia [1, 6], a circumstance that invalidates OOAH since that hypothesis does not allow the existence of Hss in Eurasia at the time in question. the species. As apparent the evolution is continuous and unidirectional through the entire tree from the oldest to the most recent Hss divergence as represented by the African Mbuti/San and the Eurasian Lund. The earliest divergence in Fig. The profiles of the Y‑DNA and mtDNA phylogenies of Hss The profiles of the Y‑DNA and mtDNA phylogenies of Hss Figure 3a–c shows the phylogenetic relationships of recent Hss as resolved in analysis of two separate sets of data, the paternally transmitted Y-DNA and the mater- nally transmitted mtDNA. The Y-DNA tree of OOEH [3, 4] is shown to the left in the figure, the mtDNA tree of OOEH in the middle [1] and the commonly acknowl- edged Y-DNA tree of OOAH (e.g. [2]) to the right. Although the three trees are superficially similar they are fundamentally different in that trees 3a and 3b are con- sistent with the out of Eurasia hypothesis whereas tree 3c is that of OOAH. y The Y-DNA tree in Fig. 3a and the mtDNA tree in 3b are both consistent with a unidirectional Hss evolution from the Eurasian root of the Hs tree to the tip of each individual Hss branch. The Y-DNA tree is based upon the largest sample that has been used to delineate the Y-DNA relationships of extant humans. The analysis [4] identified a Eurasian Y-DNA coalescence, A00, that was followed by three separate Hss exoduses, A0, A and α, from Eurasia into Africa with A0 signifying the earliest and α the most recent of these early exoduses. The Eurasian Y-DNA phy- logeny shows a continuous Eurasian span from position α to the extensive Eurasian diversification beginning at position β in the Y-DNA tree. The extensive Y-DNA study [4] behind Fig. 3a showed a minimum of five waves of Hss migration from Eura- sia into Africa while the more limited mtDNA sampling behind 3b [1] identified a minimum of three migrations with each of these coinciding with the Y-DNA results, consistent with the presence of both males (Y-DNA) and females (mtDNA) in each population migrating into Africa. As apparent a divergence between Mbuti and San prior to their migration into Africa [5] would raise the number of waves into Africa by one, as would also any The mtDNA tree in Fig. 3b mirrors the Y-DNA phy- logeny in accordance with a shared identity, male and female, within each pair of the Hss exoduses from Eurasia into Africa. Similarly the barren Eurasian branch between Fig. 3 The Y-DNA and mtDNA phylogenies of Hss with the Y-DNA tree (a) [4] and the mtDNA tree (b) [1] representing the OOEH phylogeny, and tree (c) standing for the OOAH phylogeny of both mtDNA and Y-DNA. The out of Eurasia phylogeny 1, that between Ha, Homo antecessor, and Hs, Homo sapiens, has been placed palaeontologically [16] and molecularly [17] at ≈ 850,000 YBP. As Fig. 1 shows, the Hs branch splits into a branch leading to extant humans, Hss, and another branch, Hsn, H. sapiens neanderthalensis, that gave rise to Hsnn, Neanderthals proper, and Hsnd, Denisovans. Hsnn diverged early into two branches, SH-Hsnn and Hsnn, both palaeontologically and molecularly [18] identified. SH-Hsnn inhabited northern Spain, while Hsnn reigned both in Europe and Asia, as established by extensive Eur- asian fossil finds. An extensive genomic study of extant humans that was presented a few years ago [20] identified a basal Hss divergence between a Eurasian (French) genome and the genome of the African Mbuti, but the significance of the finding for addressing Hss origin and evolution was not discussed. However, as maintained subsequently on phy- logenetic grounds [6], this basal Hss divergence compro- mised OOAH since that hypothesis rested instead upon an Hss exodus out of Africa into Eurasia by a late arising African population and not on a population that consti- tuted the earliest divergence among recent humans. In the recent study by Árnason and Hallström [1] the direction of evolution in the Hss tree was established by applying a new approach, Progressive Phylogenetic Anal- ysis, PPA, which demonstrated that the African popula- tions constituted a paraphyletic grouping. The African paraphyly invalidated the postulate of an Hss origin in Africa and a late exodus out of that continent into Eurasia since the PPA identified instead a minimum of three sep- arate waves of Hss migration from Eurasia into Africa the earliest being that of the ancestors of Mbuti/San. Thus, in i The arrowheads that lead from Hss to Hsnn* in Fig. 1 mark the phylogeny resulting from the mtDNA intro- gression that took place from Hss into Hsnn* ≈ 500,000 YBP [1 As shown in Fig. 2 the introgression joins Hss and Hsnn* on a common mtDNA branch, therewith restrict- ing the initial mtDNA branch of Hsn to SH-Hsnn and Hsnd. With the Neanderthals strictly limited to Eurasia it becomes apparent that the mtDNA introgression from Hss to Hsnn* could only take place in conjunction with Árnason Hereditas (2021) 158:36 Page 4 of 5 migration connected to the B + C branch [4] that has been tentatively indicated on the mtDNA tree. The profiles of the Y‑DNA and mtDNA phylogenies of Hss Trees (a) and (b) are consistent with a residing Eurasian Hss populations and a series of Hss exoduses from Eurasia into Africa. Position A00 in the Y-DNA tree and the corresponding position in the mtDNA tree mark the position at which the basal African and non-African lineages of extant Hss populations coalesce. Position β in tree (a) signifies the beginning of the Eurasian diversification of Hss dated to ≈ 125,000 to 120,000 YBP in the mtDNA tree [1]. The blue branch in (c) signifies a late Hss exodus out of Africa as assumed by the out of Africa hypothesis. As underlined in Figs. 1 and 2, the position of the root of the phylogeny in tree (c) is without connection to the Eurasian evolution of Hss and Hsn(Hsnn + Hsnd) Fig. 3 The Y-DNA and mtDNA phylogenies of Hss with the Y-DNA tree (a) [4] and the mtDNA tree (b) [1] representing the OOEH phylogeny, and tree (c) standing for the OOAH phylogeny of both mtDNA and Y-DNA. Trees (a) and (b) are consistent with a residing Eurasian Hss populations and a series of Hss exoduses from Eurasia into Africa. Position A00 in the Y-DNA tree and the corresponding position in the mtDNA tree mark the position at which the basal African and non-African lineages of extant Hss populations coalesce. Position β in tree (a) signifies the beginning of the Eurasian diversification of Hss dated to ≈ 125,000 to 120,000 YBP in the mtDNA tree [1]. The blue branch in (c) signifies a late Hss exodus out of Africa as assumed by the out of Africa hypothesis. As underlined in Figs. 1 and 2, the position of the root of the phylogeny in tree (c) is without connection to the Eurasian evolution of Hss and Hsn(Hsnn + Hsnd) Fig. 3 The Y-DNA and mtDNA phylogenies of Hss with the Y-DNA tree (a) [4] and the mtDNA tree (b) [1] representing the OOEH phylogeny, and tree (c) standing for the OOAH phylogeny of both mtDNA and Y-DNA. Trees (a) and (b) are consistent with a residing Eurasian Hss populations and a series of Hss exoduses from Eurasia into Africa. Position A00 in the Y-DNA tree and the corresponding position in the mtDNA tree mark the position at which the basal African and non-African lineages of extant Hss populations coalesce. The out of Eurasia phylogeny contradiction to OOAH the PPA approach yielded results that were consistent with a continuous unidirectional Eurasian evolution on the Hs branch from the divergence between H. antecessor and H. sapiens to the apical tips of extant Hss as demonstrated in Figs. 1 and 2. With respect to the phylogeny in Fig. 3c it should be noted that the OOAH tree and the out of Africa hypothesis, in addition to their earlier rebuttal [3, 4], became rejected by the recent PPA findings [1] which demonstrated the paraphyly of the African popula- tions. In contradiction to OOAH these molecular results were all in accordance with the out of Eurasia hypoth- esis and the Eurasian palaeontology of both Hss and Hsn(Hsnn + Hsnd), a topic that has been largely ignored by the adherents of OOAH. Author details 1 Author details 1 Department of Clinical Sciences Lund, Lund University, Lund, Sweden. 2 Department of Neurosurgery, Skane University Hospital in Lund, Lund, Sweden. References Á the last exodus into Africa, and position β, the initiation of the Eurasian diversification of Hss is common to both phylogenies although the span is longer in the Y-DNA phylogeny than in the mtDNA tree, a distinction that might be related to different modes of calculation. 1. Árnason Ú, Hallström B. The reversal of human phylogeny: Homo left Africa as erectus, came back as sapiens sapiens. Hereditas. 2020;157:51. https://doi.org/10.1186/s41065-020-00163-9. 1. Árnason Ú, Hallström B. 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Cann RL, Stoneking M, Wilson AC. Mitochondrial DNA and human evolu- tion. Nature. 1987;325:31–6. 7. Cann RL, Stoneking M, Wilson AC. Mitochondrial DNA and human evolu- tion. Nature. 1987;325:31–6. Author’s contributions 18. Arnason U, Xu X, Gullberg A. Comparison between the complete mito- chondrial DNA sequence of Homo and the common chimpanzee based on nonchimeric sequences. J Mol Evol. 1996;42:145–52. Conclusionsh 8. Vigilant L, Stoneking M, Harpending H, Hawkes K, Wilson AC. African populations and the evolution of human mitochondrial DNA. Science. 1991;253:1503–7. The Y-DNA and mtDNA phylogenies discussed here are consistent with a unidirectional Hs evolution from the Eurasian root of the Hs tree to each individual branch connected to Hss evolution, including the populations that migrated from Eurasia into Africa. The findings compromise the large number of results that have been interpreted in accordance with the Out of Africa hypoth- esis and the a priori assumption that the African Hss populations constituted a monophyletic assembly from which Eurasia became colonized. A crucial phylogenetic circumstance related to the rebuttal of OOAH has been the identification of the separate Hss exoduses into Africa with each of these recorded by both the female inherited mtDNA and the male inherited Y-DNA. 9. Maddison DR. African origin of human mitochondrial DNA reexamined. Syst Zool. 1991;40:355–63. 9. Maddison DR. African origin of human mitochondrial DNA reexamined. Syst Zool. 1991;40:355–63. 10. Templeton AR. Human origins and analysis of mitochondrial DNA sequences. Science. 1992;255:737. 11. Hedges BS, Kumar S, Tamura K, Stoneking M. Human origins and the analysis of mitochondrial DNA sequences. Science. 1992;255:737–9. 11. Hedges BS, Kumar S, Tamura K, Stoneking M. Human origins and the analysis of mitochondrial DNA sequences. Science. 1992;255:737–9. 12. Petr M, Hajdinjak M, Fu Q, Essel E, Rougier H, Crevecoeur I, et al. The evo- lutionary history of Neanderthal and Denisovan Y chromosomes. Science. 2020;369:1653–6. 12. Petr M, Hajdinjak M, Fu Q, Essel E, Rougier H, Crevecoeur I, et al. The evo- lutionary history of Neanderthal and Denisovan Y chromosomes. Science. 2020;369:1653–6. 13. Posth C, Wißing C, Kitagawa K, Pagani L, van Holstein L, Racimo F, et al. Deeply divergent archaic mitochondrial genome provides lower time boundary for African gene flow into Neanderthals. Nat Commun. 2017. https://doi.org/10.1038/ncomms16046. 13. Posth C, Wißing C, Kitagawa K, Pagani L, van Holstein L, Racimo F, et al. Deeply divergent archaic mitochondrial genome provides lower time boundary for African gene flow into Neanderthals. Nat Commun. 2017. https://doi.org/10.1038/ncomms16046. 14. Meyer M, Arsuaga J-L, de Filippo C, Nagel S, Aximu-Petri A, Nickel B, et al. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins. Nature. 2016;531:504–7. 14. Meyer M, Arsuaga J-L, de Filippo C, Nagel S, Aximu-Petri A, Nickel B, et al. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins. Nature. Conclusionsh 2016;531:504–7. 15. Zhu Z, Dennell R, Huang W, Wu Y, Qiu S, Yang S, et al. Hominin occupation of the Chinese loess plateau since about 2.1 million years ago. Nature. 2018;559:608–12. 15. Zhu Z, Dennell R, Huang W, Wu Y, Qiu S, Yang S, et al. Hominin occupation of the Chinese loess plateau since about 2.1 million years ago. Nature. 2018;559:608–12. Declarations 19. Meyer M, Fu Q, Aximu-Petri A, Glocke I, Nickel B, Arsuaga J-L, et al. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature. 2014;505:403–5. 19. Meyer M, Fu Q, Aximu-Petri A, Glocke I, Nickel B, Arsuaga J-L, et al. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature. 2014;505:403–5. Competing interests Competing interests The author declares that he/she has no competing interests. Competing interests The author declares that he/she has no competing interests. p g The author declares that he/she has no competing interests. 20. Mallick S, Li H, Lipson M, Mathieson I, Gymrek M, Racimo F, et al. The Simons genome diversity project: 300 genomes from 142 diverse popula- tions. Nature. 2016;538:201–6. 20. Mallick S, Li H, Lipson M, Mathieson I, Gymrek M, Racimo F, et al. The Simons genome diversity project: 300 genomes from 142 diverse popula- tions. Nature. 2016;538:201–6. The profiles of the Y‑DNA and mtDNA phylogenies of Hss Welker F, Ramos-Madrigal J, Gutenbrunner P, Mackie M, Tiwary S, Jersie- Christensen RR, et al. The dental proteome of Homo antecessor. Nature. 2020;580:235–8. 18. Arnason U, Xu X, Gullberg A. Comparison between the complete mito- chondrial DNA sequence of Homo and the common chimpanzee based on nonchimeric sequences. J Mol Evol. 1996;42:145–52. 19. Meyer M, Fu Q, Aximu-Petri A, Glocke I, Nickel B, Arsuaga J-L, et al. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature. 2014;505:403–5. 20. Mallick S, Li H, Lipson M, Mathieson I, Gymrek M, Racimo F, et al. The Simons genome diversity project: 300 genomes from 142 diverse popula- tions. Nature. 2016;538:201–6. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Acknowledgements g The author thanks Prof. Eric H. Harley, University of Cape Town, RSA, for valu- able comments and suggestions and Prof. Anatole A. Klyosov, Harvard Medical School, MA, USA, for Fig. 3a. The author also thanks The Erik Philip-Sörensens Foundation and The Royal Physiographic Society, Lund, for support. 16. de Castro JMB, Arsuaga JL, Carbonell E, Rosas A, Martínez I, Mosquera M. A Hominid from the Lower Pleistocene of Atapuerca, Spain: possible ancestor of Neandertals and modern humans. Science. 1997;276:1392–5. 16. de Castro JMB, Arsuaga JL, Carbonell E, Rosas A, Martínez I, Mosquera M. A Hominid from the Lower Pleistocene of Atapuerca, Spain: possible ancestor of Neandertals and modern humans. Science. 1997;276:1392–5. 17. Welker F, Ramos-Madrigal J, Gutenbrunner P, Mackie M, Tiwary S, Jersie- Christensen RR, et al. The dental proteome of Homo antecessor. Nature. 2020;580:235–8. 17. Welker F, Ramos-Madrigal J, Gutenbrunner P, Mackie M, Tiwary S, Jersie- Christensen RR, et al. The dental proteome of Homo antecessor. Nature. 2020;580:235–8. Author’s contributions The author read and approved the final manuscript. Author’s contributions Author’s contributions The author read and approved the final manuscript. The profiles of the Y‑DNA and mtDNA phylogenies of Hss 2016;538:201–6. Publisher’s Note Springer Nature remains neutral with regard lished maps and institutional affiliations. References 1. Árnason Ú, Hallström B. The reversal of human phylogeny: Homo left Africa as erectus, came back as sapiens sapiens. Hereditas. 2020;157:51. https://doi.org/10.1186/s41065-020-00163-9. 2. Scozzari R, et al. Molecular dissection of the basal clades in the human Y chromosome phylogenetic tree. PLoS One. 2012;7(11):1-5.e49170. https://doi.org/10.1371/journal.pone.0049170. 3. Klyosov AA, Rozhanskii IL. Re-examining the “Out of Africa” theory and the origin of Europeoids (Caucasoids) in light of DNA genealogy. Adv Anthropol. 2012;2:80–6. https://doi.org/10.4236/aa.2012.22009. 4. Klyosov AA. Reconsideration of the “Out of Africa” concept as not having enough proof. Adv Anthropol. 2014;4:18–37. https://doi.org/10.4236/aa. 2014.41004. 5. Árnason Ú. The out of Africa hypothesis and the ancestry of recent humans: cherchez la femme (et l’homme). Gene. 2016;585:9–12. 6. Árnason Ú. A phylogenetic view of the out of Asia/Eurasia and out of Africa hypotheses in the light of recent molecular and palaeontological finds. Gene. 2017;627:473–6. 7. Cann RL, Stoneking M, Wilson AC. Mitochondrial DNA and human evolu- tion. Nature. 1987;325:31–6. 8. Vigilant L, Stoneking M, Harpending H, Hawkes K, Wilson AC. African populations and the evolution of human mitochondrial DNA. Science. 1991;253:1503–7. 9. Maddison DR. African origin of human mitochondrial DNA reexamined. Syst Zool. 1991;40:355–63. 10. Templeton AR. Human origins and analysis of mitochondrial DNA sequences. Science. 1992;255:737. 11. Hedges BS, Kumar S, Tamura K, Stoneking M. Human origins and the analysis of mitochondrial DNA sequences. Science. 1992;255:737–9. 12. Petr M, Hajdinjak M, Fu Q, Essel E, Rougier H, Crevecoeur I, et al. The evo- lutionary history of Neanderthal and Denisovan Y chromosomes. Science. 2020;369:1653–6. 13. Posth C, Wißing C, Kitagawa K, Pagani L, van Holstein L, Racimo F, et al. Deeply divergent archaic mitochondrial genome provides lower time boundary for African gene flow into Neanderthals. Nat Commun. 2017. https://doi.org/10.1038/ncomms16046. 14. Meyer M, Arsuaga J-L, de Filippo C, Nagel S, Aximu-Petri A, Nickel B, et al. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins. Nature. 2016;531:504–7. 15. Zhu Z, Dennell R, Huang W, Wu Y, Qiu S, Yang S, et al. Hominin occupation of the Chinese loess plateau since about 2.1 million years ago. Nature. 2018;559:608–12. 16. de Castro JMB, Arsuaga JL, Carbonell E, Rosas A, Martínez I, Mosquera M. A Hominid from the Lower Pleistocene of Atapuerca, Spain: possible ancestor of Neandertals and modern humans. Science. 1997;276:1392–5. 17. The profiles of the Y‑DNA and mtDNA phylogenies of Hss Position β in tree (a) signifies the beginning of the Eurasian diversification of Hss dated to ≈ 125,000 to 120,000 YBP in the mtDNA tree [1]. The blue branch in (c) signifies a late Hss exodus out of Africa as assumed by the out of Africa hypothesis. As underlined in Figs. 1 and 2, the position of the root of the phylogeny in tree (c) is without connection to the Eurasian evolution of Hss and Hsn(Hsnn + Hsnd) Árnason Hereditas (2021) 158:36 Árnason Hereditas (2021) 158:36 Page 5 of 5 References 1. Árnason Ú, Hallström B. The reversal of Africa as erectus, came back as sapiens https://doi.org/10.1186/s41065-020-00 2. Scozzari R, et al. Molecular dissection o Y chromosome phylogenetic tree. PLo https://doi.org/10.1371/journal.pone.0 3. Klyosov AA, Rozhanskii IL. Re-examinin the origin of Europeoids (Caucasoids) Anthropol. 2012;2:80–6. https://doi.org 4. Klyosov AA. Reconsideration of the “Ou enough proof. Adv Anthropol. 2014;4:1 2014.41004. 5. Árnason Ú. The out of Africa hypothesi humans: cherchez la femme (et l’homm 6. Árnason Ú. A phylogenetic view of the Africa hypotheses in the light of recen finds. Gene. 2017;627:473–6. 7. Cann RL, Stoneking M, Wilson AC. Mito tion. Nature. 1987;325:31–6. 8. Vigilant L, Stoneking M, Harpending H populations and the evolution of hum 1991;253:1503–7. 9. Maddison DR. African origin of human Syst Zool. 1991;40:355–63. 10. Templeton AR. Human origins and ana sequences. Science. 1992;255:737. 11. Hedges BS, Kumar S, Tamura K, Stonek analysis of mitochondrial DNA sequen 12. Petr M, Hajdinjak M, Fu Q, Essel E, Roug lutionary history of Neanderthal and D 2020;369:1653–6. 13. Posth C, Wißing C, Kitagawa K, Pagani Deeply divergent archaic mitochondri boundary for African gene flow into N https://doi.org/10.1038/ncomms1604 14. Meyer M, Arsuaga J-L, de Filippo C, Nag Nuclear DNA sequences from the Mid hominins. Nature. 2016;531:504–7. 15. Zhu Z, Dennell R, Huang W, Wu Y, Qiu S of the Chinese loess plateau since abo 2018;559:608–12. 16. de Castro JMB, Arsuaga JL, Carbonell E M. A Hominid from the Lower Pleistoc ancestor of Neandertals and modern h 17. Welker F, Ramos-Madrigal J, Gutenbrun Christensen RR, et al. The dental proteo 2020;580:235–8. 18. Arnason U, Xu X, Gullberg A. Comparis chondrial DNA sequence of Homo and on nonchimeric sequences. J Mol Evol 19. Meyer M, Fu Q, Aximu-Petri A, Glocke I mitochondrial genome sequence of a Nature. 2014;505:403–5. 20. Mallick S, Li H, Lipson M, Mathieson I, G Simons genome diversity project: 300 tions. Nature. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Received: 10 June 2021 Accepted: 10 August 2021 Received: 10 June 2021 Accepted: 10 August 2021 Received: 10 June 2021 Accepted: 10 August 2021
W1969874909.txt	https://zenodo.org/records/1508940/files/article.pdf	fr		<i>Phyllostachys aurea</i>Riviére. Sa floraison à Montpellier	Bulletin de la Société botanique de France	1,922	public-domain	1,432	Bulletin de la Société Botanique de France ISSN: 0037-8941 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tabg17 Phyllostachys aurea Riviére. Sa floraison à Montpellier M. J. Daveau To cite this article: M. J. Daveau (1922) Phyllostachys aurea Riviére. Sa floraison à Montpellier, Bulletin de la Société Botanique de France, 69:2, 232-236, DOI: 10.1080/00378941.1922.10833432 To link to this article: http://dx.doi.org/10.1080/00378941.1922.10833432 Published online: 08 Jul 2014. Submit your article to this journal Article views: 5 View related articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tabg17 Download by: [178.17.170.99] Date: 31 May 2016, At: 20:00 232 SÉANCE DU 28 AVRIL 1922. Downloaded by [178.17.170.99] at 20:00 31 May 2016 La coloration rouge disparaît peu à peu, et devient presque nulle chez la plante entièrement développée. En 1913, sur 176 plantes, 57 avaient perdu la coloration rouge, soit environ un tiers; en 1919, sur 40 plantes, 5 seulement l'avaient perdue, soit un huitième. Ces expériences ne sont pas assez rigoureusement conduites pour permettre d'en tirer des conclusions se rapportant à la loi de Mendel. Je ne me suis décidé à les publier que dans le but de provoquer des expériences précises et prolongées sur le sujet 1 • Phyllostachys nurea Rivière. Sa floraison à Montpellier PAR M. J. DAVEAU. La rareté de la floraison chez les Bambous, cultivés ou spontanés, a toujours été un obstacle à l'étude complète de ces élégants végétaux; aussi l'apparition des fleurs êhez une espèce quelconque de ce groupe de Graminées constitue-t-elle un fait toujours intéressant à note1·. Notre confrère, M. Gadeceau, le faisait encore remarquer dans un récent article au sujet de la floraison de l'Arundinaria Hindsii Munro 2 • Il s'agit cette fois du Phyllostachys aurea Rivière a dont la floraison vient de se produire à Nimes et à 1\Iontpellier dans plusieurs propriétés. Les premières fleurs se sont montrées dès l'automne de 1921, chez un pépiniériste hien connu des viticulteurs, M. Richter, et dans quelques autres propriétés; mais partout ailleurs, au Jardin botanique, par exemple, il n'y a encore aucun indice de floraison (avril i922). Après un arrêt causé par les froids, la floraison recommence à se manifester avec la reprise de la végétation et quelques caryopses en cours de développement s'observent dans les épillets fleuris à l'automne. 1. N'ayant rien à modifier dans mon article de 1915 (t. LXII), je ne rouvrirai pas le débat, et je continuerai à rapporter le C. amaranticolor Coste et Reynier au C. purpurascens Jacquin, en vertu du principe de la priorité. 2. Revue horticole, t 920-1921 p. 367. 3. Les Bambous, p. 262 à 267, fig. 37 et 38. Downloaded by [178.17.170.99] at 20:00 31 May 2016 J. DA VEAU. - PHYLLOSTACHYS AU REA RIVIÈRE. 233 Downloaded by [178.17.170.99] at 20:00 31 May 2016 234 Sii:ANCE DU 28 AVRIL t922. On sait que le Phylloslachys aurea se distingue des autres Bambous par la structure particulière de ses chaumes. Ceux-ci présentent, surtout à leur base, des nœuds renflés, très rapprochés, canaliculés d'un côté, ce sillon couespondan t au bourgeon axillaire et alternant par conséquent d'un mérithalle à l'autre'. Comme cela se passe généralement lorsque les Bambous se préparent à fleurir, les feuilles jaunissent, tombent et sont remplacées par les inflorescences. Celles-ci sont disposées en panicules formées d'épis nombreux, mesurant 4 à 5 centimètres de longueur. Chaque épi (fig. A) est f01·mé par 3, le plus souvent 5 épillets généralement uniflores, parfois biflores, mais dans le premier cas, on trouve toujours un rudiment de fleur ou rachéole (fig. 4, 7'). Chaque épillet présente ordinaireme~( 2 glumes (quelquefois 3), 2 glumelles, 3 étamines et un ovaire surmonté d'un style à 3 stigmates légèrement serrulés-plumenx. La glume inférieure (fig. 1.) de consistance papyracée est pourvue de nervures longitudinales; elle se termine toujours par un petit limbe foliacé; plus ou moins large, articulé avec elle, souvent desséché et enroulé au moment de l'anthèse. La glume supérieure (fig. A, 2), membraneuse, beaucoup plus courte que la glume inférieure est de consistance herbacée a nervures peu accusées. Les deux glumelles, inférieure et supérieure, se distinguent des glumes par leur texture herbacée et leur nervation en damier; l'inférieure s'atténue en pointe sétacée; la supérieure est toujours bifide à son extrémité (fig. 4). Adossée à la hase de sa face dorsale, légèrement excavée, se trouve la fleur rudimentaire réduite à une sorte de rachéole (fig. 4, r). Les glumellules, quand elles existent, sont toujours rudimentaires et membraneuses-pellucides. La glume supérieure manque à l'épillet terminal; par contre, les épillets de la base ont souvent 3 glumes au lieu de 2 et les épillets intermédiaires ont parfois 2 fleurs (fig. A! a'). Dans ce L Ces nœuds renflés et superposés font rechercher ce bambou dans l'indastrie, en particulier pour les manches d'ombrelles, de parapluies, les cannes, etc. Downloaded by [178.17.170.99] at 20:00 31 May 2016 J. DAVEAU. - PHYLLOSTACHYS AUREA RIVII;RE. 235 dernier cas, les glumelles supérieures sont dépourvues. de rachéole, ce qui semble bien démontrer que celle-ci n'est qu'une fleur avortée. L'androcée (fig. B etC) se compose de 3 étamines à filets très allongés et à anthères basifixes; le pistil présente un ovaire allon~é, presque cylindrique, surmonté d'un style et de 3 stigmates plumeux dont 2. sont soudés à la base : en réalité 2 stigmates dont l'tm est profondément bifide (fig. C). Avant l'anthèse, les anthèt·es se montrent à peine au sommet de la fleur (fig. A, a') les stigmates étant repliés sur eux-mêmes comme on le voit figure B. Au moment de t:anthèse, les filets s'allongent considérablement et l'anthère retombe suivant une direction verticale; cette disposition a pour effet de favoriser la pollinisation anémophile, bien que les anthères soient basifixes. En effet, contrairement à l'organisation généralè des Graminées dont les anthères sont dorsifixes et versatiles, les Bambous semblent faire exception à cette règle. On l'a vu plus haut pour le Phyllostachys aurea; il en est de même, comme nous avons pu le vérifier pour le P. fle.ruosa; les A tundinaria Simoni, Hindsii, J(hasiana, nzac1·osperma; Clntsquea Gaudi- chaudi, Quila; Planotia elata, et probablement chez d'autres Bambusacées. Bentham et Hooker 1 disent les anthères dorsifixes et très rarement basifixes, mais ces auteurs ne précisent pas dans quel groupe se produit cette exception. Rivière 2 écrit : « L'anthère est linéaire, biloculaire et versatile; c'est· à-dire « vacillante, parce qu'elle est attachée vers le milieu de sa partie dorsale .... >> Il est vrai que plus loin (p. 278), l'auteur décrivant le Phyllostachys flexuosa dit que les anthères « sont suspendues par la partie échancrée de l'une des extrémités », ce qui revient à dire qu'elles sont basifixes. La figure qui accompagne cette description (fig. 40·41) ne laisse du reste aucun doute à cet égard. Le genre Phyllostachys a été décrit pour la première fois par Siebold et Zuccarini 3 et cette description est reproduite 1. Genera Plantarum, Ill, Caractères géneraua: des Graminées, 2. L. c., p. 59. 3. In Abhandl. der München Acad., II, cL 3, p. 74.5, V, fig. 3. 236 SÉANCE DU 28 AVRIL 1922. Downloaded by [178.17.170.99] at 20:00 31 May 2016 dans Endlicher 1 ; les étamines y sont décrites comme il suit : « Stamina 3 libera, primum inclusa demum longe e.xserta, filamel!la fili(onrâa, antherœ basi emarginatœ sa,qittatœ apice inte,qrœ acutœ, Dl}Il.Su AFFIX.E. » Quant aux stigmates, tous les auteurs consultés se bornent à décrire 3 stigmates; nulle part, il n'est fait mention de la structure particulière dont nous avons- parlé et qu'on peut voir sur la planche jointe it eette note (fig. C). Le Châtaignier dans les Alpes-Maritimes M. J. AH BOST. PAR Au cours de notre préeédent travail sur la végétation de la Côte d'Azur 2 , sur la foi des anciennes cartes géologiques et l'assertion d'une publication antérieure à la nùtre, nous signalions le Châtaignier sur les calcaires dolomitiques de SaintVallier (Alpes-Maritimes). Or, il résulte des renseignements qu'a hien voulu nous fournir M. le Professeur D• Guibhard, l'éminent géologue, que « la petite châtaigneraie d' Arhoin ou du Piton (près S' Vallier) délimite au contraire exactement, au milieu des calcaires blancs tithoniques. un r~panchement labradori tique, sans même déborder sur les grès nummulitiques voisins ». Le substratum est donc nettement siliceux. Recherches sur l'embryogénie des Solanacées (Suite 3) .1 R M. 1\ EN É S 0 U È (; ES . HYOSCYAM:tES HYOSCYAMUS NIGER L., H. ALBUS L. L'étude embryogénique des Jusquiames peut ètre conduite assez aisément. Les ovules, dès les premiers stades de la séminogenèse, peuvent être séparés de la jeune capsule et inclus 1. Genera Plantarum, suppl. III, p. 58, n" 90.J.j2. 2. Bull. Soc. bot. de France, LX VIll, p. 273, i 921. 3. Voir plus haut, p. 163.
W4246850394.txt	https://www.biodiversitylibrary.org/partpdf/89158	de		Shalow on Arctic Birds Die Vögel der Arktis Herman Schalow	The Auk/The auk	1,905	public-domain	1,012	Vol.XXII cae Recent Literature. 103 grallatorial passeriform bird,’ obtained by Coloman Katona on the shores of Lake Jippe in East Atrica, and here described as Charadriola singular:s, gen. et. sp. nov. The terminal third of the tibia is unfeathered, scaled, and reticulated as in the plovers. In other respects this new type is much like the South African genus Macronyx, having the same long hind claw and other features, but a slenderer bill. The bird is illustrated by three text-figures, giving full-length figures of the male and female, the hind limb (natural size), and an enlarged figure of the tibia and proximal portion of the tarsus, to show the plover-like scutellation. In a notice of this paper in a recent number of ‘ The Ibis’ (Oct. 1904, p. 653), however, the bird here described is said to be not anew discovery, but a redescription of ZTmetothylacus tenellus Cabanis, and “that it is sof an aquatic species!”? Cabanis first described and figured the species as Macronyx tenellus (J. f. O. 1878, pp. 205, 220, pl. iii, fig. 2), and later (¢b7d., 1879, p. 438) made it the type of a new genus 7mefothylacus, on the basis mainly of the nakedness of the lower half of the tibia and its scaly covering. Sharpe, in 1885 (Cat. Birds Brit. Mus., X, p. 618), referred the species to Anthus and cites the already considerable literature relating to it—J. A. A. Shalow on Arctic Birds.2— The area embraced in the present elaborate memoir is limited to the seas and islands north of the Arctic Circle. Some fifteen pages of introductory matter (pp. 81-96) treat of the faunal relations of the region, and the views of leading writers thereon, and define the limits of the region under consideration. The bibliography of the subject takes the next fifteen pages (pp. 96-110), the titles being arranged chronologically, beginning with Martens, 1675, and including a portion of the literature of 1904. A systematic enumeration of the species, subspecies and higher groups follows. The 270 species and subspecies are next treated (pp. 114-271) in due sequence under the subheading ‘ Die Geographisch Verbreitung der das nordliche Polarmeer bewohnenden Vogel,’ under which is given for each the synonymy, bibliographic references, and a detailed account of its manner of occurrence in the region. Following this, the main body of the work, is an analysis of the distribution of the species and subspecies in the different islands, with reference to their faunal relations,—whether circumpolar, palearctic, or nearctic, and whether known to breed or not at the various islands in question. 1 An Extraordinary Discovery in Ornithology. By Dr. Julius v. Madarasz, Ann. Mus. Nat. Hungarici, II, 1904, pp. 396-398. ? Die V6gel der Arktis. Von Herman Schalow in Berlin. Fauna Arctica, eine Zusammenstellung der arktische Tierformen, mit besonderer Beriicksichtigung des Spitzbergen-Gebietes auf Grund der Ergebnisse der Deutchen Expedition in das Nordliche Eismeer im Jahr 1898. Band IV, Lieferung I, Verlag von Gustav Fischer in Jena. 1904. — 4to, pp. 81-288. n 104 Votes and News. Auk Jan. The region is limited, as already said, to the area north of the Arctic Circle, and includes the following eleven islands and groups of Islands: (1) Bear Island, with 28 species and subspecies, of which 17 breed on the island. (2) Spitzbergen Archipelago, with 50, of which 25 breed, while 18 of the 50 are circumpolar. (3) Franz Joseph Land, 28, 16, and possibly 18, of which breed, while 18 are circumpolar. (4) Nova Zembla, 49, of which 18 are circumpolar. (5) Kolgnew, 51, of which 42, or 84 percent, breed, and 16 are circumpolar. (6) Dolgoi, 23, of which 18 are circumpolar and 18 breed. (7) Waigat, with 49, of which 29 breed, and 17 are circumpolar. (8) New Siberia, 50, of which 19 are circumpolar, 23 palzarctic, and 8 nearctic, and 37 breed. (9) Wrangel Island, 28, of which 15 breed, I2 are circumpolar, 10 ‘ pacific-nearctic,’ and 6 palearctic. (10) Herald Island, 23, of which 14 breed, 8 are circumpolar, 11 ‘ pacific-nearctic,’ and 4 ‘Europeo-Asiatic.’ (11) Greenland, 161, of which 20 are circumpolar, 83 palearctic, and 58 nearctic, with 60 that breed. Of these 56 are common to both East and West Greenland; West Greenland has 153, of which 20 are circumpolar, 60 nearctic, and 73 palearctic; East Greenland, 64, of which 17 are circumpolar, 9 nearctic, and 38 palearctic. (12) Jan Mayen, 52, 18 of which are circumpolar, 4 nearctic, and 30 palearctic, and 19 have been found nesting. The ornithology of these different islands is very unequally known, some, like Jan Mayen, being very well known ornithologically, while others, like Dolgoi, Kolgnew, Waigat, etc., are very imperfectly known, so that the statistics for some of them include some more or less doubtful species. As will be seen, however, from the foregoing, in this critical examination of the arctic ornis the author has brought together a vast amount of exceedingly important and interesting information.— J. A. A. NOTES AND NEWS. Dr. SAMUEL W. Woopuouss, a Corresponding Fellow of the American Ornithologists’ Union, died at his home in Philadelphia on October 23, 1904. Although for many years retired from active work, Dr. Woodhouse’s early life was full of adventure and his name was prominent among men of science and travel fifty years ago. Samuel Washington Woodhouse was born in Philadelphia, June 27, 1821, the son of Com. Samuel Woodhouse, U. S. N., and H. Matilda Roberts. The family came from Northumberland, England, his grandfather emigrating to America in 1776. Dr. Woodhouse was educated at private schools in Philadelphia and at 1905. "Shalow on Arctic Birds." The Auk 22, 103–104. https://doi.org/10.2307/4069914. View This Item Online: https://www.biodiversitylibrary.org/item/54102 DOI: https://doi.org/10.2307/4069914 Permalink: https://www.biodiversitylibrary.org/partpdf/89158 Holding Institution Smithsonian Libraries and Archives Sponsored by Smithsonian Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 1 April 2024 at 12:32 UTC
https://openalex.org/W1995635219	https://zenodo.org/records/1548929/files/article.pdf	English	null	Working for the Individual	The annals of the American Academy of Political and Social Science/The Annals	1,913	public-domain	2,544	BY DAVID C. PEYTON, BY DAVID C. PEYTON, General Superintendent, Indiana Reformatory, Jeffersonville, Ind. He who blazes the way in any field of pioneer work must suffer suspicion, criticism and ridicule on the altar of public opinion which always contests the acceptance of new ideas until it is proved that the principles thus enunciated are true. Yet public opinion serves the very necessary purpose of staying the hand of the adventurer and dreamer who would selfishly exploit ideas and schemes that give no hope of ultimate usefulness. However, from the earliest time the line of duty in these matters has been so clear that few men, who have had an abiding faith in the genuine good that would ultimately come to humanity, have hesitated to render the service. The indi- vidual fall of a few men is of no consequence when compared with the great benefit accruing to society from action based upon truly scien- tific principles. Those of us here venturing to lend our endorsement to certain principles of pioneer labor in the field of conservation-the greatest of all its divisions, that of defective or sub-normal man-do so in all modesty, recognizing that any or all the truth and worth of our efforts is the result of our opportunity of observation and the evolution and enlargement of the thoughts of other and abler men. S h h b i f h i l i i l f h So much has been written of the essential principles of the great work of reclaiming the criminal, or law violator, that it is difficult to separate the new from the old. Our grateful nation would be impoverished indeed should it attempt fully to repay in love and gratitude such noble spirits as Brockway, Henderson, McClaughry, Butler and the late lamented Dr. Wines, and the hosts of others who have given so abundantly and unselfishly of their intellects and energies. i d h i hi h 66) Every generation and every epoch in history has brought advancement in all lines of human good, and our people and our time have not been weighed in the balance and found wanting. We have given to posterity the indeterminate sentence and parole laws, and their author has lived to see them universally accepted. BY DAVID C. PEYTON, How- ever, there is yet so much to be done, and the time in which to do it 66) at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from 67 so short that we should not even hesitate, much less stop. Let us here and now resolve to search out new and additional truths and, if needs be, pay the price of self-sacrifice in order to establish them on the pedestal of man’s eternal good. p g Is crime a disease? Not exactly; no. May it not better be regarded as an evidence of defective or undeveloped brain? Through medical science the world learned that insanity is a physical and not a spiritual ill, and kindness and scientific treatment supplanted harshness and unscientific methods. So also, crime is a problem for the neurologist and the psychologist, and the field of research is the brain and the mind. We know that certain divisions of the brain preside over intellect-volition, thought, memory-and another division over muscular co-ordination, and still another over the vital centers. Whether the mind or intellect is the brain acting, or whether the mind acts through the brain, it would seem that the process is inseparable from the organ. We should recognize that crime, like disease, has natural causes, and before we can intelligently treat the offender we must know his genesis. We account for some deformities by reason of an inflammatory interference with certain brain cells: we also have the precocious child as a result of abnormal, or early, excessive development of certain brain cells. May not science yet disclose that crime is but the natural result of a defective or undevel- oped brain ? Who will undertake to point out a sharp line of demarca- tion between the normal and abnormal, or tell where sanity leaves off and insanity begins ? Let him who can arbitrarily and with certainty fix these lines, point out to us the fundamentals of crime. Who can tell what the secret chambers of science may yet have in store for us ? We have the curable and the incurable diseases. There is such an apparent intimate relation between crime and defect that we are not surprised that the field has for a long time been attractive to many scientists, such as Lombroso, who considers epilepsy as forming, with atavism, the sub-stratum upon which is based the criminal world. at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from BY DAVID C. PEYTON, L t li l f h i Let us venture a little further into the speculative. If the mind is not the brain acting, but is rather to be considered synonymous with the spiritual part of man, where is its abiding place in cases of insanity or imbecility? If conscience is the moral attribute of mind that sits in judgment on and approves or disapproves our actions, what are its limitations-its influence over physical brain function? at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from 68 After all, it is the integrity of the mind that determines the integrity of the life. Let us return to the material and consider some of the causes of crime in order that we may more intelligently individualize the treatment rather than the punishment of the criminal. For the purposes under consideration we may classify most cases under two general heads, &dquo;heredity&dquo; and &dquo;environment.&dquo; Practically all causative influences may be properly considered under one or the other. The whole problem has been clouded with innumerable theories. As Dr. Healy so well says in a splendid article in the July Review: &dquo; It is even more important to note that many of the most competent students, especially those who have dealt largely with life rather than books, feel that the matter is anything but closed; the problem anything but solved.&dquo; Again the same writer strikes an important key-note when he says: &dquo;Emerging as students from the conglomerate material offered us by many authors, and attempt- ing a survey of the field with fair-minded shrewdness, where does it seem we can turn for relief from the futility of our failures? One sees certainly no immediate help from any attempt at theorizing.&dquo; He is quite right. We must approach this question in the same sane and practical way as any other physical and scientific problem. Let us view the actions of the offender as the natural outgrowth of an undeveloped or abnormal central nervous system. Medical and psychological science must earnestly and intelligently work out scientific individualization and correct classification, after having determined as nearly as possible the physical and intellectual strength of the criminal. We cannot hope to control and improve the offender unless we know him, and to know him we must study him-his conduct, his physical and mental make-up, the general ensemble of his qualities. Where can this best be done? at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from BY DAVID C. PEYTON, First in the larger field of personal contact and individual handling, and second, in the psychological and pathological laboratory. The more carefully we study him the more convinced are we that we are dealing with a defective or unde- veloped, and possibly undevelopable, brain. In many cases there has been a disturbance or complete arrest of development, account- ing for the fact that probably one-third of those entering a reforma- tory, while physically ranging in ages from sixteen to thirty years, are mentally under twelve years of age. This of itself furnishes ample evidence of the necessity for a longer period of institutional at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from 69 treatment than is too often understood or practiced. Essential fea- tures of any method of treatment must be firmness, unquestioned justice, patience and kindly interest. This thought is well expressed by Dr. Henderson in his summary of the discussions of the congress at Washington, in 1910. To return the criminal to organized society before he is fitted to meet its requirements is to weaken society’s confidence in our methods of treatment, and to make of the offender a recidivist. When we learn to analyze scientifically the irregularities of the defective intellect, the matter of employment and discipline will both become easier. The problem is as endless as the human race, and the matter as inexhaustible as nature itself. It is important that we observe the same diligence in the restric- tion of the spread of crime as we do in the prevention of the spread of contagious disease. In considering the preventive or restrictive measures let us first think of more thoroughly safeguarding our marriage laws; and, second, the humane prevention of procreation by the positively defective and criminal type, the feasibility of which has been so well demonstrated by Dr. Sharp. It is impossible to read the findings of Lombroso and others on heredity and not appre- ciate the necessity for positive control of such procreation. The moral and intellectual constitution must be rebuilt. The criminal’s physical condition must also be improved and his somatic defects corrected as far as possible. The wonderful good accruing from the gymnasium, the drill room and other kinds of physical exercise does not stop with the bodily improvement, but the stimulation to the brain and mind gives a decided improvement in mental acumen. at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from BY DAVID C. PEYTON, In securing control and obedience of the prisoner we must convince him that we are not only just with him, but that we are actually interested in him. It must be borne in mind also that by reason of his defect he does not have a normal view of things. This does not mean that he must not be made to conform to rules. The very nature of his life as a member of society and his failure to respect the regulations of society, one of which was his probable failure to support those having a natural and legal right to look to him, make it incumbent upon the state to take custodial care of him and bring about compulsory support. In order to do this the state assumes the obligation to afford him the necessary opportunity. To do this he should be given his entire earnings over and above the cost of raw material entering into the product he creates. It is useless to advo- at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from 70 cate that his maintenance should first be deducted, for no reforma- tory, attempting to carry out the purpose for which it was created, can ever hope to be self-sustaining. When the state takes into custody and locks up one of its derelicts and affords society protection against his offenses, society has gotten all to which it is entitled and has no right to appropriate the prisoner’s earnings, thus pauperizing his family, which is one of the causes of crime. y In this connection it is interesting to consider the results of Lombroso’s investigations in the matter of employment, where he shows the relative increase of crime and recidivism as we leave agriculture and replace it with the manufacturing industries, the agricultural employment having the lowest percentage of crime. From the foregoing it seems that the importance of training prisoners in agriculture-intensive farming-by the best instructor to be furnished by some such great school as Purdue University, can not be over-estimated. This instruction should be given on as extensive a scale as is consistent with the welfare of the inmates and the inter- ests of the state. at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from BY DAVID C. PEYTON, The unsatisfactory employment of prisoners, along with other features of disappointment in the general treatment of penal and reformatory problems, has resulted in a state of uncer- tainty and unrest, causing us to seek other and newer methods, and the most promising outlook is the truly scientific field. The State of Indiana is fortunate in having a governor, and the Indiana Reformatory a board of trustees, who have given much thought and consideration to the advancement of such sensible and practicable things that tend to the general uplift and improvement of the inmates. Recognizing that the work of a reformatory is neces- sarily along scientific and educational lines, the Indiana Reformatory has secured one of our distinguished educators to become associated with us in our work here. He is a gentleman who has for some years been at the head of the department of education and psychology in Depauw University, this state, and the entering into this work by such men as he augurs much for the advancement of the work. The t tli d d i th l t h lf i th l The startling advance made in the last half century in the general world of science, by the exact methods of laboratory investigation, has suggested the feasibility of their application to the problems of penal science. The psychological laboratory is no longer an experi- ment. The work done by Mansterburg, of Harvard University, and by others in the field proves conclusively the potency of this method at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from 71 in determining mental qualities, in detecting psychic defects, and in estimating the general state of the central nervous system. Incred- ible as it may appear, there is no penal or reformatory institution for the care of males where psychological methods formulated on scientific laboratory experiments are in use, or even where a comprehensive scheme of psychic findings has any place in the general conduct of the management of criminals. Despite the remarkable advance along the whole range of scientific and cultural activities the idea of punishment for crime is in essence singularly archaic, harking back to the old punitive system of the Mosaic law. True, the refor- matory idea has been extant for some years and much good has been accomplished, but, in the light of modem scientific investigation, even greater advancement is to be expected. BY DAVID C. PEYTON, g p We would not have maudlin sentiment usurp the place of even strabismic justice, but would approach the subject from the stand- point of broad humanism and treat it as a problem of physical science. As scientific problems can be elucidated only by scientific methods, we are now on the threshold of instituting in the Indiana Reformatory the first effort in the methods suggested. We feel fully the immensity of the undertaking. We are leaving the familiar ground, strewn as it is with many failures and disappointments, and, guided by the refulgent star of science, we dare to follow her into the unknown, confident in her unerring instinct to guide us to our goal, certain that she alone can now lead us aright. at UNIV CALIFORNIA SAN DIEGO on April 12, 2015 ann.sagepub.com Downloaded from
https://openalex.org/W4237293390	http://www.sfu.ca/biology/wildberg/NewCWEPage/papers/JonkeretalMolEcol2013.pdf	English	null	Peer Review #2 of "Genetic structure of the threatened West-Pannonian population of Great Bustard (Otis tarda) (v0.1)"	null	2,016	cc-by	10,580	Abstract Cultural transmission of migratory traditions enables species to deal with their environ- ment based on experiences from earlier generations. Also, it allows a more adequate and rapid response to rapidly changing environments. When individuals break with their migratory traditions, new population structures can emerge that may affect gene ﬂow. Recently, the migratory traditions of the Barnacle Goose Branta leucopsis changed, and new populations differing in migratory distance emerged. Here, we investigate the popu- lation genetic structure of the Barnacle Goose to evaluate the consequences of altered migratory traditions. We used a set of 358 single nucleotide polymorphism (SNP) mark- ers to genotype 418 individuals from breeding populations in Greenland, Spitsbergen, Russia, Sweden and the Netherlands, the latter two being newly emerged populations. We used discriminant analysis of principal components, FST, linkage disequilibrium and a comparison of geneﬂow models using MIGRATE-N to show that there is signiﬁcant popu- lation structure, but that relatively many pairs of SNPs are in linkage disequilibrium, suggesting recent admixture between these populations. Despite the assumed traditions of migration within populations, we also show that genetic exchange occurs between all populations. The newly established nonmigratory population in the Netherlands is char- acterized by high emigration into other populations, which suggests more exploratory behaviour, possibly as a result of shortened parental care. These results suggest that migratory traditions in populations are subject to change in geese and that such changes have population genetic consequences. We argue that the emergence of nonmigration probably resulted from developmental plasticity. Keywords: admixture, cultural evolution, migration modelling, population genetics, SNP, speciation Received 27 March 2013; revision received 18 September 2013; accepted 20 September 2013 Received 27 March 2013; revision received 18 September 2013; accepted 20 September 2013 Molecular Ecology (2013) 22, 5835–5847 Molecular Ecology (2013) 22, 5835–5847 doi: 10.1111/mec.12548 Genetic consequences of breaking migratory traditions in barnacle geese Branta leucopsis R. M. JONKER,† R. H. S. KRAUS,‡ Q. ZHANG,§ P. VAN HOOFT, K. LARSSON,¶ H. P. VAN DER JEUGD,** R. H. J. M. KURVERS,†† S. E. VAN WIEREN, M. J. J. E. LOONEN,‡‡ R. P. M. A. CROOIJMANS,§§ R. C. YDENBERG,¶¶ M. A. M. GROENEN§§ and H. H. T. PRINS Resource Ecology Group, Wageningen University, Droevendaalsesteeg 3a, 6708 PB Wageningen, The Netherlands, †Department of Animal Behaviour, University of Bielefeld, Morgenbreede 45, 33615 Bielefeld, Germany, ‡Conservation Genetics Group, Senckenberg Research Institute and Natural History Museum, Clamecystraße 12, 63571 Gelnhausen, Germany, §Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 China, ¶Kalmar Maritime Academy, Linnaeus University, 391 82 Kalmar, Sweden, Dutch Centre for Avian Migration and Demography, NIOO-KNAW, Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands, ††Leibniz Institute of Freshwater Ecology and Inland Fisheries, M€uggelseedamm 310, 12587 Berlin, Germany, ‡‡Arctic Centre, University of Groningen, Aweg 30, 9718 CW Groningen, The Netherlands, §§Animal Breeding and Genomics Centre, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands, ¶¶Department of Biological Sciences, Centre for Wildlife Ecology, Simon Fraser University, Burnaby, BC, Canada V5A 1S6 Received 27 March 2013; revision received 18 September 2013; accepted 20 September 2013 Correspondence: Rudy M. Jonker, E-mail: mrjonker@gmail.com Introduction Here, we study the consequences of these dramatic changes in migratory behaviour on the population genetic structure of the Barnacle Goose. We use genetic samples from the ﬁve main global populations, and we make use of the avail- ability of a new set of 358 single nucleotide polymor- phism (SNP) markers with genomewide coverage (Jonker et al 2012b) 2011) and can be found in species ranging from insects to birds to mammals. The mode of transmission of migratory behaviour plays a crucial role in the evolu- tion of migration and in how migration changes in response to environmental change. Generally, there are two distinct modes of transmission of migration (Sutherland 1988), genetic (e.g. Helbig 1991; Pulido & Berthold 2010) and cultural (Sutherland 1998; Guttal & Couzin 2010). 2011) and can be found in species ranging from insects to birds to mammals. The mode of transmission of migratory behaviour plays a crucial role in the evolu- tion of migration and in how migration changes in response to environmental change. Generally, there are two distinct modes of transmission of migration (Sutherland 1988), genetic (e.g. Helbig 1991; Pulido & Berthold 2010) and cultural (Sutherland 1998; Guttal & Couzin 2010). In the case of culturally transmitted migration, social or kin groups follow a migratory route acquired by tradition (Raveling 1979; Nelson 1995; Palacın et al. 2011) or selected by majority decisions (Prins 1996). Offspring generally travel where their parents have travelled and breed where they were born (Van der Jeugd et al. 2002). Traditions and natal philopatry can limit gene ﬂow between colonies or populations, thereby increasing the rate of divergence (Friesen et al. 2007; Riesch et al. 2012). Culturally transmitted migra- tion is widespread and is especially prominent in long- lived species because longevity allows an individual to proﬁt from acquired information. Learning from par- ents is especially beneﬁcial, as their behaviour demon- strably produced offspring and was thus successful. Overlapping generations and a relatively stable envi- ronment (McNamara & Dall 2011) are a prerequisite for cultural transmission. The more generations over- lap, the greater the beneﬁt for individuals that learn migration (or any other behaviour) culturally as compared to individuals that do not, because there are more informed generations available for naive individuals. Introduction Long-distance migration, in which individuals travel seasonally between breeding and nonbreeding locations, is a widespread phenomenon (Milner-Gulland et al. Correspondence: Rudy M. Jonker, E-mail: mrjonker@gmail.com © 2013 John Wiley & Sons Ltd Correspondence: Rudy M. Jonker, E-mail: mrjonker@gmail.com © 2013 John Wiley & Sons Ltd 5836 R. M. JONKER ET AL. In geese, parental care extends up to nearly a year. These close family bonds and cultural inheritance of migratory behaviour are assumed to have caused ‘the closest kind of inbreeding’ (Mayr 1942 p.242) among Arctic or sub-Arctic birds, with the emergence of many differ- ent races as a result (Mayr 1942; Hochbaum 1955; Kear 1970; Baker 1978; Owen 1980). An example of this mechanism of cultural transmission is that of the lesser white-fronted goose Anser erythropus. Eggs of this spe- cies were placed in nests of barnacle geese upon which the young adopted the migration strategy of their foster parents (Von Essen 1991). Recently, the migration sys- tem of the Barnacle Goose has gone through a number of striking changes. In the middle of the twentieth cen- tury, there were three Arctic breeding populations (Greenland, Spitsbergen and Russia) with distinct ﬂy- ways (Fig. 1). The Arctic populations have rapidly increased and expanded their breeding ranges within the Arctic over the past few decades (Fox et al. 2010). New breeding colonies established on Gotland, Sweden, in the 1970s (Larsson et al. 1988). Later in the 1980s and 1990s, the number of colonies increased in the Baltic region as well as in the southwest of the Netherlands in the 1980s. The populations breeding in Russia, Sweden and the Netherlands all winter mainly in the Nether- lands. Additionally, the barnacle geese migrating from the Netherlands to Russia have since the 1990s delayed their commencement of spring migration by approxi- mately one month, although there was considerable vari- ation in commencement of migration before the 1990s already. The cause of this delay is subject to debate as both competition for food (Eichhorn et al. 2008) and predation danger (Jonker et al. 2010) are proposed. Recently, the population wintering in Scotland and breeding on Spitsbergen have also started to delay the commencement of spring migration (Loonen et al. unpublished data). This delay in migration has the potential to affect the cultural transmission of migratory behaviour, possibly causing the establishment of new migration routes or breeding areas. Introduction Migratory routes or schedules are, however, by no means ﬁxed/unalterable across generations (Berthold & Helbig 1992; Pulido 2007), and in both genetic and cul- tural transmissions, mechanisms have evolved that allow for changes in migratory behaviour. Interestingly, those species able to adjust their migratory behaviour rapidly are the ‘cultural’ migrants (Sutherland 1998) as traditions change more quickly than genes. A change in migratory tradition can result in the emergence of new migratory routes or new populations, and the question arises how a change in migration tradition affects the population genetic structure of a species. We expect that the discovery of novel breeding areas or migratory routes (and subsequent establishment of new breeding populations) may lead to genetic differentiation due to subsequent natal philopatry, whereas the mixing of pre- viously (culturally) separated breeding areas or ﬂyways may lead to population admixture. Here we study how a change in migratory traditions affected the popula- tion genetic structure of a long-distance migrant, the Barnacle Goose Branta leucopsis, which has gone through several major changes in migration. GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5837 Fig. 1 Map of Barnacle Goose populations. Colours correspond with the colours in the other ﬁgures. The separate migratory ﬂyways are indicated in grey. GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5837 Fig. 1 Map of Barnacle Goose populations. Colours correspond with the colours in the other ﬁgures. The separate migratory ﬂyways are indicated in grey. Table 1 Overview of origin of samples of individuals Table 1 Overview of origin of samples of individuals Location Lat/lon Number of individuals Greenland 70.4° N/22.3° W 5 Spitsbergen 78° N/12° E 117 Russia 70° N/50° E 107 Sweden 57.27° N/18.45° E 55 The Netherlands 51.6° N/4.2° E 134 Spitsbergen (SP), Russia (RU), Sweden (SE) and the Netherlands (NL). The Swedish and Russian popula- tions use the Netherlands as wintering area, but mainly use the north of the Netherlands for wintering, whereas the Dutch population mainly uses the southwest of the Netherlands as breeding area. All samples were col- lected at the breeding site during the moult after breed- ing, and the vast majority of individuals were sampled as adult (388 of 418). For Greenland, tissue samples col- lected from the foot sole were used. These were col- lected in the 1970s and were obtained from the collection of the Zoological Museum in Amsterdam. For all other populations, we used ethanol-preserved whole-blood samples. Spitsbergen (SP), Russia (RU), Sweden (SE) and the Netherlands (NL). The Swedish and Russian popula- tions use the Netherlands as wintering area, but mainly use the north of the Netherlands for wintering, whereas the Dutch population mainly uses the southwest of the Netherlands as breeding area. All samples were col- lected at the breeding site during the moult after breed- ing, and the vast majority of individuals were sampled as adult (388 of 418). For Greenland, tissue samples col- lected from the foot sole were used. These were col- lected in the 1970s and were obtained from the collection of the Zoological Museum in Amsterdam. For all other populations, we used ethanol-preserved whole-blood samples. single nucleotide polymorphisms (SNPs) (Jonker et al. 2012b) and the program Genome Studio (Illumina Inc.) for allele calling (clustering) for each SNP individually. single nucleotide polymorphisms (SNPs) (Jonker et al. 2012b) and the program Genome Studio (Illumina Inc.) for allele calling (clustering) for each SNP individually. Samples from Spitsbergen were collected in 2007 from the colonies near Nordenskioldkysten, Ny-Alesund and Longyearbyen. General population genetic analyses We tested all SNP markers for deviation from Hardy– Weinberg equilibrium (HWE) in each population sepa- rately using the package Adegenet 1.2-8 (Jombart 2008) in R (R Development Core Team 2013). We calculated pairwise FST for all populations with pp.fst from the package hierfstat (Goudet 2005) in R. We tested the sig- niﬁcance of these observed FST values using a bootstrap procedure using 100 000 bootstraps using boot.ppfst from the same package and calculated 95% conﬁdence intervals of these FST values. © 2013 John Wiley & Sons Ltd GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5837 Russian samples originated from the colony of the Kolokolkova Bay near Tobseda (Van der Je- ugd et al. 2003) and were collected in 2007 and 2008. Swedish samples were collected from the Gotland popu- lation (Larsson et al. 1988) in 2009. Samples from the Netherlands were collected in the Krammersche Slikken in 2007 and 2008 (Table 1). All samples were collected following national and institutional rules. Because barnacle geese have an average life expectancy of 8–15 years (depending on population), the collected sam- ples represent a cross-section from these populations covering up to one decade before the moment of sam- pling. We isolated DNA using Proteinase-K and the Gen- tra Systems Puregene DNA puriﬁcation kit, described in detail elsewhere (Kraus et al. 2011a; Jonker et al. 2012b). General population genetic analyses Study populations and sampling Study populations and sampling We used previously collected samples of the ﬁve main populations of the Barnacle Goose: Greenland (GL), © 2013 John Wiley & Sons Ltd © 2013 John Wiley & Sons Ltd GeneFlow model selection We used the program MIGRATE-N (Beerli 2009) to com- pare different models of gene ﬂow among the popula- tions (Beerli 2012) to test different scenarios on the evolution of migratory ﬂyways of the Barnacle Goose and obtain insight into whether some populations have stronger traditions (i.e. lower relative emigration) or are difﬁcult to immigrate into because of differences in life history. We excluded samples from Greenland in these analyses because they were collected before the Swed- ish and Dutch populations emerged, which makes infer- ence of gene ﬂow between these populations from present-day data questionable. We ran two DAPC analyses. First, we assigned each individual a priori to its population of origin (a priori population assignment) and obtained for each individ- ual the probability of assignment to their populations of sampling. This allowed us to test which of the prior populations an individual could be assigned to best and showed us whether individuals recently moved from one population to another, indicating admixture. Thereafter, we used the ﬁnd.clusters function, to deter- mine the number of clusters and assign each individual to a cluster without providing any a priori population assignment. Similar to the a priori population assign- ment, we then obtained a probability of assignment to each cluster for each individual a posteriori (a posteriori population assignment). This removes the effect of assigning populations a priori on the eventual assign- ment to clusters and offers an unbiased interpretation of population structure. We deﬁned seven candidate models constraining the presence and directionality of gene ﬂow between the four populations: Spitsbergen, Russia, Sweden and the Netherlands. Model 1 allowed gene ﬂow between all possible population pairs (full island model). In model 2, there was no gene ﬂow between Spitsbergen and any other population, except to and from the Neth- erlands. Two-way gene ﬂow was possible among Rus- sia, Sweden and the Netherlands. Model 3 was similar to model 2, but did not allow gene ﬂow from Spitsber- gen to the Netherlands. Model 4 reﬂects the situation before the emergence of the additional populations in the 1970s. At that time, only the Russian and Spitsber- gen (and Greenland) populations were present, which are assumed to have had some exchange. From the Rus- sian population, the Swedish and Dutch populations emerged, which is reﬂected in our model by allowing gene ﬂow between these populations. Discriminant analysis of principal components We used discriminant analysis of principal components (DAPCS) (Jombart et al. 2010) to analyse the popula- tion structure. This method allows identiﬁcation of genetic clusters and unravelling complex population We used the Illumina Golden Gate genotyping assay on an Illumina BeadXpress with VeraCode TM technology to genotype each individual for 384 © 2013 John Wiley & Sons Ltd 5838 R. M. JONKER ET AL. see how many of the LD pairs were within and between chromosomes. To test for inbreeding as a cause for LD, we calculated the average inbreeding coefﬁcient using dyadic maximum-likelihood estimator (DyadML) (Milligan 2003) using COANCESTRY, which was lower than 0.06 for all populations. structures. Because DAPC is not sensitive to underlying family structures, we did not remove related individuals from the analysis. We used the percentage of successful assignment as a measure of group differentiation, cor- rected for the number of retained principal components (function a.score in R-package Adegenet version 1.3-5). Because using too many principal components leads to overﬁtting of the model, the successful assignment is corrected for the number of principal components. We determined the optimal number of retained principal components using the function optim.a.score (with 20 simulations per principal component). In our study, we retained 50 principal components cumulatively explain- ing 45% of the variance. structures. Because DAPC is not sensitive to underlying family structures, we did not remove related individuals from the analysis. We used the percentage of successful assignment as a measure of group differentiation, cor- rected for the number of retained principal components (function a.score in R-package Adegenet version 1.3-5). Because using too many principal components leads to overﬁtting of the model, the successful assignment is corrected for the number of principal components. We determined the optimal number of retained principal components using the function optim.a.score (with 20 simulations per principal component). In our study, we retained 50 principal components cumulatively explain- ing 45% of the variance. GeneFlow model selection Model 5 is differ- ent from model 4 in that there is no direct gene ﬂow from Russia to the Netherlands. This model represents the situation that there is gene ﬂow between Russia and Sweden, and between Sweden and the Netherlands. Model 6 reﬂects gene ﬂow between Russia and the Netherlands and Sweden, without allowing any gene ﬂow between Sweden and the Netherlands. In model 7, one-way gene ﬂow from the Netherlands to Sweden is added to those possible in model 6 (Table 4 and Fig. S1 (Supporting information) for a visual representation of the seven models). © 2013 John Wiley & Sons Ltd in which mLmodeli is the maximum likelihood of modeli and Pn j mLmodelj is the sum of the maximum likelihoods of all the other models. Results The number of polymorphic SNPs for the entire sample (all populations combined) was 358. In the Greenland samples, only 282 SNPs were polymorphic, whereas the other populations had more than 350 polymorphic SNPs. The vast majority of polymorphic SNPs did no deviate signiﬁcantly from Hardy–Weinberg equilibrium (GL: 100%, SP: 96%, RU: 95%, SV: 98%, NL: 95% a = 0.05). Calculations of pairwise FST values reveal sig niﬁcant population structure. Although the FST values are low (between 0.006 and 0.035), they are signiﬁcantly different from 0 (Table 2). The FST analyses show tha Greenland is most separated from the other populations with values ranging from 0.028 to 0.034. However, the conﬁdence intervals of these estimates are much larger compared with those from other populations, so we need to be cautious with such conclusions because o the low sample size of this population. The Spitsbergen population is least differentiated from the Russian pop ulation (Fig. 2, FST = 0.020) and equally differentiated from the Swedish and Dutch populations (Fig. 2, FST respectively, 0.026 and 0.027). The Swedish population and the Russian population are the two least differenti ated populations with an FST of 0.006. The FST values from the Dutch population indicate that the Russian Table 2 FST values between population pairs. All calculated between brackets indicate the lower and upper 95% conﬁdence Greenland Spitsber Greenland — Spitsbergen 0.029 (0.016–0.043) — Russia 0.028 (0.015–0.043) 0.020 (0. Sweden 0.035 (0.020–0.051) 0.026 (0. Netherlands 0.035 (0.021–0.049) 0.027 (0. © 2013 John Wiley & Sons Ltd GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 583 GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5839 5839 models using: models using: and Dutch populations are similarly differentiated (FST = 0.015) compared with the Dutch and Swedish populations (FST = 0.018). BF ¼ e HMmodeli HMmodelj in which model i is the model with the lowest harmonic mean (HM) of the marginal likelihood and model j is each of the other models. This difference between two harmonic means is denoted as dHM (Table 4). Subse- quently, we calculated the probability of each model using Discriminant analysis of principal components The DAPC analysis, which explained 45% of the variance in our data of prior clusters, conﬁrms the presence of the genetic structure as indicated by the FST analysis. Both the FST values and the PCA plot indicate that Spitsbergen is relatively differentiated from the other populations. Furthermore, these results show that Sweden and Russia are closer to each other than to the Dutch cluster (Fig. 3A). The probability of assignment of individuals to their original sampling locality (Fig. 4A) shows that the individuals from Greenland were all assigned to the Greenland clusters with proba- bilities close to 1. For Spitsbergen, most individuals (113 of 117) were assigned to the Spitsbergen cluster, but some individuals had a high probability of being assigned to the Russian genetic cluster (3 of 117). In the Russian population, most individuals had the highest probability of being assigned to the Russian cluster (93 of 107), but a relatively large proportion of individuals show a substantial probability (>0.3) of assignment to the Dutch (5 of 107) and Swedish clusters (8 of 107). The results for the Swedish population show that most were assigned to Sweden (42 of 55), and many of the individuals were assigned to the Russian (8 of 55). Also, there were a number of individuals for which the prob- ability of assignment to Sweden and Russia differed lit- tle (3 of 55). Only few Swedish individuals had high probabilities of assignment to the Dutch cluster (2 of 55). Of the Dutch population, most individuals (124 of 134) were assigned with high probabilities to the Dutch cluster, and number of individuals had high assignment probabilities to the Russian cluster (10 of 134). Only very few individuals (2 of 134) had some assignment probability (>0.3) to the Swedish genetic cluster. Probmodeli ¼ mLmodeli Pn j mLmodelj in which mLmodeli is the maximum likelihood of modeli and Pn j mLmodelj is the sum of the maximum likelihoods of all the other models. Table 2 FST values between population pairs. All calculated FST values were signiﬁcant at the P < 0.0001 threshold. Values in between brackets indicate the lower and upper 95% conﬁdence limits Greenland Spitsbergen Russia Sweden Greenland — Spitsbergen 0.029 (0.016–0.043) — Russia 0.028 (0.015–0.043) 0.020 (0.016–0.023) — Sweden 0.035 (0.020–0.051) 0.026 (0.021–0.031) 0.006 (0.004–0.008) — Netherlands 0.035 (0.021–0.049) 0.027 (0.022–0.031) 0.015 (0.012–0.017) 0.018 (0.014–0.021) Linkage disequilibrium We used COANCESTRY (Wang 2011), with standard set- tings unless mentioned differently, to calculate related- ness between all individuals in our data set. Of the pairs that had relatedness higher than 0.2, we randomly removed one individual to remove possible family sub- structure from the data set. Then we tested for each pair of SNPs whether there was signiﬁcant linkage dis- equilibrium using LD from the R-package Genetics (Warner & Leisch 2002) to estimate ‘D’. Only those markers polymorphic within each population were tested. Bonferroni correction for multiple pairwise com- parisons thus resulted in a different P-value threshold (0:05= nðn1Þ 2 , with n being the number of polymor- phic loci) per population because in some populations, more SNPs were polymorphic than in others. To test for physical linkage as explanation for linkage disequi- librium, we have mapped the LD pairs on the genome (SNP positions as inferred from comparisons with the chicken genome, for details see Jonker et al. (2012b)) to We compared the models using Bayes factors (Beerli & Palczewski 2010; Beerli 2012), which are marginal likelihoods over the complete parameter range (Newton & Raftery 1994). We ranked the Bayes factors of all © 2013 John Wiley & Sons Ltd Results The number of polymorphic SNPs for the entire sample (all populations combined) was 358. In the Greenland samples, only 282 SNPs were polymorphic, whereas the other populations had more than 350 polymorphic SNPs. The vast majority of polymorphic SNPs did not deviate signiﬁcantly from Hardy–Weinberg equilibrium (GL: 100%, SP: 96%, RU: 95%, SV: 98%, NL: 95%, a = 0.05). Calculations of pairwise FST values reveal sig- niﬁcant population structure. Although the FST values are low (between 0.006 and 0.035), they are signiﬁcantly different from 0 (Table 2). The FST analyses show that Greenland is most separated from the other populations with values ranging from 0.028 to 0.034. However, the conﬁdence intervals of these estimates are much larger compared with those from other populations, so we need to be cautious with such conclusions because of the low sample size of this population. The Spitsbergen population is least differentiated from the Russian pop- ulation (Fig. 2, FST = 0.020) and equally differentiated from the Swedish and Dutch populations (Fig. 2, FST, respectively, 0.026 and 0.027). The Swedish population and the Russian population are the two least differenti- ated populations with an FST of 0.006. The FST values from the Dutch population indicate that the Russian The a posteriori DAPC analysis (Fig. 3B and 4B) shows that most individuals (111 of 117) of the Spitsbergen population were assigned to the same cluster (cluster 2), where the ones assigned to other clusters were also Table 2 FST values between population pairs. All calculated FST values were signiﬁcant at the P < 0.0001 threshold. Values in between brackets indicate the lower and upper 95% conﬁdence limits Greenland Spitsbergen Russia Sweden Greenland — Spitsbergen 0.029 (0.016–0.043) — Russia 0.028 (0.015–0.043) 0.020 (0.016–0.023) — Sweden 0.035 (0.020–0.051) 0.026 (0.021–0.031) 0.006 (0.004–0.008) — Netherlands 0.035 (0.021–0.049) 0.027 (0.022–0.031) 0.015 (0.012–0.017) 0.018 (0.014–0.021) Table 2 FST values between population pairs. All calculated FST values were signiﬁcant at the P < 0.000 between brackets indicate the lower and upper 95% conﬁdence limits © 2013 John Wiley & Sons Ltd 5840 R. M. JONKER ET AL. 0.00 0.01 0.02 0.03 0.04 0.05 FST (+/– 95% CI) GL−SP GL−RUS GL−SWE GL−NL SP−RUS SP−SWE SP−NL RUS−SWE RUS−NL SWE−NL Fig. 2 FST statistics between all population pairs with 95% con- ﬁdence intervals. GL = Greenland, SP = Spitsbergen, RUS = Russia, SWE = Sweden, NL = The Netherlands. Linkage disequilibrium We detected linkage disequilibrium between SNPs in all but the Greenland population. The absence of LD in the Greenland population is probably due to low sam- ple size. In the Spitsbergen population, 63 pairs of SNPs (0.05%) showed linkage disequilibrium. Also in the Rus- sian population (40 pairs, 0.03%), the Swedish popula- tion (33 pairs, 0.03%) and the Dutch population (130 pairs, 0.10%), a higher proportion of SNPs than would be expected by chance were in linkage disequilibrium (Table 3). Of the 247 unique LD pairs, 45 were within Fig. 2 FST statistics between all population pairs with 95% con- ﬁdence intervals. GL = Greenland, SP = Spitsbergen, RUS = Russia, SWE = Sweden, NL = The Netherlands. d = 2 GL NL RU SP SE Eigenvalues (A) d = 2 1 2 3 4 5 6 Eigenvalues (B) Fig. 3 Scatter plot of prior and posterior clusters. In the left panel (3A), the individuals are plotted with a priori-deﬁned populations (GL: Greenland, SP: Spitsbergen, RU: Russia, SE: Sweden, NL: The Netherlands). In the right panel (3B), the individuals are assigned to populations a posteriori, that is, after determining the number of clusters by the program, instead of forcing into known popula- tions. Cluster 2 corresponds largely with Spitsbergen and cluster 1 corresponds to Russia and Sweden together. In cluster 6, individ- uals from all populations are present, but mainly from the Netherlands; clusters 3, 4 and 5 almost completely consist of individuals from the Netherlands. The colours in Fig. 3A correspond with the colours in Fig. 4A, and the colours of Fig. 3B correspond with the colours of Fig. 4B. The bar graph insets indicate the amount of variance explained by the two discriminant eigenvalues used for plot- ting. Both plots have the same scale on both axes, as indicated by the d = 2 in both graphs. Ellipses are inertia ellipses calculated by the variance of both pc-axes and represent 67% of the variance. d = 2 1 2 3 4 5 6 Eigenvalues (B) d = 2 GL NL RU SP SE Eigenvalues (A) (B) Fig. 3 Scatter plot of prior and posterior clusters. In the left panel (3A), the individuals are plotted with a priori-deﬁned populations (GL: Greenland, SP: Spitsbergen, RU: Russia, SE: Sweden, NL: The Netherlands). Results assigned to other populations in the a priori assignment procedure. The individuals of the Russian and Swedish population were also assigned mostly to one cluster (cluster 1, 147 of 162). Cluster 6 consists of individu- als originating from Russia (4), Sweden (7) and the Netherlands (69). Most remaining individuals (45) from the Netherlands are assigned to clusters 3, 4 and 5. These clusters contain, except for one Swedish individ- ual, no individuals from other populations than the Dutch population. Linkage disequilibrium In the right panel (3B), the individuals are assigned to populations a posteriori, that is, after determining the number of clusters by the program, instead of forcing into known popula- tions. Cluster 2 corresponds largely with Spitsbergen and cluster 1 corresponds to Russia and Sweden together. In cluster 6, individ- uals from all populations are present, but mainly from the Netherlands; clusters 3, 4 and 5 almost completely consist of individuals from the Netherlands. The colours in Fig. 3A correspond with the colours in Fig. 4A, and the colours of Fig. 3B correspond with the colours of Fig. 4B. The bar graph insets indicate the amount of variance explained by the two discriminant eigenvalues used for plot- ting. Both plots have the same scale on both axes, as indicated by the d = 2 in both graphs. Ellipses are inertia ellipses calculated by the variance of both pc-axes and represent 67% of the variance. © 2013 John Wiley & Sons Ltd GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5841 GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5841 0.0 0.5 1.0 0.0 0.5 1.0 Probability of assignment (A) GL Probability of assignment (B) Netherlands Sweden Russia Spitsbergen GL Netherlands Sweden Russia Spitsbergen Fig. 4 Stacked bar graph of assignment probabilities per individual. In the upper panel (Fig. 4A), populations of the individuals were deﬁned a priori. The probability of assignment indicates the probability that each individual was assigned to its a priori set popula- tion. In the lower panel (Fig. 4B), no populations were deﬁned a priori. Instead individuals were assigned to one of the clusters that were detected by the software. Both Fig. 4A,B consist of 418 stacked bars, in which each bar is one individual and the order of the individuals is the same in both graphs. If an individual was assigned to multiple clusters, bars were stacked. 0.0 0.5 1.0 Probability of assignment (A) GL Netherlands Sweden Russia Spitsbergen 0.0 0.5 1.0 Probability of assignment (B) p g GL Spitsbergen Spitsbergen Netherlands Netherlands Fig. 4 Stacked bar graph of assignment probabilities per individual. In the upper panel (Fig. 4A), populations of the individuals were deﬁned a priori. The probability of assignment indicates the probability that each individual was assigned to its a priori set popula- tion. In the lower panel (Fig. 4B), no populations were deﬁned a priori. Migration rate model comparison Our comparison of candidate models of gene ﬂow between populations showed that the full geneﬂow model (model 1) ﬁtted our data best (Table 4). The large difference in harmonic mean of the marginal likeli- hood between the models results in a probability of 1 (1–0.11027) for this model. Hence, we further only Linkage disequilibrium Instead individuals were assigned to one of the clusters that were detected by the software. Both Fig. 4A,B consist of 418 stacked bars, in which each bar is one individual and the order of the individuals is the same in both graphs. If an individual was assigned to multiple clusters, bars were stacked. report results for model 1. For convenience, we scaled all geneﬂow measures relative to the smallest, which is MSE?SP (Migration from Sweden (SE) to Spitsbergen (SP), unscaled M: 922). Consequently, all further conclu- sions from this analysis are based on relative differ- ences in gene ﬂow. A number of patterns become clear from these geneﬂow measures (Fig. 5). The geneﬂow measures to Sweden (MSP?SE, MRU?SE, MNL?SE) are all relatively high (average: 2.08), whereas the geneﬂow measures from Sweden are relatively low (average: 1.17). For the Netherlands, the pattern is the opposite. The geneﬂow measures to the Netherlands (MSP?NL, MRU?NL, MSE?NL) are relatively low (average: 1.30), whereas the geneﬂow measures from the Netherlands are relatively high (average: 1.93). The geneﬂow mea- sures from Spitsbergen (MSP?RU, MSP?SE, MSP?NL) are relatively higher (average: 1.75) than geneﬂow measures to Spitsbergen (average: 1.43). Finally, the geneﬂow measures from Russia (MRU?SP, MRU?SE, MRU?NL) are similar (average 1.53) to the geneﬂow measures to Russia (average: 1.57). Table 3 Number of pairs of markers in linkage disequilibrium (LD) and associated P-value thresholds (0.05/((nn-1)/2)) for LD. The number of polymorphic SNPs in each population is n. The number of individuals is the number that remained in the analysis after removing the closely related individuals chromosomes. The median distance between these within chromosome LD pairs was 2.7 Mb (Fig. S2–S6, Nr of pairs in LD P-value threshold n (SNPs) # of individuals Greenland 0 1.262E-06 282 5 Spitsbergen 63 8.048E-07 353 112 Russia 40 8.002E-07 354 103 Sweden 33 8.140E-07 351 49 Netherlands 130 8.094E-07 351 79 chromosomes. The median distance between these within chromosome LD pairs was 2.7 Mb (Fig. S2–S6, Supporting information). Discussion The ﬁrst sign of the ﬁrst quartet and the second of the second quartet indicate estimation of theta for populations 1, 2, etc. An asterisk indicates that that particular migration rate was estimated by the model, and a 0 indicates that no migration was allowed. For example, for model 2: no migration from Russia and Sweden to Spitsbergen was estimated, and no migration from Spitsbergen to Russia and Sweden was estimated. For each model, we used 4 heated chains with 1, 1.5, 3, 1 000 000 heating scheme. The sampling increment in the prior was set to 20, the number of steps dis- carded (burn-in) was 2.000.000, and the number of steps analysed was 5.000. Prior thetas were generated from a uniform distribution ranging from 0 to 15, and prior migration rates were generated from a uniform distribution ranging from 0 to 4000. These settings resulted in converged posterior distributions with a clear maximum for each estimate (Supplementary File 1 for model 1). 0.0 0.5 1.0 1.5 2.0 2.5 Relative migration rate RU → SP SWE → SP NL → SP SP → RU SWE → RU NL → RU SP → SWE RU → SWE NL → SWE SP → NL RU → NL SWE → NL To Spitsbergen To Russia To Sweden To The Netherlands Colour of origin population: Spitsbergen Russia Sweden The Netherlands Fig. 5 Migration rates with conﬁdence intervals. The modes of the scaled migra- tion rates with 95% conﬁdence intervals are shown. Colours represent the source population. Dashed lines separate the populations of destination. Relative migration rate differentiated populations. However, the presence of linkage disequilibrium among a number of the SNPs suggests admixture (Hartl & Clark 2007) between the populations. The alternative explanations for linkage disequilibrium, physical linkage, low recombination rate or selection for particular SNPs, are unlikely to explain this. Because the minimum physical distance between the SNPs ranged from a 100–200 kb (Jonker et al. 2012b), it is unlikely this could have caused linkage disequilib- rium, which is also shown by the distance between LD pairs within chromosomes (Fig. S6, Supporting information). Differences in recombination rates between the populations are unlikely, and linkage dis- equilibrium caused by selective sweeps would have resulted in a nonrandom pattern of distribution of LD pairs over the genome, which is not the case (Fig. S2–S5, Supporting information). Discussion Our FST analysis and the structure analysis of the DAPC show that the ﬁve populations of barnacle geese are © 2013 John Wiley & Sons Ltd 5842 R. M. JONKER ET AL. Table 4 Bayes factors model comparison of migration models Model Model parameters Harmonic mean (HM) dHM Probability Model 1 * ** 5899 0 1 Model 2 00 0* 0* **** 7428 1530 0 Model 3 00 0* 0* 0* 6537 639 0 Model 4 00 ** 0* 0* 7076 1177 0 Model 5 00 *0 0* 00 8178 2279 0 Model 6 00 ** 00 00 8191 2293 0 Model 7 00 0* 00 7819 1920 0 In this model comparison, four populations were used: (i) Spitsbergen, (ii) Russia, (iii) Sweden, (iv) the Netherlands. Model parame- ters code as follows: the ﬁrst four signs indicate migration to the ﬁrst population from populations 1, 2, 3 and 4. The second four signs indicate migration to the second population from 1,2,3 and 4. The ﬁrst sign of the ﬁrst quartet and the second of the second quartet indicate estimation of theta for populations 1, 2, etc. An asterisk indicates that that particular migration rate was estimated by the model, and a 0 indicates that no migration was allowed. For example, for model 2: no migration from Russia and Sweden to Spitsbergen was estimated, and no migration from Spitsbergen to Russia and Sweden was estimated. For each model, we used 4 heated chains with 1, 1.5, 3, 1 000 000 heating scheme. The sampling increment in the prior was set to 20, the number of steps dis- carded (burn-in) was 2.000.000, and the number of steps analysed was 5.000. Prior thetas were generated from a uniform distribution ranging from 0 to 15, and prior migration rates were generated from a uniform distribution ranging from 0 to 4000. These settings resulted in converged posterior distributions with a clear maximum for each estimate (Supplementary File 1 for model 1). In this model comparison, four populations were used: (i) Spitsbergen, (ii) Russia, (iii) Sweden, (iv) the Netherlands. Model parame- ters code as follows: the ﬁrst four signs indicate migration to the ﬁrst population from populations 1, 2, 3 and 4. The second four signs indicate migration to the second population from 1,2,3 and 4. © 2013 John Wiley & Sons Ltd Discussion The probability for a Russian individual immigrating into the Spitsber- gen population was much lower at 0.00014 and 0.0008 in the other direction, with fewer birds returning (9–39%). No evidence for exchange between the Russian and Greenland populations based on resightings was found (Black et al. 2007). These data illustrate that dis- persal events occur, creating opportunities for interpop- ulation breeding pairs to establish. Our genetic analysis allow for much more ﬁne-grained analysis of exchange rates between populations than based on ring resigh- tings and suggest that exchange rates are generally higher than previously assumed. Additionally, our MIGRATE-N analysis indicates that a full island model (bidirectional gene ﬂow between all populations) is best supported, while dispersal between all populations, and especially between the three ﬂy- ways, is very unlike traditionally inherited migration systems (Mayr 1942; Anderson et al. 1992). The exchange between the ﬂyways could in time lead to a pattern of general lack in ﬂyway structure already pres- ent in Mallard Anas platyrhynchos (Kraus et al. 2011b, 2013) depending on overall migration rates. The rela- tively large emigration from and low immigration to the Netherlands suggests high exploration rates for individuals from this population and a certain con- straint to immigrate into this population. Potentially, the difference in seasonally migratory behaviour of the Dutch population, as compared to all the others (the Dutch population is the only nonmigratory population), reduces the chance of settling permanently in the Dutch population, because adults who already adopted a migratory tradition may not be likely to lose it. Further, population differences in the timing of breeding and moult (Van der Jeugd et al. 2009) may reduce the ﬁtness of individuals that settle into a new population because these events can have strong ﬁtness consequences (Prop et al. 2003), and individuals have shown to not easily adjust the timing of moult (Loonen & Follestad 1997). Both the DAPC analysis and the FST analysis showed that the genetic distance between the Spitsbergen popu- lation and the Russian, Swedish and Dutch populations is larger than among the last three populations, with the Russian population being closest to Spitsbergen of these three. This suggests that the Swedish and Dutch population recently diverged from the Russian popula- tion. Moreover, the result that the FST between Russia and Sweden is the smallest of all pairwise comparisons indicates that most exchange of individuals occurs between Russia and Sweden. Discussion Linkage disequilibrium in the Spitsbergen population could potentially have been caused by small population size in the past, while link- age disequilibrium in the Dutch and Swedish popula- tions could have been caused by small populations of founders. In the Russian population, such a cause is © 2013 John Wiley & Sons Ltd GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5843 unlikely because of the large population size in the past. However, our explanation of admixture is consistent with the assignment probabilities in the DAPC analysis that showed that in most populations a considerable number of individuals have a high assignment probabil- ity to a population different from their sampling popula- tion and with the gene ﬂow estimated by MIGRATE-N. the strong connection between the Swedish and Russian populations. Van Der Jeugd & Litvin (2006) documented long-distance dispersal from the Baltic based on ring resightings and recoveries and estimated that 6.6% of all Baltic juveniles dispersed over long distances, mainly to the Russian population. At the same time, the three identiﬁed groups of Dutch barnacle geese in the a posteriori DAPC analysis may lead one to believe that minor mixing with individuals of a captive origin (Lensink 1996), or possibly hybridisation with cackling geese Branta hutchinsi (HvdJ pers obs) have occurred. If this were the case, however, the FST between the Dutch population and the other popula- tions would be expected to be higher than FST values among the other populations. In either case, this popula- tion structure within the Dutch population is an interest- ing observation that warrants further research in the future. g y These ﬁndings suggest that over the past few genera- tions, individuals have moved from one population to another, which implies that a number of individuals broke with their migratory traditions. We argue that this is a recent event, because otherwise linkage dis- equilibrium would already have disappeared. Evidence for recent population exchange via dispersal also comes from (sparse) data of ring recoveries. Based on ring recoveries, exchange rates between the Spitsbergen and Greenland populations were estimated at (0.0010 and 0.0011 (probability of dispersal to destination popula- tion, taking into account source population speciﬁc sur- vival and recapture probabilities, see for more details Black et al. 2007), but most individuals that dispersed (40–90%) returned to their population of origin after one or several years (Black et al. 2007). © 2013 John Wiley & Sons Ltd Discussion Subse- quently, among 1127 individuals, there was no apparent population structure, in contrast to what they expected based on natal philopatry and dispersal distances. Although a large part of their individuals studied hardly dispersed from the natal area, there was a long tail in the distribution of dispersal distances, which would explain the lack of population structure in their analysis. This suggests that only few broken traditions can make a population appear panmictic, a phenome- non compatible with the often quoted ‘one migrant per generation’ rule (Mills & Allendorf 1996). Similarly, a study on lesser kestrels Falco naumanni that combined capture–recapture and genetic analysis using microsat- ellites showed that very small-scale natal philopatry (<100 km) did not lead to genetic differentiation (Alcaide et al. 2009). Contrastingly, in greater snow geese Chen caerulescens atlantica, it was shown, using AFLP markers, that even on this small spatial scale, genetic differentiation occurred as a result of natal philopatry (Lecomte et al. 2009). g Changes of traditions in populations have been reported before, but not always have population genetic consequences. In herring Clupea harengus, the removal of adult individuals as a result of overﬁshing caused young individuals to explore new spawning grounds, whereas they would normally spawn at the traditional locations used by the older individuals (Corten 2002). The spawning ground was the tradition, and hatched larvae disperse easily, after which young herring join existing schools (McQuinn 1997). Consequently, the loss or acquisition of traditions would not likely have popu- lation genetic consequences. Also, in Canada geese Branta canadensis, nonmigratory populations have emerged. In some cases, this can be attributed to the re-introduction programmes from captive bred geese, which lacked traditions of migration (Mowbray et al. 2002). It has also been shown that young Canada geese made so-called reversal migration, which could play an important role in exchange between populations (Ravel- ing 1976). Some populations of Paciﬁc ﬂyway brant geese Branta bernicla nigricans also lost their migratory behaviour. Unlike the Dutch population of barnacle geese that remain year-round in their traditional wintering habitat, they remain in their breeding area year-round (Ward et al. 2009), most likely because of increasing temperatures in the breeding area in winter. Thus, despite long-standing ideas of strong natal philopatry and high winter site ﬁdelity in geese, in recent years, a variety of dramatic changes in migratory behaviour have been witnessed in several species of geese. Discussion This is also supported by the DAPC analysis, which shows that many birds sam- pled in Sweden have a high probability of being assigned to the Russian population. During wintering in the Netherlands, individuals from the Russian and Swedish populations mainly reside in the north of the Netherlands, while individuals from the Dutch popula- tion mainly reside in the south as indicated from the ring recoveries (Jonker et al. 2012a). This could explain The seemingly high emigration rate from the Dutch population could be caused by the very short duration of parental care in this nonmigratory population (Jonker et al. 2012a). Whereas parents in the (migratory) Russian population provide parental care until approximately early March, the parents in the (nonmigratory) Dutch population provide parental care only until the end of November. As juveniles are suggested to venture on exploratory trips after being released from the family (Baker 1978), this increases the exploratory potential for © 2013 John Wiley & Sons Ltd 5844 R. M. JONKER ET AL. likely to accompany its new mate to her population. Interestingly, Lecomte et al. (2009) showed that in greater snow geese, distances of 10–30 km at the rearing site resulted in differentiation even though the pairing took place almost 3000 km away, indicating that pair- ing mostly occurred between individuals of the same rearing site. the Dutch population. Because linkage disequilibrium due to admixture disappears very rapidly (halving each generation), our results suggest that this mixing did not start longer than a few generations ago, which ﬁts the timescale of the behavioural changes in migration and parental care duration (Jonker et al. 2010, 2011). More- over, the convergence of populations, resulting from the reduced cultural transmission of migration, probably reversed the ongoing differentiation of populations caused by the strong natal philopatry and conserva- tism of migratory traditions in populations. This race- forming is thought to be exceptionally high in geese (Mayr 1942), although the genetic consequences of migratory traditions have been rarely studied in detail and on the large spatial scale as in our study. Harrison et al. (2010) studied whether the strong natal philopatry affected the dispersal distance between parents and off- spring within one ﬂyway of the light-bellied brent geese Branta bernicla hrota. They show that dispersal distances were indeed smaller than expected based on random dispersal distances, conﬁrming natal philopatry. © 2013 John Wiley & Sons Ltd References Alcaide M, Serrano D, Tella JL, Negro JJ (2009) Strong philopa- try derived from capture-recapture records does not lead to ﬁne-scale genetic differentiation in lesser kestrels. Journal Of Animal Ecology, 78, 468–475. Anderson MG, Rhymer JM, Rohwer FC (1992) Philopatry, dis- persal, and the genetic structure of waterfowl populations. In: Ecology and Management of Breeding Waterfowl (eds Batt BDJ, Afton AD, Anderson MG, Ankney CD, Johnson DH, Kadlec JA, Krapu GL), pp. 365–395. University of Minnesota Press, Minneapolis, US. Baker RR (1978) The Evolutionary Ecology of Animal Migration. Holmes & Meier Publishers, New York. Beerli P (2009) How to use migrate or why are markov chain monte carlo programs difﬁcult to use?. In: Population Genetics for Animal Conservation (eds Bertorelle G, Bruford MW, Hauffe HC, Vernesi C, Rizzoli A), pp. 42–79. Cambridge University Press, Cambridge UK. Finally, cultural transmission mechanisms are most likely to evolve in occasionally ﬂuctuating environ- ments in which changes persist for a number of gener- ations (Feldman et al. 1996). This suggests that geese, which primarily transmit migration strategy culturally, have evolved in an environment that has frequently changed before with intermediate periods of stability, bringing ‘new’ traditions into populations following events of environmental change. Following this argu- mentation, we hypothesize that the loss and acquisi- tion of migration behaviour in geese has occurred before. In the Netherlands, there is currently a debate about the status of nonmigratory geese because geese are not ‘supposed’ to be nonmigratory with conse- quent persecution measures. We show that traditions in populations are not ﬁxed and loss of traditions may lead to gene ﬂow between traditional migration routes or lead to the emergence of nonmigration. We argue that this is not probably the result of a newly evolved trait, but of developmental plasticity. It is hoped that this insight will lead to a less conservative attitude towards wild populations in a rapidly changing environment. Beerli P (2012) Tutorial: Comparison of Gene Flow Models Using Bayes Factors. Comparison of gene ﬂow models using Bayes Factors, Tutorial. Beerli P, Palczewski M (2010) Uniﬁed framework to evaluate panmixia and migration direction among multiple sampling locations. Genetics, 185, 313–326. Berthold P, Helbig AJ (1992) The genetics of bird migration: stimulus, timing, and direction. Ibis, 134, 35–40. Black JM, Prop J, Larsson K (2007) Wild Goose Dilemmas. Branta Press, Groningen, The Netherlands. References Choudhury S, Black JM (1994) Barnacle geese preferentially pair with familiar associates from early life. Animal Behav- iour, 48, 81–88. Corten A (2002) The role of “conservatism” in herring migra- tions. Reviews in Fish Biology and Fisheries, 11, 339–361. Eichhorn G, Drent RH, Stahl J, Leito A, Alerstam T (2008) Skip- ping the Baltic: the emergence of a dichotomy of alternative spring migration strategies in Russian barnacle geese. Journal of Animal Ecology, 78, 63–72. Feldman M, Aoki K, Kumm J (1996) Individual versus social learning: evolutionary analysis in a ﬂuctuating environment. Anthropological Science, 104, 209–232. Fox AD, Ebbinge BS, Mitchell C et al. (2010) Current estimates of goose population sizes in western Europe, a gap analysis and an assassment of trends. Ornis Svecica, 20, 115–127. Friesen VL, Burg TM, McCoy KD (2007) Mechanisms of popu- lation differentiation in seabirds. Molecular Ecology, 16, 1765–1785. GENETIC CONSEQUENCES OF BREAKING MIGRATORY TRADITIONS 5845 This is publication 5517 of the Netherlands Institute of Ecology (NIOO-KNAW). This is publication 5517 of the Netherlands Institute of Ecology (NIOO-KNAW). migration or vice versa. Their analysis suggested that the short-distance migrants recently diverged from the long-distance migrant via a founder event (although Jacobsen & Omland (2011) note that the species tree in this genus is not yet completely understood). They argued that the difference in the experienced year- round environment of the short-distance migrant led to altered natural selection, resulting in divergence between the two species, which supported the theory of speciation by developmental plasticity. In our case, we argue for the opposite effect of this developmental plas- ticity, that is, because of the reduced transmission of migratory traditions and the increased exploration, pos- sibly leading to increased gene ﬂow between popula- tions, the likely ongoing differentiation between the different populations of barnacle geese might have been reversed. © 2013 John Wiley & Sons Ltd Discussion Studying the population genetic consequences in these systems with migratory change, as well as in sys- tems without much migration changes have taken place (Humphries et al. 2009), may provide insights into the generality of our ﬁndings. An important aspect in understanding gene ﬂow between goose populations is the pair formation in winter. As the individuals in our study from the Russian, Swedish and Dutch populations winter in the Netherlands, there is considerable potential for exchange between these populations. Pairs between individuals from the same population will be most common as barnacle geese prefer to mate with individ- uals familiar from earlier in life (Choudhury & Black 1994). However, pair formation with unfamiliar mates can and does occur (Kurvers et al. 2013). Because of the strong female natal philopatry in geese (Van der Jeugd et al. 2002), a male from another population is most We hypothesize that our results provide an elegant example of the emergence of novelty as a result of developmental plasticity as proposed by West-Eberhard (2003, 2005). Developmental plasticity means that the phenotype of individuals develops during their lifetime as a result of a particular sensitivity to their environ- ment. Our results are comparable with Kondo et al. (2008) who compared two recently diverged long-dis- tance and short-distance migrant Neotropical species to test which of these two species was the ancestral type, that is, whether short-distance migration emerged from long-distance migration as a result of a loss of Acknowledgements Many thanks to Bert Dibbits, whose assistance was invaluable in the laboratory. Blood samples were collected as part of project BIRDHEALTH, funded by the Netherlands Organiza- tion for Scientiﬁc Research (NWO) on the occasion of the Inter- national Polar Year. Jouke Prop, G€otz Eichhorn, Helmut Kruckenberg, Alexander Kondratyev and several students assisted in sample collection. We thank Kees Roselaar for access to the museum samples from the Zoological Museum Amsterdam. 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https://openalex.org/W4235661071	https://www.qeios.com/read/9C4I6Z/pdf	English	null	Osmolality Unit	Definitions	2,020	cc-by	61	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Osmolality Unit National Cancer Institute National Cancer Institute Qeios ID: 9C4I6Z · https://doi.org/10.32388/9C4I6Z Source National Cancer Institute. Osmolality Unit. NCI Thesaurus. Code C67426. A unit for concentration measurement of osmotically active particles expressed as a number of osmoles of solute per kilogram of solvent. Qeios ID: 9C4I6Z · https://doi.org/10.32388/9C4I6Z 1/1
https://openalex.org/W2255952494	https://ecodag.elpub.ru/ugro/article/download/297/291	Russian	null	PERSPECTIVE DIRECTIONS OF THE FISHING DEVELOPMENT INTERSKO-CASPIAN FISH INDUSTRIAL SUBREGION	Ûg Rossii: èkologiâ, razvitie	2,015	cc-by	4,991	PERSPECTIVE DIRECTIONS OF THE FISHING DEVELOPMENT INTERSKO-CASPIAN FISH INDUSTRIAL SUBREGION 1Abdusamadov A.S., 1Musayev P.G., 1Grigorian O.P., 1Barkhalov R.M., 1Akhmayev E.A., 1 Taibov P.S. Daghestan branch of FGUP «CaspSIF» Makhachkala, Russia, 367022, 104 Abubakarova St. 1Abdusamadov A.S., 1Musayev P.G., 1Grigorian O.P., 1Barkhalov R.M., 1Akhmayev E.A., 1 Taibov P.S. Daghestan branch of FGUP «CaspSIF» Makhachkala, Russia, 367022, 104 Abubakarova St. ABSTRACT. Aim. To work out perspective of stable fishing development in Tersko-Caspian fish industrial subregion. Location. Tersko-Caspian subregion. Methods. Analysis of own and literature materials according to the state of reserves and catches of the main food fish species in the region of investigation. Results. Proposals on the main perspective directions of fish industry and also the improvement of thecnoironment of the water biological resources, permilting to increase effectiveness of their natural reproduction of fish are worked out. Main conclusions. In the perspective the organization of sea fishery of common sprat, herrings, grey mullet and aterines in the Russian part of the Middle Caspiy will promote the creation of stable fishery and fish processing pro- duction in the region. Keywords: reserves, fishery, fishing, Tersko-Caspian fish industrial subregion, sturgeon, sprat, herrings, grey m lets, aterine, catadromous and river fish Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Общие вопросы General problems 22001144,, № №33,, сс 3366--4433 22001144,, № №33,, рррр.. 3366--4433 1Абдусамадов А.С., 1Мусаев П.Г., 1Григор 1Бархалов Р.М.,1Ахмаев Э.А., 1Таи 1Дагестанский филиал ФГУП «Кас Махачкала, Россия, 367022 ул. Абубакаро PERSPECTIVE DIRECTIONS OF THE FISHING DEVELOPMENT INTERSKO-CASPIAN FISH INDUSTRIAL SUBREGION 1Абдусамадов А.С., 1Мусаев П.Г., 1Григорьян 1Бархалов Р.М.,1Ахмаев Э.А., 1Таибов 1Дагестанский филиал ФГУП «КаспНИ Махачкала, Россия, 367022 ул. Абубакарова, д PERSPECTIVE DIRECTIONS OF THE FISHING DEVELOPMENT INTERSKO-CASPIAN FISH INDUSTRIAL SUBREGION 1Абдусамадов А.С., 1Мусаев П.Г., 1Григорьян О.П., 1Бархалов Р.М.,1Ахмаев Э.А., 1Таибов П.С. 1Дагестанский филиал ФГУП «КаспНИРХ», Махачкала, Россия, 367022 ул. Абубакарова, д. 104 Общие вопросы General problems Babajan V.K. Metodicheskie rekomendacii po primeneniju sovremennyh metodov ocenki obshhego dopustimog ulova (ODU) M.: VNIRO. 1985. 57 s. ( ) Berdichevskij L.S. Biologicheskie osnovy racional'noj jekspluatacii rybnyh zapasov [Biological basis of rational explo tation of fish stocks.]. Tr. VNIRO. T. 67. 1969. ( ) Berdichevskij L.S. Biologicheskie osnovy racional'noj jekspluatacii rybnyh zapasov [Biological basis of rational exploi- tation of fish stocks.]. Tr. VNIRO. T. 67. 1969. Kanat'ev S.V., Pomogaeva T.V., Reznikov V.P. Perspektivy morskogo tralovogo promysla obyknovennoj kil'ki v Ros- sijskoj chasti Srednego Kaspija [Prospects sea trawling for sprat common in the Russian part of the Middle Caspian. materials]. Materialy Mezhdunarodnoj nauchno-prakticheskoj konferencii «Sohranenie biologi- cheskih resursov Kaspija. Astrahan': Izd-vo AGTU. 2014. S. 62-71. Kanat'ev S.V., Pomogaeva T.V., Reznikov V.P. Perspektivy morskogo tralovogo promysla obyknovennoj kil'ki v Ros- sijskoj chasti Srednego Kaspija [Prospects sea trawling for sprat common in the Russian part of the Middle Caspian. materials]. Materialy Mezhdunarodnoj nauchno-prakticheskoj konferencii «Sohranenie biologi- cheskih resursov Kaspija. Astrahan': Izd-vo AGTU. 2014. S. 62-71. p j Mahmudbekov A.A., Doroshkov P.K. Sel'di Kaspija. Baku: VNIRO. 1956. 75 s. p j Nikol'skij G.V. Teorija dinamiki stada ryb kak biologicheskaja osnova racional'noj jekspluatacii i vosproizvodstva ryb- nyh resursov. - M.: Pishhevaja promyshlennost'. 1974. 448 s. p j Nikol'skij G.V. Teorija dinamiki stada ryb kak biologicheskaja osnova racional'noj jekspluatacii i vosproizvodstva ryb- nyh resursov. - M.: Pishhevaja promyshlennost'. 1974. 448 s. Nikol'skij G.V. Teorija dinamiki stada ryb kak biologicheskaja osnova racional'noj jekspluatacii i vosproizvodstva ryb- nyh resursov. - M.: Pishhevaja promyshlennost'. 1974. 448 s. y j p y Omarov M.O., Abdusamadov A.S., Stoljarov I.A., Ahmedov M.R., Mirzoev M.Z., Aligadzhiev A.D., Magomedov K.A., Pushbarnjek Je.B., Abusheva K.S., Halilbegov P.H. Sostojanie zapasov i prognoz vylova ryb na 2003 g. v zapadno-kaspijskom rajone [Stock status and forecast catch fish for 2003 in the West Caspian area.]. Rybo- hozjajstvennye issledovanija na Kaspii. Rezul'taty NIR za 2001 g. Astrahan': Izd-vo KaspNIRH, 2002. S.318- 330. Omarov M.O., Abdusamadov A.S., Stoljarov I.A., Ahmedov M.R., Mirzoev M.Z., Aligadzhiev A.D., Magomedov K.A., Pushbarnjek Je.B., Abusheva K.S., Halilbegov P.H. Sostojanie zapasov i prognoz vylova ryb na 2003 g. v zapadno-kaspijskom rajone [Stock status and forecast catch fish for 2003 in the West Caspian area.]. Rybo- hozjajstvennye issledovanija na Kaspii. Rezul'taty NIR za 2001 g. Astrahan': Izd-vo KaspNIRH, 2002. S.318- 330. Резюме. Рассмотрены вопросы состояния запасов основных промысловых видов рыб и перспективам их рационального использования в Терско-Каспийском рыбохозяйственном подрайоне. Общие вопросы General problems Установлено, что в ус- ловиях критического состояния запасов анчоусовидной кильки необходимо использование другого перспек- тивного объекта - обыкновенной кильки, а также морских сельдей и кефалей. Предложены меры по решению существующих проблем в рыбной отрасли региона. Разработаны предложения по основным перспективным направлениям рыболовства, а также улучшению среды обитания водных биологических ресурсов, позво- ляющие повысить эффективность их естественного воспроизводства рыб Ключевые слова: запасы, промысел, рыболовство, Терско-Каспийский рыбохозяйственный подрайон, осе ровые рыбы, кильки, сельди, кефали, атерина, полупроходные и речные рыбы. В успешном развитии рыболовства главное значение имеет сырьевая база, т.е. со- стояние запасов водных биологических ресурсов (ВБР). При этом устойчивое использо- вание запасов ВБР зависит от организации рационального промысла и регулированию рыболовства (Бердичевский, 1969; Никольский, 1974; Бабаян, 1985 и др.). Материалы по современному состоянию запасов рыб в Терско-Каспийском рыбохозяйственном подрай- оне и перспективам их промысла приведены в работах ряда авторов (Омаров и др., 2002; Абдусамадов и др., 2003; Абдусамадов, Пушбарнэк, 2003; Абдусамадов, Мирзоев, 2003; Абдусамадов, 2004; 2008 и др.), но они требуют новых исследований, анализа и дополне- ния. Проведенные нами в 2010-2014 гг. исследования по оценке состояния запасов ВБР, прогнозе и перспективам их вылова, мониторингу состояния среды обитания показывают следующее. Рыбохозяйственный комплекс республики базируется на рыболовстве и товарной аквакультуре. Суммарный годовой улов рыбы в республике снизился в 10 раз по отноше- нию к уровню 1990 г., табл.1. Наиболее значительное сокращение произошло по вылову кильки и производству прудовой рыбы. Таблица 1 ц Объемы вылова рыбы и производства товарной рыбы по Республики Дагестан в 1990-2013 гг. ц бъемы вылова рыбы и производства товарной рыбы по Республики Дагестан в 1990-2013 гг. REFERENCES Abdusamadov A.S. Perspektivy razvitija pribrezhnogo rybolovstva v zapadno-kaspijskom regione Rossii [Prospects for the development of coastal fisheries in the western Caspian region of Russia]. Rybnoe hozjajstvo. 2004, №6. S. 8-10. Abdusamadov A.S. Perspektivy razvitija pribrezhnogo rybolovstva v zapadno-kaspijskom regione Rossii [Prospects for the development of coastal fisheries in the western Caspian region of Russia]. Rybnoe hozjajstvo. 2004, №6. S. 8-10. Abdusamadov A.S. Problemy i prioritetnye napravlenija sohranenija biologicheskih resursov zapadnoj chasti Sred- nego i Severnogo Kaspija [Challenges and priorities for the conservation of biological resources of the west- ern part of the Middle and North Caspian]. Izvestija Dagestanskogo gosudarstvennogo pedagogicheskogo universiteta. Estestvennye i tochnye nauki. 2008. №1. S.30—34. Abdusamadov A.S. Problemy i prioritetnye napravlenija sohranenija biologicheskih resursov zapadnoj chasti Sred- nego i Severnogo Kaspija [Challenges and priorities for the conservation of biological resources of the west- ern part of the Middle and North Caspian]. Izvestija Dagestanskogo gosudarstvennogo pedagogicheskogo universiteta. Estestvennye i tochnye nauki. 2008. №1. S.30—34. y y Abdusamadov A.S., Abdurahmanov G.M., Karpjuk M.I. Sovremennoe sostojanie i jekologo-jekonomicheskie pers- pektivy razvitija rybnogo hozjajstva zapadno-kaspijskogo regiona Rossii. M.: Nauka. 2004. 350 s. s il. Abdusamadov A.S., Mirzoev M.Z. Analiz sostojanija rybnogo hozjajstva Agrahanskogo zaliva i perspektivy ego vo- zrozhdenija. [Analysis of the state of fisheries Agrakhan Gulf and the prospects for its revival] Sb. statej Mezhdunarodnoj konferencii «Rybohozjajstvennaja nauka na Kaspii: zadachi i perspektivy». –Astrahan': Izd- vo KaspNIRH, 2003.S. 15 – 19. Abdusamadov A.S., Omarov M.O., Stoljarov I.A., Ahmedov M.R., Mirzoev M.Z., Aligadzhiev A.D., Pushbarnjek Je.B., Abusheva K.S. Sostojanie zapasov i perspektivy promysla presnovodnyh ryb v zapadno-kaspijskom rajone. Rybohozjajstvennye issledovanija na Kaspii. [Status and prospects of fisheries stocks of freshwater fish in the West Caspian area. Fisheries research in the Caspian Sea] Rezul'taty NIR za 2002 g. Astrahan': Izd-vo KaspNIRH, 2003. S. 307-325. Abdusamadov A.S., Pushbarnjek Je.B. Sostojanie zapasov i perspektivy promysla v zapadno-kaspijskom rajone morskih sel'dej, obyknovennoj kil'ki i kefalej. Rybohozjajstvennye issledovanija na Kaspii [Stock status and prospects of fisheries in the area of West Caspian sea herring, sprat and ordinary mullet. Fisheries research in the Caspian Sea]. Rezul'taty NIR za 2002 g. Astrahan': Izd-vo KaspNIRH, 2003. S. 356-366. p ] y g p Asejnova A.A., Hodorevskaja R.P., Abdusamadov A.S. Sovremennoe sostojanie zapasov obyknovennoj kil'ki Clu- peonella cultriventris caspia V Kaspijskom more [Current state of the common stocks of sprat Clupeonella cultriventris caspia in the Caspian Sea.]. Jug Rossii: jekologija, razvitie. 2012. №4.S. 32-39. 36 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Table 1 e of fish catch and production of trade fish in the Republic of Daghestan in 1990-2013. Volume of fish catch and production of trade fish in the Republic of Daghestan in 1990-2013. Объекты лова Годы 1990 2000 2009 2012 2013 Улов рыбы, всего: 34253,1 19076,97 5019,24 4973,04 4624,68 в т.ч. килька 28939,0 16494,0 1521,0 1003,0 850,48 частиковые рыбы 1816,9 2029,0 1364,0 1593,0 2005,32 кефаль 0 2,235 850,0 551,5 574,86 сельдь 337,0 81,0 3,0 184,3 112,52 осетровые 17,9 24,74 - - 0 37 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 производство товарной рыбы 3142,3 445,99 1281,24 1641,24 1057,5 в т.ч. осетровой - - 113,225 53,0 24 Осетровые рыбы. Состояние запасов осетровых рыб в Каспийском море про- должает оставаться критическим. Главной целью является сохранение и восстановление их запасов. Для этого должны решаться задачи по борьбе с браконьерством и наращива- нию масштабов искусственного воспроизводства до приемной емкости моря. С 1961 года осетровые рыбы не являются объектами промышленного рыболовства в Дагестане. Полупроходные и речные рыбы. Подавляющее большинство рыбодобывающих организаций осуществляют прибрежный промысел полупроходных и речных видов рыб (семейства карповые, окуневые, сомовые, щуковые) в северной зоне дагестанского побе- режья Каспийского моря. Вылов данной группы рыб составляет около 2 тыс. т в год, при квоте 2,2-2,3 тыс. т. Промысел осуществляют, в основном, предприятия из прибрежных сел Тарумовского и Кизлярского районов. Фактический вылов частиковых рыб значи- тельно выше официального уровня и составляет, по экспертным оценкам, около 5-6 тыс. т в год. д В Терско-Каспийском районе полупроходные и озерно-речные виды рыб, обра- зующие сложный многовидовой биоценоз прибрежного промысла, за последние 70 лет претерпели существенные изменения численности и биомассы (Абдусамадов и др., 2004). Несмотря на имеющиеся негативные явления (неучтенный вылов, браконьерство), запасы по большинству видов частиковых рыб стабильны, рис.1. Это связано с высоким уровнем естественного воспроизводства таких видов, как сазан, щука, кутум, карась, красноперка и др. благодаря многоводности рек Терек и Сулак в последние годы. 0 2 4 6 8 10 12 14 16 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Годы Запас, тыс. т Рис. 1. Динамика промысловых запасов полупроходных и речных видов рыб в Терско-Каспийском рыбохозяйственном подрайоне в 2005-2014 гг. Fig. 1.Dinamics of fishery reserves of species of fish in Tersko-Caspian fish industrial subregion in 2005-2014. Рис. 1. Динамика промысловых запасов полупроходных и речных видов рыб в Терско-Каспийском рыбохозяйственном подрайоне в 2005-2014 гг. Fig. Table 1 1.Dinamics of fishery reserves of species of fish in Tersko-Caspian fish industrial subregion in 2005-2014. Современное состояние запасов пресноводных рыб в регионе позволяют вести промысел в стабильном режиме с объемом изъятия до 3,0 тыс. т. На ближайшую пер- спективу, до 2015 г., в случае сокращения доли неучтенного и браконьерского вылова и введения в промысел структурных изменений, эта величина может быть увеличена до 4,0 тыс. т. В этой группе рыб в последние годы наблюдаются структурные изменения, связанные с флуктуацией численности и промысловых запасов отдельных видов. Например, наблюда- ется существенное увеличение численности производителей судака на нерестилищах, соответственно - урожайности молоди и промыслового запаса. Аналогичная картина на- блюдается в популяции рыбца, который в прежние годы практически не был отражен в статистике лова. В 2014 году его вылов существенно превысил рекомендуемую величину. 38 Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 В перспективе ожидается значительное увеличение промыслового запаса за счет высоко- урожайных поколений 2013-2014 гг., несмотря на высокий уровень эксплуатации про- мыслом. Проблемы: недостаточные объемы рыбоводно-мелиоративных работ в дельтовых водоемах рек Терек, Сулак, Самур и др., вследствие чего наносится большой ущерб эко- логической системе, в т.ч. и водным биологическим ресурсам, потери которых исчисля- ются до 12-15 тыс. т ежегодно. Критическая ситуация в Кизлярском заливе (основном рыбопромысловом районе), вследствие обмеления и зарастания растительностью. За период с 2000 по 2013 год значительно изменилась структура сырьевой базы отрасли. Основу её до 2008 г. составляли кильки, доля которых в общих уловах водных биологических ресурсов в среднем составляла около 75 %. В настоящее время доля килек снизилась до 25-30%, а доля полупроходных и речных видов рыб при этом увеличилась до 55%. Запасы морских видов рыб (кильки, сельди, кефали, атерина) в Каспии за бо- лее чем вековой период активной промысловой эксплуатации и вследствие влияния при- родных и антропогенных факторов претерпели значительные изменения. В этой группе рыб до 1960-х годов главное значение в промысле имели морские сельди (долгинская, большеглазый, каспийский пузанки), дававшие до 50-70% общего вылова рыбы в регио- не. (Махмудбеков, Дорошков, 1956). Максимального уровня – до 30,0 тыс. т и более - уловы достигали в 1910-1930-е годы, а в последующем, вплоть до закрытия морского промысла в 1960 г., наблюдалось снижение уловов сельдей, что было вызвано сокраще- нием запасов под воздействием экологических факторов (падение уровня моря, уменьше- ние кормовой базы, ухудшение условий для нереста и др.), а также переэксплуатацией промыслом. р Начиная с 1960-х гг. Table 1 в Дагестане, как и в других прикаспийских регионах СССР, активное развитие получил промысел анчоусовидной и большеглазой килек. В середине 1970-х гг. вылов их промышленностью Дагестана достигал максимальных величин – 70 тыс. т. Основу морского промысла вплоть до начала 2000-х годов составляли кильки, до- ля которых в общей массе вылова рыб по Республике Дагестан составляла 80-90%. В на- стоящее время удельный вес килек в уловах рыб снизился до 18-20%, а доля уловов с ис- пользованием судов еще ниже – 10-12%. Исследования КаспНИРХ и проводимый Дагестанским филиалом мониторинг промысла показывает, что в настоящее время основную часть промыслового запаса килек составляет обыкновенная килька - 82,8 %. На долю анчоусовидной и большеглазой килек, на которых базировался промысел до недавнего времени, приходится до 17,2 %. Критическое состояние на килечном промысле, как известно, связано с много- кратным сокращением запасов анчоусовидной и большеглазой килек вследствие их мас- совой гибели в 2001 году по всей акватории обитания в море. На фоне гибели рыб про- изошло стихийное проникновение гребневика мнемиопсиса, являющегося пищевым кон- курентом килек, поедающего при этом также икру и личинок последних. Соответственно, с начала 2000-х годов уловы килек на Каспии также начали со- кращаться и к настоящему времени они составляют около 0,5 тыс. т. Лов осуществляют лишь два судна. Среднесуточные уловы на одно судно составляют 2-3 т. Промысел килек по традиционной технике лова в Среднем и Южном Каспии стал нерентабельным, рис. 2. В то же время в Каспийском море обитает перспективный объект морского про- мысла – обыкновенная килька. Запасы обыкновенной кильки находятся в удовлетвори- тельном состоянии, позволяя организовать ее эффективный промысел с выловом до 57 тыс. т (Асейнова и др., 2012; Канатьев и др., 2014). Можно считать, что данный вид явля- ется основным перспективным объектом на Каспии в современных условиях. р р у ФГУП «КаспНИРХ», на основании материалов многолетних исследований, пока- зал возможность организации специализированного промысла обыкновенной кильки у побережья Дагестана. В качестве орудия лова рекомендован разноглубинный трал. В то ФГУП «КаспНИРХ», на основании материалов многолетних исследований, пока- зал возможность организации специализированного промысла обыкновенной кильки у побережья Дагестана. В качестве орудия лова рекомендован разноглубинный трал. В то 39 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. Table 1 № 3, 2014 же время, отсутствие флота (до 10 ед. среднетонажных судов), приспособленного для тралового лова, и другие организационные причины не позволяют развивать данное на- правление рыболовства. По объемам вылова и перспективам развития килечному лову в настоящее время на Каспии альтернативы нет. Организация морского промысла обыкно- венной кильки позволит создать устойчивое производство рыбодобывающих и перераба- тывающих предприятий отрасли в регионе. 0 1000 2000 3000 4000 5000 6000 7000 8000 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Годы Улов килек, т Рис. 2. Уловы килек в Каспийском море судами в 2004- 2013 гг. Fig. 2. Sprat catches in the Caspian sea by vehicles in 2004-2013. Рис. 2. Уловы килек в Каспийском море судами в 2004- 2013 гг. Fig. 2. Sprat catches in the Caspian sea by vehicles in 2004-2013. В сложившейся на промысле килек обстановке в Дагестане в последние годы оп- ределенное развитие получил прибрежный лов обыкновенной кильки и кефалей. Напри- мер, уловы морских видов рыб в прибрежных водах Дагестана ставными неводами и раз- личными видами сетей (за исключением морского килечного лова с использованием су- дов) в последние шесть лет возросли с 400 т в 2008 г. до 1400 т в 2013 г. Относительно стабильны также уловы обыкновенной кильки ставными неводами у побережья моря вдоль Аграханского полуострова (рис. 3). Ежегодно, на лову задейст- вовано 4-5 килечных неводов со средним уловом 60-70 т на один невод за сезон лова (март-апрель). Возможно существенно увеличить вылов обыкновенной кильки ставными неводами за счет улучшения организации лова, увеличения количества неводов и др. Да- гестанским филиалом ФГУП «КаспНИРХ» в 2014 году проводятся исследования по целе- сообразности продления срока лова обыкновенной кильки ставными неводами с 10 мая до 31 мая. В перспективе необходимо увеличить количество неводов до 20-30 ед. с выло- вом до 3-х тыс. т кильки. 0 50 100 150 200 250 300 350 400 450 500 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Годы Улов кильки, т Рис. 3. Динамика уловов обыкновенной кильки ставными неводами на Дагестанском побережье в 2003- 2013 гг. Fig. 3. Common sprat catches dinamics by sweep-net on Daghestan coast in 2003-2013. Рис. 3. Динамика уловов обыкновенной кильки ставными неводами на Дагестанском побережье в 2003- 2013 гг. Fig. 3. Common sprat catches dinamics by sweep-net on Daghestan coast in 2003-2013. 40 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Как отмечено выше, морские сельди (долгинская сельдь, каспийский и больше- глазый пузанки) до 1960-х годов являлись главными промысловыми объектами у побе- режья Дагестана. Предпринимаемые с 1980-х годов попытки по возрождению сельдяного лова с использованием береговых закидных неводов не увенчались успехом. Более того, уловы с 2001 года начали неуклонно снижаться. При этом показатели сетного лова сель- дей у побережья удовлетворительные и составляют 5-7 кг/сеть/сутки. Морские сельди составляют существенный резерв промысла - 11,8 тыс. т. Перспективным объектом промысла из морских рыб является также атерина. Вы- лов атерины осуществляется у побережья моря вдоль Аграханского полуострова в каче- стве прилова при промысле обыкновенной кильки ставными неводами. Возможный вы- лов определен в объеме 7 тыс. т. Недостаточно используемыми объектами морского промысла являются кефали. Промысел их осуществляется с июня по октябрь обкидными и ставными сетями. Наибо- лее перспективными участками являются районы у побережья Дагестана от Сулакской бухты до конца побережья Аграханского полуострова, акватория у о. Чечень и Кизляр- ский залив с зарослевой зоной. В последние четыре года наблюдается рост объема вылова, рис. 4. Максимальный вылов – 850 т - отмечен в 2009 году. Увеличить вылов кефали до 2-х тыс. т представляет- ся вполне реальным в ближайшие годы. В республике Дагестан имеются большие перспективы в области развития спор- тивного и любительского рыболовства. Важным вопросом в регулировании данного на- правления рыболовства является его доступность, которая определяется свободой досту- па граждан к водным объектам. Для осуществления спортивного и любительского рыбо- ловства может быть использовано более 100 внутренних водоемов озерного типа, 530 км береговой линии Каспийского моря, более 100 рек. Природно-климатические условия, водные ресурсы позволяют выращивать десят- ки тыс. т ценной рыбы. Это осетровые, форель, карп, белый амур, толстолобики и др. Не- обходимо использовать потенциал развития товарной аквакультуры, в т.ч. товарного осетроводства с использованием геотермальных источников, форелеводства в горных водохранилищах, озерах и реках, прудового рыбоводства, марикультуры и др. 0 100 200 300 400 500 600 700 800 900 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Годы Улов кефали,т Рис. 4. Динамика уловов кефалей на дагестанском побережье в 2003- 2013 гг. Fig. 4. Grey-mullet catches dynamics on Daghestan coast in 2003-2013. Рис. 4. Динамика уловов кефалей на дагестанском побережье в 2003- 2013 гг. Fig. 4. Grey-mullet catches dynamics on Daghestan coast in 2003-2013. Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Существуют следующие основные проблемы, препятствующие эффективному развитию рыбохозяйственного комплекса Республики Дагестан: Существуют следующие основные проблемы, препятствующие эффективному азвитию рыбохозяйственного комплекса Республики Дагестан: - сокращение запасов анчоусовидной кильки вследствие воздействия стихийно проникшегося в Каспий азово-черноморского вселенца - гребневика мнемиопсиса, а так- же крупномасшатбной гибели килек в Среднем и Южном Каспии весной-летом 2001 г.; 41 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 - отсутствие современного добывающего флота и береговой инфраструктуры для приема, хранения и переработки ВБР; - отсутствие современного добывающего флота и береговой инфраструктуры дл приема, хранения и переработки ВБР; - отсутствие современного добывающего флота и береговой инфраструктуры для приема, хранения и переработки ВБР; - отсутствие инвестиций, вызванное неустойчивостью сырьевой базы, а также не- рачностью и высокой долей теневого сектора данного направления бизнеса; - отсутствие инвестиций, вызванное неустойчивостью сырьевой базы, а также н прозрачностью и высокой долей теневого сектора данного направления бизнеса; - недостаточные объемы рыбоводно-мелиоративных работ в дельтовых водоемах рек Терек, Сулак, Самур и др., вследствие чего наносится большой ущерб экологической системе, в т.ч. и водным биологическим ресурсам, потери которых исчисляются до 12-15 тыс. т ежегодно; - недостаточные объемы рыбоводно-мелиоративных работ в дельтовых водоемах рек Терек, Сулак, Самур и др., вследствие чего наносится большой ущерб экологической системе, в т.ч. и водным биологическим ресурсам, потери которых исчисляются до 12-15 тыс. т ежегодно; - слабое развитие товарной аквакультуры при особо благоприятных природно- климатических условиях республики и др. - слабое развитие товарной аквакультуры при особо благоприятных природно- климатических условиях республики и др. Для реализации имеющегося потенциала рыбохозяйственного комплекса Респуб- лики Дагестан полагаем целесообразным решить следующие первоочередные проблемы в отрасли: 1. С целью сохранения и восстановления запасов особо ценных и ценных видов биоресурсов Каспия – терских и сулакских стад осетровых, кутума и др. провести модер- низацию рыбоводных заводов с внедрением современных технологий, в т.ч. оборотного водоснабжения, создания условий для содержания и формирования маточных стад рыб и др. р 2. Принять меры по активному развитию прибрежного рыболовства за счет ос- воения запасов малоиспользуемых объектов промысла – килек, сельдей, кефалей, атери- ны и др. р 3. Развивать береговую инфраструктуру рыбопромышленного комплекса, в т.ч. осуществить реконструкцию махачкалинского рыбного порта, содействовать приобрете- нию малотоннажного флота с холодильными мощностями, рыбопереработки и пр. ф р р р р 4. Использовать потенциал развития товарной аквакультуры, в т.ч. Общие вопросы General problems Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 товарного осетроводства с использованием геотермальных источников, форелеводства в горных водохранилищах, озерах и реках, прудового рыбоводства, марикультуры и др. р р р ру р р у ур р 5. Развивать любительское и спортивное рыболовство на водных объектах с обес- печением его социальной доступности. 6. Разработать основные направления развития рыболовства, позволяющие ис- пользовать запасы перспективных объектов водных биологических ресурсов в Каспий- ском море и создавать на этой основе устойчиво растущее производство рыбопромысло- вых и рыбоперерабатывающих предприятий отрасли. В перспективе организация морского промысла обыкновенной кильки, сельдей, кефали и атерины в российской части Среднего Каспия будет способствовать созданию устойчивого производства рыбопромысловых и рыбоперерабатывающих предприятий отрасли в регионе. В перспективе организация морского промысла обыкновенной кильки, сельдей, кефали и атерины в российской части Среднего Каспия будет способствовать созданию устойчивого производства рыбопромысловых и рыбоперерабатывающих предприятий отрасли в регионе. БИБЛИОГРАФИЧЕСКИЙ СПИСОК Астрахань: Изд во АГТУ. 2014. С. 62 71. Махмудбеков А.А., Дорошков П.К. Сельди Каспия. - Баку: ВНИРО. - 1956. - 75 с. у Махмудбеков А.А., Дорошков П.К. Сельди Каспия. - Баку: ВНИРО. - 1956. - 75 с. у у Никольский Г.В. Теория динамики стада рыб как биологическая основа рациональной эксплуатации и воспро- изводства рыбных ресурсов. М.: Пищевая промышленность. 1974. – 448 с. Омаров М.О., Абдусамадов А.С., Столяров И.А., Ахмедов М.Р., Мирзоев М.З., Алигаджиев А.Д., Магомедов К.А., Пушбарнэк Э.Б., Абушева К.С., Халилбегов П.Х. Состояние запасов и прогноз вылова рыб на 2003 г. в западно-каспийском районе. Рыбохозяйственные исследования на Каспии. Результаты НИР за 2001 г. – Астрахань: Изд-во КаспНИРХ, 2002. С.318-330. ИНФОРМАЦИЯ ОБ АВТОРАХ Абдусамадов Ахма Саидбегович – доктор биологических наук, (8722)635519, Дагестанский филиал ФГУП «КаспНИРХ», М Р 367022 Аб б 104 il d k i @ il Абдусамадов Ахма Саидбегович – доктор биологических наук, (8722)635519, Дагест Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru Абдусамадов Ахма Саидбегович – доктор биологических наук, (8722)635519, Дагестанский филиал ФГУП «КаспНИРХ», Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru Мусаев П.Г. – заведующий лабораторией промысловой ихтиологии, (8722)635519, Дагестанский филиал ФГУ «КаспНИРХ», Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru Григорьян О.П. – заместитель директора по научной работе, (8722)635519, Дагестанский филиал ФГУП «КаспНИРХ», Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru Григорьян О.П. – заместитель директора по научной работе, (8722)635519, Даге Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru Бархалов Р.М. – кандидат биологических наук, заведующий сектором полупроходных и речных рыб, (8722)635519, Дагестанский филиал ФГУП «КаспНИРХ», Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru p y@ Ахмаев Э.А. – заведующий сектором естественного воспроизводства рыб, (8722)635519, Дагестанский фили ФГУП «КаспНИРХ», Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru Таибов П.С. – заведующий сектором морских рыб, (8722)635519, Дагестанский филиал ФГУП «КаспНИРХ», Махачкала, Россия, 367022 ул. Абубакарова, д. 104, e-mail: dokaspiy@mail.ru БИБЛИОГРАФИЧЕСКИЙ СПИСОК Абдусамадов А.С. Перспективы развития прибрежного рыболовства в западно-каспийском регионе Росси Рыбное хозяйство. 2004, №6. С. 8-10. бдусамадов А.С. Перспективы развития прибрежного рыболовства в западно-каспийском регионе России. Рыбное хозяйство. 2004, №6. С. 8-10. Абдусамадов А.С. 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С. 15 – 19. р р Абдусамадов А.С., Омаров М.О., Столяров И.А., Ахмедов М.Р., Мирзоев М.З., Алигаджиев А.Д., Пушбарнэк Э.Б., Абушева К.С. Состояние запасов и перспективы промысла пресноводных рыб в западно- 42 Общие вопросы General problems Общие вопросы Юг России: экология, развитие. № 3, 2014 The South of Russia: ecology, development. № 3, 2014 каспийском районе. Рыбохозяйственные исследования на Каспии. Результаты НИР за 2002 Астрахань: Изд-во КаспНИРХ, 2003. С. 307-325. каспийском районе. Рыбохозяйственные исследования на Каспии. Результаты НИР за 2002 Астрахань: Изд-во КаспНИРХ, 2003. С. 307-325. Абдусамадов А.С., Пушбарнэк Э.Б. Состояние запасов и перспективы промысла в западно-каспийском районе морских сельдей, обыкновенной кильки и кефалей. Рыбохозяйственные исследования на Каспии. Результаты НИР за 2002 г. Астрахань: Изд-во КаспНИРХ, 2003. С. 356-366. Асейнова А.А., Ходоревская Р.П., Абдусамадов А.С. Современное состояние запасов обыкновенной киль Clupeonella cultriventris caspia В Каспийском море. Юг России: экология, развитие. 2012. №4.С. 32-39 Бабаян В.К. Методические рекомендации по применению современных методов оценки общего допустимо улова (ОДУ) М.: ВНИРО. 1985. 57 с. Бердичевский Л.С. Биологические основы рациональной эксплуатации рыбных запасов. Тр. ВНИРО. Т. 67. 1969. Канатьев С.В., Помогаева Т.В., Резников В.П. Перспективы морского тралового промысла обыкновенной кильки в Российской части Среднего Каспия. Материалы Международной научно-практической конференции «Сохранение биологических ресурсов Каспия. Астрахань: Изд-во АГТУ. 2014. С. 62-71. Махмудбеков А А Дорошков П К Сельди Каспия - Баку: ВНИРО - 1956 - 75 с кильки в Российской части Среднего Каспия. Материалы Международной научно-практической конференции «Сохранение биологических ресурсов Каспия. Астрахань: Изд-во АГТУ. 2014. С. 62-71. Махмудбеков А.А., Дорошков П.К. Сельди Каспия. - Баку: ВНИРО. - 1956. - 75 с. конференции «Сохранение биологических ресурсов Каспия. INFORMATION ABOUT AUTHORS Abdusamatov Ahma Saidbegovich - doctor of biological Sciences, (8722)635519, Dagestan Makhachkala, Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru Makhachkala, Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru , , , , p y@ Musayev, P. G. - head of the laboratory of fishery ichthyology, (8722)635519, Dagestan branch of FSUE "CaspNIRKh", Ma hachkala, Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru y@ Musayev, P. G. - head of the laboratory of fishery ichthyology, (8722)635519, Dagest hachkala, Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru Grigorian O. P. - Deputy Director on scientific work, (8722) 635519, Dagestan branch of FSUE "CaspNIRKh", Makhachkala, Russia 367022 street Abubakarov D 104 e-mail: dokaspiy@mail ru Grigorian O. P. - Deputy Director on scientific work, (8722) 635519, Dagestan branch of FSUE "CaspNIRKh", Makhachkala, R i 36 022 Ab b k D 104 il d k i @ il Grigorian O. P. - Deputy Director on scientific work, (8722) 635519, Dagesta Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru Barkalow R. M. - candidate of biological Sciences, head of the sector of semi-anadromous and fluvial fish, (8722)635519, Dages- tan branch of FSUE "CaspNIRKh", Makhachkala, Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru Akmaev E. A. - head of the sector of natural reproduction of fish, (8722)635519, Dage hachkala, Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru p , ( ) , g hachkala, Russia, 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru hachkala, Russia, 367022 street Abubakarov, D. 104, e mail: dokaspiy@mail.ru Taibov P.F. - head of the sector of marine fish, (8722)635519, Dagestan branch of FSUE "CaspNIRKh", Makhachkala, Russia, p y@ Taibov P.F. - head of the sector of marine fish, (8722)635519, Dagestan branch of FSUE "CaspNIRKh", Makhachkala, Russ 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru Taibov P.F. - head of the sector of marine fish, (8722)635519, Dagestan branch of 367022 street Abubakarov, D. 104, e-mail: dokaspiy@mail.ru 43
https://openalex.org/W4393256949	https://www.businessperspectives.org/images/pdf/applications/publishing/templates/article/assets/19842/PPM_2024_01_Soares.pdf	English	null	Impact of research and development expenses on the profitability of assets: The case of textile and clothing industry in Portugal	Problems and perspectives in management/Problems & perspectives in management	2,024	cc-by	9,973	“Impact of research and development expenses on the profitability of assets: The case of textile and clothing industry in Portugal” “Impact of research and development expenses on the profitability of assets: The case of textile and clothing industry in Portugal” “Impact of research and development expenses on the profitability of assets: The case of textile and clothing industry in Portugal” p p p p y The case of textile and clothing industry in Portugal” AUTHORS Ricardo de Moraes e Soares Alexandre Morais Nunes Paula Heliodoro Vanda Martins ARTICLE INFO Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro and Vanda Martins (2024). Impact of research and development expenses on the profitability of assets: The case of textile and clothing industry in Portugal. Problems and Perspectives in Management, 22(1), 702-715. doi:10.21511/ppm.22(1).2024.55 DOI http://dx.doi.org/10.21511/ppm.22(1).2024.55 RELEASED ON Thursday, 28 March 2024 RECEIVED ON Friday, 12 January 2024 ACCEPTED ON Tuesday, 12 March 2024 LICENSE This work is licensed under a Creative Commons Attribution 4.0 International License JOURNAL "Problems and Perspectives in Management" ISSN PRINT 1727-7051 ISSN ONLINE 1810-5467 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 48 NUMBER OF FIGURES 0 NUMBER OF TABLES 2 © The author(s) 2024. This publication is an open access article. © The author(s) 2024. This publication is an open access article. © The author(s) 2024. This publication is an open access article. Vanda Martins, Specialist in Accounting and Taxation, Professor, Accounting and Finance Department, Higher School of Business Sciences, Polytechnic Institute of Setúbal, Portugal. businessperspectives.org businessperspectives.org Problems and Perspectives in Management, Volume 22, Issue 1, 2024 Ricardo de Moraes e Soares (Portugal), Alexandre Morais Nunes (Portugal), Paula Heliodoro (Portugal), Vanda Martins (Portugal) Keywords clothing, efficiency, innovation, industry, manufacturing, textile clothing, efficiency, innovation, industry, manufacturing, textile JEL Classification C67, L67, O14 JEL Classification JEL Classification C67, L67, O14 Paula Heliodoro, Ph.D. in Management, Professor, Accounting and Finance Department, Higher School of Business Sciences, Polytechnic Institute of Setúbal, Portugal. BUSINESS PERSPECTIVES LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine www.businessperspectives.org This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Conflict of interest statement: Author(s) reported no conflict of interest Abstract The study aims to examine the financial efficiency of the textile and clothing industries in Portugal using official statistical data. The main objective is to assess the relation- ship between spending on research and development and return on assets. The study analyzes the performance of various subsectors of the textile and clothing industries, presenting the relationship between investments in research and development and the operating return on assets over various economic periods. The study adopted data envelopment analysis, classifying decision-making units based on average efficiency levels. The results highlight sectors of manufacture of textiles for technical and indus- trial use, manufacture of other textiles, production of outerwear, and manufacture of workwear as the most efficient. In contrast, sectors of manufacture of clothing and accessories, manufacture of knitwear, and leather clothing show lower levels of effi- ciency. From 2003 to 2022, the textile industry exhibited the highest levels of financial efficiency, with an above-average ratio between spending on research and development and return on assets. However, sectors of knitwear manufacturing and textile finishing have maintained a more or less constant level of financial efficiency. The analysis high- lights the need for targeted interventions to increase the financial efficiency of different subsectors within the textile and clothing industries. It is evident that there are varying levels of financial efficiency across these sectors, and the need for benchmarking peri- ods can help identify areas for improvement and set achievable goals. Received on: 12th of January, 2024 Accepted on: 12th of March, 2024 Published on: 28th of March, 2024 Received on: 12th of January, 2024 Accepted on: 12th of March, 2024 Published on: 28th of March, 2024 © Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro, Vanda Martins, 2024 Ricardo de Moraes e Soares, Ph.D. in Public Administration, Professor, Accounting and Finance Department, Higher School of Business Sciences, Polytechnic Institute of Setúbal, Portugal. (Corresponding author) Alexandre Morais Nunes, Ph.D. in Public Administration, Professor, Public Administration Department, Higher Institute of Social and Political Sciences, University of Lisbon, Portugal. Ricardo de Moraes e Soares (Portugal), Alexandre Morais Nunes (Portugal), Paula Heliodoro (Portugal), Vanda Martins (Portugal) 1. LITERATURE REVIEW AND HYPOTHESES patterns, industry reforms, new practices, and in- creased research and development spending are im- perative (Basto et al., 2021). In the European Union, the textile and clothing industry constitutes approximately 4.0% of pro- duction and 7.0% of employment in manufac- turing. In Portugal, similar to the EU, the indus- try faces challenges such as global competition (European Commission, 2003), low innovation (Montenegro, 2011), and low research levels (Sousa, 2020). Maintaining employment and production levels (Ferreira, 2018), ensuring quality (Cardoso & Ferreira, 2013), adding value (Pereira, 2021), ac- quiring new skills (Merle & Fermam, 2023), and pursuing modernization (Nikam, 2003) are key priorities for the industry. Jajri and Ismail (2006) explored the relationship between new technologies and the growth of Malaysia’s manufacturing sector. Their analysis concentrated on the sector’s evolution in technical efficiency. The results suggest that, on average, the food, wood, chemicals, and iron products sectors showcase the highest technical efficiency. In con- trast, the food and textiles sectors exhibit the low- est efficiency in resource allocation, indicating po- tential areas for improvement in these industries. Khalil (2011) scrutinized the relative efficiency of Pakistan’s textile sector using data envelop- ment analysis (DEA). The findings revealed sub- par technical efficiency across all production units, necessitating the adoption of more efficient mea- sures. The study concluded that addressing the rising pollution levels associated with textile pro- duction processes is imperative for the sustainable development. The abolition of import quotas in 2005 instigated a need in the Portuguese sector to adapt to new circumstances and accept the rules of the interna- tional market (Silva, 2013). The European textile and clothing industry, primarily comprising small and medium-sized companies, holds a value ex- ceeding 200 billion euros. In Portugal, its signifi- cance is underlined by tradition (Nogueira, 2020), empirical knowledge (Santos, 2011), quality, and creativity (Proença et al., 2008). Rakhmawan et al. (2012) explored the efficien- cy relationship between performance indicators and productivity in the textile industry through data envelopment analysis. The sector exhibited a 40% efficiency level and an average productivity growth of 2.40%. The study inferred that enhanc- ing efficiency distinctly and positively influences the overall productivity of the textile sector. The Portuguese textile and clothing industry, piv- otal for the country’s economy, generates income through exports and internal trade. INTRODUCTION The financial efficiency of the textile and clothing sector in Portugal is currently a highly relevant area of research, particularly for industrial economics and business management. Studying the efficiency dynam- ics of the subsectors of the textile and clothing industry contributes to a theoretical understanding of economic activities. It can also aid the decision-making process of those involved in terms of drawing up and implementing public policies that are more beneficial to the develop- ment of this sector. The topic of financial efficiency is indeed a funda- mental issue, especially when it comes to the allocation of scarce re- sources that are used by the various segments of the textile and cloth- ing industry and how it relates to the performance and sustainability of companies (Elisa, 2023). Research into the issue involves analyzing the multifaceted factors involved in efficiency, such as technological innovation, levels of in- 702 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 vestment in research and development (R&D), the types of production methods adopted, and market dynamics (Liu & Xia, 2018). It is interesting to analyze the implications of financial efficiency on the economic and political choices of companies and decision-makers. Examining possible relationships between efficiency, competitiveness, and sustainability of resource allocation and providing informa- tion both at the organizational and political levels are vital. Analyzing efficiency in the textile and cloth- ing industries aims to generate knowledge that can strengthen theoretical understanding and practical results in industrial economics and business management. http://dx.doi.org/10.21511/ppm.22(1).2024.55 1. LITERATURE REVIEW AND HYPOTHESES Renowned for product quality, particularly textiles (Puime- Guillén et al., 2021), Portugal exports to global markets, with a focus on European countries (Nagy, 1999). Quality and innovation have significant- ly enhanced the competitiveness of the industry (Montagna & Carvalho, 2018), contributing to its ability to offer diverse opportunities and economic growth. In pursuit of innovation, the Portuguese textile industry has embraced advanced technolo- gies and sustainable practices (Costa, 2022). Faced with global competition and evolving consumption In assessing the technical efficiency of the Chinese manufacturing industry in relation to energy ex- penditure, Fang et al. (2013) employed the DEA model. The study identified the textile sector as having the highest technical efficiency. In compari- son, the machinery and electronics manufacturing industry exhibited a notable increase in efficiency when environmental variables were not considered. 703 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 Pérez et al. (2017) evaluated the technical effi- ciency of the Chilean textile and clothing indus- try. They revealed diverse efficiency levels related to energy expenditure, greenhouse gas emissions, and geographical location. The study, employing DEA analysis, identified industries in Coquimbo, La Araucania, and Aysen as the most efficient, while those in Tarapaca, Antofagasta, and Biobio exhibited the lowest technical efficiency. Pérez et al. (2017) evaluated the technical effi- ciency of the Chilean textile and clothing indus- try. They revealed diverse efficiency levels related to energy expenditure, greenhouse gas emissions, and geographical location. The study, employing DEA analysis, identified industries in Coquimbo, La Araucania, and Aysen as the most efficient, while those in Tarapaca, Antofagasta, and Biobio exhibited the lowest technical efficiency. and production levels. Simultaneously applying Z-Score and data envelopment analysis (DEA) methodologies, the results indicated variable and disparate efficiency in the relationship between fi- nancial performance and production levels. Du et al. (2022) assessed the impact of innovation on manufacturing industry efficiency by exploring the relationship between innovation and develop- ment expenditure, personnel expenditure, reve- nue from sales of new products, and the number of patent applications. Findings indicated low levels of innovation and technological evolution, result- ing in overall low technical efficiency. However, increased investment in innovation, particularly in cities like Xianning, Xiantao, and Tianmen, is identified as a means to enhance the efficiency of the manufacturing sector. Li and Tsai (2018) examined the efficiency of the textile/clothing pipeline, spanning from the pro- duction of fibers, fabrics, and clothing to end con- sumers. 1. LITERATURE REVIEW AND HYPOTHESES They delved into the sector’s cost calcula- tion processes and the retail capacity of companies. The findings underscored that efficiency levels are notably influenced by these factors. Financial efficiency in the textile and clothing in- dustries depends on multiple factors, including training expenses, education level, capital costs, company size, wages, innovation, technology, and research and development spending (Fahmy- Abdullah et al., 2018). The analysis of 1,010 com- panies in Malaysia revealed a high level of techni- cal efficiency. The study suggests that the textile industry should enhance its approach to high-tech production by fostering stronger connections be- tween production, research, education, motiva- tion, and wage levels. Darji and Dahiya (2023) evaluated the operational efficiency of textile production units in India by examining the relationship between performance indicators and production factors. Findings indi- cated notably low operational performance, sug- gesting that companies should consider reducing the size of production factors to enhance efficiency. Regarding assessing financial efficiency, Zenzerovic et al. (2023) focused on the relationship between re- turn on assets and human capital efficiency. The ob- jective was to categorize technical efficiency based on accounting indicators. Results indicated that service sectors outperformed industrial ones due to a more efficient relationship between EBITDA and human capital efficiency. At a sectoral level, wholesale trade, retail, repair, information, communication, and ed- ucation sectors demonstrated higher technical effi- ciency, while construction, water-related activities, sanitation, waste management, remediation, accom- modation, and catering displayed lower efficiency. Sueyoshi and Goto (2019) highlighted the textile and clothing industry’s financial efficiency by con- sidering the interaction between operational and environmental outcomes. The industry in Japan exhibited high proficiency in resource alloca- tion and energy management, emphasizing the substantial potential for efficiency improvement through increased investments in innovation and emerging technologies. Industry efficiency assessment involves analyz- ing employee numbers, optimizing working hours through increased assets, evaluating unproductive machines, and adjusting raw material consump- tion (Sudri et al., 2019). Findings indicate variable technical efficiency among production units. Efficiency is characterized by the ability to mini- mize resource waste, achieving optimal results with the least amount of resources. Efficiency is measured by comparing what was produced to what could have been produced with the same re- sources (Mello et al., 2005). It mathematically rep- resents the relationship between outputs (desired objectives) and inputs (allocated resources), em- phasizing the optimal utilization of resources to attain specified goals. 1. LITERATURE REVIEW AND HYPOTHESES H1: Research and development (R&D) costs do not show a relationship with the return on assets for all financial years, with financial efficiency equal to one (eλ = 0). H1: Research and development (R&D) costs do not show a relationship with the return on assets for all financial years, with financial efficiency equal to one (eλ = 0). H2: Research and development (R&D) costs show some level of relationship with the return on assets for all financial years, with financial efficiency equal to one (0 > eλ < 1). For this study, efficiency varies between zero (inef- ficient) and one (efficient) and is obtained through the distance between the inefficient DMU and the efficient DMU (Shewell & Migiro, 2016), which translates into input surpluses or output deficits. The input-oriented model and efficiency are de- scribed as follows: H3: Research and development (R&D) costs show a perfect relationship with the return on as- sets for all financial years, with financial ef- ficiency equal to one (eλ = 1). 1 1 , s r r r m i i i U Y Efficiency V X = = = ∑ ∑ (1) (1) 1. LITERATURE REVIEW AND HYPOTHESES Popker and Raju (2020) explored the financial effi- ciency of the textile and clothing industries by ex- amining the link between financial performance 704 http://dx.doi.org/10.21511/ppm.22(1).2024.55 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 ted fabrics; I.5: Manufacture of made-up textile articles; I.6: Carpet and rug manufacturing; I.7: Manufacture of nonwovens and related products; I.8: Manufacture of nonwovens and related prod- ucts; I.9: Manufacture of textiles for technical and industrial use; I.10: Manufacture of other textiles; and II.1: Leather garment manufacturing; II.2: Making work clothes; II.3: Manufacture of other outerwear; II.4: Underwear manufacturing; II.5: Manufacture of other clothing articles and ac- cessories; II.6: Manufacture of fur articles; II.7: Manufacture of knitted and crocheted hosiery; II.8: Manufacture of other knitted and crocheted garments). The weights of the inputs (R&D spend- ing and gross value added) and their relationship with the outputs (operating return on assets, eco- nomic return on assets, and net return on assets) were calculated for each of the sectors. When analyzing efficiency, it is vital not to con- flate it with effectiveness (Mello et al., 2005). Effectiveness pertains to what is produced, dis- regarding the resources allocated. In essence, ef- ficiency optimizes resources for results, while ef- fectiveness concentrates on results without con- sidering the resources invested in achieving the objectives. Despite the small number of studies on the subject under analysis, this study aims to deepen the de- bate on innovation in the textile and clothing sec- tors and analyze the possible levels of efficiency of Portuguese companies, providing a new perspec- tive on the performance of investments and, above all, their profitability. The paper explores the effi- ciency of Portugal’s textile and clothing sectors from 2003 to 2022, focusing on the correlation between R&D spending and the profitability of companies’ assets. The following hypotheses are established: The study applied nonparametric DEA approach, and no explicit relationship was established be- tween inputs and outputs. The ability to transform inputs into outputs was measured as an efficiency index, with the subsector being more efficient (Ar & Baki, 2007) in a given year, the more outputs it obtains (results) with the fewest possible inputs (Sangeetha, 2020). H1: Research and development (R&D) costs do not show a relationship with the return on assets for all financial years, with financial efficiency equal to one (eλ = 0). http://dx.doi.org/10.21511/ppm.22(1).2024.55 2. METHODS where r U – weight of financial returns on assets; rY – the level of financial returns on assets; iV – weight of efficiency of research and development spending; i X – the level of efficiency of research and development spending; s – number of sec- tors related to financial returns on assets; and m – number of sectors related to efficiency of research and development spending. where r U – weight of financial returns on assets; rY – the level of financial returns on assets; iV – weight of efficiency of research and development spending; i X – the level of efficiency of research and development spending; s – number of sec- tors related to financial returns on assets; and m – number of sectors related to efficiency of research and development spending. This case study applies statistical data relating to the financial statements of companies in the tex- tile and clothing sector of Portugal. Data are avail- able on the official websites of the Bank of Portugal and the National Statistics Institute for 2003–2022. The data were organized into two sectors of eco- nomic activity (I: Textiles and II: Clothing) and then into eighteen subsectors (I.1: Preparation and spinning of textile fibers; I.2: Textile weaving; I.3: Textile finishing; I.4: Manufacture of knit- The procedure measures the efficiency of resource utilization (financial returns on assets and re- search and development expenditure) in each of 705 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 turns on assets. The results for 2003 to 2007, 2010, 2013 to 2016, and 2018 to 2022 indicated techni- cal inefficiency (0 > eλ < 1). However, in 2017, the relationship between R&D spending and return on assets was efficient, portraying a positive im- pact of R&D investments on technical efficiency (eλ = 1). the subsectors of the textile and clothing industry. The numerator signifies the weighted total finan- cial return on assets in all subsectors, while the de- nominator represents the weighted total efficiency of research and development expenditure in all subsectors. Efficiency is calculated by dividing the weighted total return on assets by the weighted total effi- ciency of research and development expenditures. 2. METHODS The results provide insights into how effectively resources are being used to generate financial re- turns in relation to the research and development expenditures of the various subsectors, taking in- to account the model’s constraints and limitations (Barbosa & Fuchigami, 2018). Moving on to textile finishing (DMU I.3), the results indicated an average efficiency level of 41.43%, signifying inefficiency. In specific years (2006, 2008, 2009, 2011, and 2012), the relation- ship between R&D spending and return on as- sets was zero (eλ = 0). Conversely, technical inef- ficiency was evident (0 > eλ < 1) for 2003 to 2005, 2007, 2010, 2013 to 2019, 2021, and 2022. However, in 2020, the data indicated an efficient relation- ship between R&D spending and return on assets (eλ = 1). The study calculations were carried out using Excel software. The DEA methodology was selected for analyzing the efficiency of the textile and clothing industry due to its flexibility in selecting inputs and outputs and model’s orientation. The research preference is to maintain inputs (level of expendi- ture on innovation) and increase outputs (results through the efficiency of net fixed applications). In the manufacturing of made-up textile arti- cles (DMU I.5), the average efficiency level was 45.46%, indicating inefficiency in allocating R&D spending in relation to the return provided by as- sets. In 2011 and 2012, the relationship between the return on assets and R&D spending was zero (eλ = 0), suggesting that R&D spending did not lead to improved levels of return on investment. For 2003 to 2010, 2013 to 2020, and 2022, ineffi- ciency was observed (0 > eλ < 1). However, for 2021, a technically efficient relationship between R&D spending and return on assets was observed (eλ = 1). 3. RESULTS For the underwear manufacturing sector (DMU II.4), the results showed an average technical ef- ficiency of 36.90%, indicating inefficiency. In 2009 and 2012, there was zero efficiency (eλ = 0), while in 2003–2008, 2010, 2011, and 2013–2020, there was inefficient technical efficiency between R&D spending and return on assets (0 > eλ < 1). However, the 2021 and 2022 results suggested that both periods were technically efficient (eλ = 1), designating these intervals as benchmarking periods where R&D spending contributed efficiently to the return on assets. For the manufacturing of other textile sectors (DMU I.10), the results indicated an average ef- ficiency level of 65.96%, portraying inefficiency. Between 2003 and 2016 and between 2018 and 2021, the relationship between R&D spending and the return on invested assets was technically inef- ficient (eλ < 1). Conversely, the results for the 2017 and 2018 economic periods suggested technical efficiency (eλ = 1). In the manufacturing of other articles and cloth- ing accessories (DMU II.5), the results indicated an average technical efficiency level of 28.82%, mak- ing it the fourth-most inefficient sector in terms of the relationship between R&D spending and re- turn on assets. 2008, 2012, and 2013 were, accord- ing to the model, years of zero efficiency (eλ = 0). In other words, there was no relationship between R&D spending and investment returns. 2003 to 2007, 2009 to 2011, and 2014 to 2021 correspond- ed to periods of technical inefficiency (0 > eλ < 1). 2022 was a reference period, i.e., the relationship between R&D spending and the return on assets was technically efficient (eλ = 1). Moving to leather garment manufacturing (DMU II.1), the data suggested an average level of tech- nical efficiency in the ratio of R&D spending to return on assets of 19.58%, indicating inefficiency. 2003–2010, 2013, 2019, 2021, and 2021 showed zero technical efficiency (eλ = 0). Conversely, for 2015 to 2018, the data suggested inefficient technical efficiency between R&D spending and return on assets (0 > eλ < 1). For 2016, 2017, and 2021, the re- lationship between R&D spending and return on assets was technically efficient (eλ = 1). For the manufacturing of fur articles (DMU II.6), an average technical efficiency of 29.87% was found, indicating inefficiency. 2003, 2007–2009, 2011, and 2022 were periods of zero efficiency (eλ = 0). 3. RESULTS The results shed light on the dynamic interplay be- tween R&D investments and operational perfor- mance across different economic periods (Table 1). For DMU I.1, encompassing textile fiber prepa- ration and spinning, the average participation of R&D spending in efficiency results was 14.36%. Notably, during certain economic periods (2003, 2006–2010, 2013, and 2019–2021), R&D spending exhibited negligible influence on operational, eco- nomic, and net returns on invested assets (eλ = 0). In contrast, 2015 and 2017 marked efficient periods, indicating a significant positive impact of R&D spending on overall efficiency (eλ = 1). Analyzing the manufacturing of rope and nets (DMU I.7), the data showed an average techni- cal efficiency of 54.98%, indicating inefficiency in the activity. In 2003, 2004, 2006 to 2008, and 2010 to 2019, technical inefficiency was observed (0 > eλ < 1). However, 2005, 2009, 2020, and 2022 were technically efficient (eλ = 1), signifying that R&D spending positively contributed to the efficiency of the sector’s return on assets. In the textile weaving sector (DMU I.2), the av- erage efficiency level was 31.22%, indicating inef- ficiency. Zero contribution of R&D spending to efficiency was observed in 2008, 2009, 2011, and 2012 (eλ = 0), suggesting that during these years, R&D spending did not significantly contribute to the sector’s operational, economic, and net re- In the manufacturing of non-woven fabrics and articles (DMU I.8), the average technical efficiency level was 61.31%, indicating inefficiency in allocat- ing R&D spending. In 2003, 2004, 2006 to 2014, 2016, 2018, 2019, and 2022, technical inefficiency was observed (0 > eλ < 1). Conversely, 2005, 2015, 2017, 2020, and 2021 were technically efficient 706 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 (eλ = 1), designating these periods as benchmark DMUs with an efficient relationship between R&D spending and return on invested assets. research and development (0 > eλ < 1). 2015, 2018, and 2022 corresponded to technically efficient intervals (eλ = 1), where the relationship between R&D spending and asset profitability was efficient. Moving to technical and industrial textile manu- facturing (DMU I.9), the average technical effi- ciency level was 74.81%, indicating inefficiency. 2003 and 2004, 2006 to 2014, 2016 and 2017, 2019, and 2022 were technically inefficient (0 > eλ < 1). However, 2005, 2015, and 2018 were technically efficient (eλ = 1), designating these years as benchmark periods. 3. RESULTS For 2007 to 2010, zero technical efficiency was found in the sector (eλ = 0). From 2003 to 2006, 2011 to 2015, and 2017 to 2021, the results indicated a relationship between R&D spending and return on assets that was inefficient (0 > eλ < 1). While 2016 and 2022 reflected periods of technical efficiency (eλ = 1), it was observed that R&D spending efficiently con- tributed to the return on assets. Certain sectors, including the manufacture of knit- ted fabrics (DMU I.4), textile finishing (DMU I.3), the manufacture of carpets and rugs (DMU I.6), and the manufacture of other knitted garments (DMU II.8), consistently maintained technical ef- ficiency over the 2003–2022 period. However, de- spite highlighting a degree of technical efficiency, these sectors remain heavily influenced by exter- nal factors such as market evolution, pricing dy- namics, and consumer preferences, underscoring the multifaceted nature of industry performance beyond innovation alone. This nuanced under- standing is crucial for industry stakeholders to navigate dynamic market landscapes and make informed strategic decisions. In the textile industry, the period with the highest level of technical efficiency was 2017 (82.76%), and the lowest was 2011 (15.47%). For the clothing in- dustry, the data indicated that the period with the highest level of technical efficiency in the alloca- tion of R&D spending was 2021 (73.11%), and the lowest efficiency was in 2010 (7.67%). The results on the average efficiency of the tex- tile and clothing industry sub-sectors (Table 2) show that hypothesis 2 is accepted (0 > eλ < 1), while hypotheses 1 (eλ = 0) and 3 are rejected (eλ = 1). The results suggest that there is some level of relationship between R&D costs and return on assets, i.e., they have contributed in some way to the efficiency of the return on as- sets of the sub-sectors of the Portuguese textile and clothing industry. The DEA methodology serves to rank DMUs by average efficiency levels. Table 2 presents the es- timated technical efficiency for each subsector, of- fering a clear classification based on performance. The DEA model analysis highlights the textile industry as a leader in technical efficiency, excel- ling in optimizing the correlation between R&D spending and returns on invested assets. Textile subsectors consistently demonstrate superior fi- nancial performance, evidenced by in high return on assets (ROA), return on equity (ROE), and re- turn on liabilities and assets (RLA). 3. RESULTS The economic years 2004 to 2006, 2010, 2012, 2013, and 2015 to 2020 were technically in- efficient periods (0 > eλ < 1), as the relationship between R&D spending and return on assets was inefficient. The years 2014 and 2021, on the oth- er hand, were technically efficient (eλ = 1) and represented reference periods. For the manufacturing of work clothes (DMU II.2), the data pointed to an average technical ef- ficiency of 61.77%, indicating inefficiency. 2003 to 2013, 2015 to 2017, 2019, 2021, and 2022 showed an inefficient relationship between R&D spend- ing and return on assets (0 > eλ < 1). Meanwhile, the financial years 2014, 2018, and 2020 were effi- cient periods (eλ = 1), designating these periods as benchmarking intervals in which R&D spending efficiently influenced the return on assets. For the manufacturing of knitted and crocheted hosiery (DMU II.7), the average technical efficien- cy of R&D spending was 23.77%, indicating an in- efficient sector. For 2003, 2006 to 2008, 2010, 2015, 2018 to 2020, zero technical efficiency was found (eλ = 0). In 2004, 2005, 2011, 2012, 2014, 2016, Analyzing the manufacturing of other outerwear (DMU II.3), the data suggested an average tech- nical efficiency of 63.19% (inefficient). In 2003 to 2014, 2016, 2017, and 2019 to 2021, there was tech- nical inefficiency in the allocation of spending on 707 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 of other textiles (DMU I.10), the manufacture of other outerwear (DMU II.3), and the manufacture of workwear (DMU II.2). Conversely, sectors with lower average technical efficiency levels encompass the manufacture of other wearing apparel (DMU II.5), knitted and crocheted hosiery (DMU II.7), leather goods (DMU II.1), and the preparation and spinning of textile fibers (DMU I.1). These sectors exhibit lower R&D expenditure levels, affecting returns on assets employed in production. 2017, 2021, and 2022, the results suggested periods of technical inefficiency (0 > eλ < 1). On the other hand, 2009 and 2013 reflected moments of techni- cal efficiency in the allocation of R&D spending in relation to the profitability of the sector’s assets (eλ = 1). For the manufacturing of other knitted garments (DMU II.8), the results suggested a level of techni- cal efficiency, on average, in the allocation of R&D spending for the purposes of return on assets of 37.25%, indicating inefficiency. 3. RESULTS This under- scores the industry’s prowess in leveraging inno- vation for financial success and positions it as a benchmark for effective resource allocation and operational efficiency. Throughout the series, it can be seen, by industry sub-sector and economic year (Table 1), that the efficiency results are variable, which allows the hypotheses to be accepted and rejected simulta- neously. In 2003, hypothesis 1 was accepted in 4 DMUs, while hypothesis 2 was accepted in 14 DMUs. Hypothesis 3 was rejected. In 2004, hy- pothesis 1 was accepted in 1 DMU, and hypoth- esis 2 was accepted in 17 DMUs. Hypothesis 3 was rejected for all DMUs. As for 2005, hypoth- esis 1 was accepted in 1 DMU, and hypothesis 2 was accepted in 14 DMUs. The hypothesis was accepted in 3 DMUs. In 2006, hypothesis 1 was Table 2 highlights key findings, revealing that ac- tivities with the highest average technical efficien- cy include the manufacture of textiles for techni- cal and industrial use (DMU I.9), the manufacture 708 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Average efficiency ranking for the textile and clothing industry for the period 2003–2022 Rank Economic activities Average 1 I.9-Manufacture of textiles for technical and industrial use 74.81% 2 I.10-Manufacture of other textiles 65.96% 3 II.3-Manufacture of other outerwear 63.19% 4 II.2-Making work clothes 61.77% 5 I.8-Manufacture of nonwovens and articles made from nonwovens 61.31% 6 I.7-Manufacture of rope and netting 54.98% 7 I.5-Manufacture of made-up textile articles 45.46% 8 I.4-Manufacture of knitted fabrics 43.35% 9 I.3-Textile finishing 41.43% 10 I.6-Carpet and rug manufacturing 37.74% 11 II.8-Manufacture of other knitted and crocheted garments 37.25% 12 II.4-Underwear manufacturing 36.90% 13 I.2-Textile weaving 31.22% 14 II.6-Manufacture of fur articles 29.87% 15 II.5-Manufacture of other clothing articles and accessories 28.82% 16 II.7-Manufacture of knitted and crocheted hosiery 23.77% 17 II.1-Leather garment manufacturing 19.58% 18 I.1-Preparation and spinning of textile fibers 14.36% Total average efficiency 42.88% erage efficiency ranking for the textile and clothing industry for the period 2003–2022 For 2018, hypothesis 1 was accepted in 1 DMU, hy- pothesis 2 was accepted in 14 DMUs, and hypothe- sis 3 was accepted in 3 DMUs. In 2019, hypothesis 1 was accepted in 3 DMUs, and hypothesis 2 was ac- cepted in 15 DMUs. Hypothesis 3 was rejected. In 2020, hypothesis 1 is accepted in 3 DMUs, hypoth- esis 2 was accepted in 11 DMUs, and hypothesis 3 in 4 DMUs. In 2021, hypothesis 1 was accepted in 1 DMU, hypothesis 2 in 10 DMUs, and hypothesis 3 in 7 DMUs. Finally, in 2022, hypothesis 1 was ac- cepted in 2 DMUs, hypothesis 2 was accepted in 8 DMUs, and hypothesis 3 was accepted in 8 DMUs. accepted in 4 DMUs, while hypothesis 2 was ac- cepted in 14 DMUs. Hypothesis 3 was rejected. In 2007, hypothesis 1 was accepted in 5 DMUs, and hypothesis 2 was accepted in 13 DMUs. Hypothesis 3 was rejected. As for 2008, hypothesis 1 was accepted in 9 DMUs, and hypothesis 2 was accepted in 9 DMUs. Hypothesis 3 was rejected. In 2009, hypothesis 1 was accepted in 8 DMUs, hypothesis 2 in 8 DMUs, and hypothesis 3 in 2 DMUs. In 2010, hypothesis 1 was accepted in 4 DMUs and hypothesis 2 in 14 DMUs. Hypothesis 3 was rejected. In 2011, hy- pothesis 1 was accepted in 6 DMUs, and hypoth- esis 2 was accepted in 12 DMUs. Hypothesis 3 was rejected. ge % % % % % % % % % % % % % % % % % % Years Average DMU 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 Textile industry 0.00% 0.00% 2.56% 5.36% 0.00% 23.30% 100.00% 7.49% 100.00% 13.06% 0.00% 0.00% 0.00% 30.24% 14.36% 0.00% 4.73% 0.00% 0.00% 25.20% 33.72% 34.73% 44.79% 100.00% 60.36% 42.66% 35.40% 75.41% 51.67% 31.22% 0.00% 13.68% 0.00% 0.00% 63.19% 82.63% 48.79% 71.94% 90.28% 53.50% 55.73% 100.00% 61.61% 34.91% 41.43% 5.70% 18.19% 20.70% 17.44% 55.86% 54.10% 62.43% 80.01% 79.12% 56.62% 44.20% 44.69% 100.00% 100.00% 43.35% 40.92% 35.26% 0.00% 0.00% 0.84% 22.10% 40.35% 44.43% 61.55% 45.43% 35.66% 38.12% 100.00% 92.85% 45.46% 0.00% 20.12% 0.00% 0.00% 17.71% 100.00% 44.83% 30.48% 67.19% 45.72% 76.76% 29.86% 72.04% 100.00% 37.74% 100.00% 49.16% 22.61% 15.40% 35.17% 42.04% 64.17% 54.51% 43.16% 21.00% 46.63% 100.00% 100.00% 100.00% 54.98% 10.99% 89.71% 41.25% 65.29% 15.53% 69.81% 100.00% 88.38% 100.00% 81.45% 45.86% 100.00% 100.00% 67.45% 61.31% 46.48% 67.90% 56.63% 46.20% 62.57% 88.08% 100.00% 97.60% 86.32% 100.00% 97.05% 72.30% 93.88% 84.22% 74.81% 42.46% 40.25% 10.96% 20.19% 58.60% 58.91% 59.57% 80.44% 100.00% 93.99% 95.99% 64.65% 78.76% 100.00% 65.96% 24.66% 33.90% 15.47% 16.99% 33.47% 57.47% 65.49% 60.01% 82.76% 57.11% 54.05% 58.50% 78.17% 76.13% – Clothing industry 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 15.12% 100.00% 100.00% 76.42% 0.00% 0.00% 100.00% 0.00% 19.58% 67.48% 26.34% 45.84% 26.69% 68.06% 100.00% 79.59% 76.88% 89.47% 100.00% 73.04% 100.00% 55.17% 66.54% 61.77% 18.99% 8.97% 26.24% 32.11% 71.93% 88.07% 100.00% 65.84% 86.83% 100.00% 68.81% 52.12% 74.80% 100.00% 63.19% 0.00% 15.73% 0.59% 0.00% 25.23% 27.47% 49.05% 37.26% 53.65% 68.78% 46.25% 42.86% 100.00% 100.00% 36.90% 10.27% 5.97% 6.35% 0.00% 0.00% 23.92% 32.19% 48.06% 51.38% 38.80% 40.35% 45.83% 57.79% 100.00% 28.82% 0.00% 4.37% 0.00% 12.17% 49.95% 100.00% 73.72% 20.01% 11.99% 75.91% 50.81% 44.35% 100.00% 0.00% 29.87% 100.00% 0.00% 24.79% 8.91% 100.00% 23.21% 0.00% 51.29% 9.31% 0.00% 0.00% 0.00% 59.81% 87.34% 23.77% 0.00% 0.00% 32.70% 3.56% 41.30% 60.48% 74.20% 100.00% 78.87% 90.51% 55.56% 2.02% 37.27% 100.00% 37.25% 24.59% 7.67% 17.06% 10.43% 44.56% 52.89% 52.98% 62.42% 60.19% 68.80% 41.85% 35.90% 73.11% 69.24% – nd clothing sector for the period 2003–2022 Years Average DMU 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 Textile industry 0.00% 0.00% 2.56% 5.36% 0.00% 23.30% 100.00% 7.49% 100.00% 13.06% 0.00% 0.00% 0.00% 30.24% 14.36% 0.00% 4.73% 0.00% 0.00% 25.20% 33.72% 34.73% 44.79% 100.00% 60.36% 42.66% 35.40% 75.41% 51.67% 31.22% 0.00% 13.68% 0.00% 0.00% 63.19% 82.63% 48.79% 71.94% 90.28% 53.50% 55.73% 100.00% 61.61% 34.91% 41.43% 5.70% 18.19% 20.70% 17.44% 55.86% 54.10% 62.43% 80.01% 79.12% 56.62% 44.20% 44.69% 100.00% 100.00% 43.35% 40.92% 35.26% 0.00% 0.00% 0.84% 22.10% 40.35% 44.43% 61.55% 45.43% 35.66% 38.12% 100.00% 92.85% 45.46% 0.00% 20.12% 0.00% 0.00% 17.71% 100.00% 44.83% 30.48% 67.19% 45.72% 76.76% 29.86% 72.04% 100.00% 37.74% 100.00% 49.16% 22.61% 15.40% 35.17% 42.04% 64.17% 54.51% 43.16% 21.00% 46.63% 100.00% 100.00% 100.00% 54.98% 10.99% 89.71% 41.25% 65.29% 15.53% 69.81% 100.00% 88.38% 100.00% 81.45% 45.86% 100.00% 100.00% 67.45% 61.31% 46.48% 67.90% 56.63% 46.20% 62.57% 88.08% 100.00% 97.60% 86.32% 100.00% 97.05% 72.30% 93.88% 84.22% 74.81% 42.46% 40.25% 10.96% 20.19% 58.60% 58.91% 59.57% 80.44% 100.00% 93.99% 95.99% 64.65% 78.76% 100.00% 65.96% 24.66% 33.90% 15.47% 16.99% 33.47% 57.47% 65.49% 60.01% 82.76% 57.11% 54.05% 58.50% 78.17% 76.13% – Clothing industry 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 15.12% 100.00% 100.00% 76.42% 0.00% 0.00% 100.00% 0.00% 19.58% 67.48% 26.34% 45.84% 26.69% 68.06% 100.00% 79.59% 76.88% 89.47% 100.00% 73.04% 100.00% 55.17% 66.54% 61.77% 18.99% 8.97% 26.24% 32.11% 71.93% 88.07% 100.00% 65.84% 86.83% 100.00% 68.81% 52.12% 74.80% 100.00% 63.19% 0.00% 15.73% 0.59% 0.00% 25.23% 27.47% 49.05% 37.26% 53.65% 68.78% 46.25% 42.86% 100.00% 100.00% 36.90% 10.27% 5.97% 6.35% 0.00% 0.00% 23.92% 32.19% 48.06% 51.38% 38.80% 40.35% 45.83% 57.79% 100.00% 28.82% 0.00% 4.37% 0.00% 12.17% 49.95% 100.00% 73.72% 20.01% 11.99% 75.91% 50.81% 44.35% 100.00% 0.00% 29.87% 100.00% 0.00% 24.79% 8.91% 100.00% 23.21% 0.00% 51.29% 9.31% 0.00% 0.00% 0.00% 59.81% 87.34% 23.77% 0.00% 0.00% 32.70% 3.56% 41.30% 60.48% 74.20% 100.00% 78.87% 90.51% 55.56% 2.02% 37.27% 100.00% 37.25% 24.59% 7.67% 17.06% 10.43% 44.56% 52.89% 52.98% 62.42% 60.19% 68.80% 41.85% 35.90% 73.11% 69.24% – Problems and Perspectives in Management, Volume 22, Issue 1, 2024 Table 2. In 2012, hypothesis 1 was accepted in 7 DMUs and hypothesis 2 in 11 DMUs. Hypothesis 3 is rejected. This analysis provides a detailed overview of the technical efficiency levels within various subsec- tors of the textile and clothing industries, empha- sizing the relationship between R&D spending and return on assets. The findings reveal fluctu- ating efficiency levels across different economic periods, highlighting areas of inefficiency and opportunities for improvement. Understanding these dynamics is crucial for industry stakehold- ers, policymakers, and investigators to develop targeted strategies, enhance overall industry per- formance, and foster sustainable growth. In 2013, hypothesis 1 was accepted in 3 DMUs, hypothesis 2 in 14 DMUs, and hypothesis 3 in 1. In 2014, hypothesis 1 was accepted in 1 DMU, hypothesis 2 was accepted in 14 DMUs, and hy- pothesis 3 was accepted in 3 DMUs. In 2015, hy- pothesis 1 was accepted in 1 DMU, hypothesis 2 in 13 DMUs, and hypothesis 3 in 4 DMUs. For 2016, hypothesis 1 was rejected, hypothesis 2 was accepted in 16 DMUs, and hypothesis 3 was accepted in 2 DMUs. For 2017, hypothesis 1 was rejected, hypothesis 2 was accepted in 13 DMUs, and hypothesis 3 was accepted in 5 DMUs. http://dx.doi.org/10.21511/ppm.22(1).2024.55 4. DISCUSSION The Portuguese textile and clothing industry is a crucial sector of the national economy, integrat- ing tradition, artisanship, and innovation. Over 710 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 the years, the sector has faced a number of chal- lenges, including international competition, the need to modernize production technology, and changing consumer preferences. The results of the study attest to the principle that Portuguese companies face vari- ous challenges and are in line with the conclusions of the aforementioned studies, especially as they pro- vide a perspective on the efficiency and performance of the textile and clothing industry in Portugal. novation and modernization (CIP, 2017). For Darji and Dahiya (2023), the textile and clothing sector has always shown low levels of modernization and efficiency regardless of the model adopted. The re- sults of this study show that the financial efficiency of the Portuguese textile and clothing industry is clearly low, which means that it needs to invest heav- ily in order to modernize. In addition, the industry needs to adopt innovative procedures and research methodologies to acquire new knowledge. As Darji and Dahiya (2023) discovered, the existing techno- logical levels in the textile sector represent challenges for achieving better efficiency results. The results indicate the efficiency levels of the textile and clothing subsectors in a new international con- text. Due to their low efficiency, it is essential to un- derstand and increase the financial efficiency levels of companies. The imperative is to maintain com- petitiveness and, at the same time, give new impetus to the sector’s growth. Like Jajri and Ismail (2006), the results attest to the low financial efficiency of the Portuguese industry. On the other hand, the results also align with Khalil’s (2011) observations, which emphasize the inefficiency of production units and the need to adopt innovative and efficient produc- tion tools. One of the sector’s problems stems from the need to increase the current levels of efficiency of research and development (R&D) spending in relation to the profitability of investments. According to the doc- trine, industrial development is truly related to the ability of companies to adopt new, more efficient production technologies that foster the competi- tiveness of manufacturing companies and increase financial results (ATP, 2019). According to the es- timated results, which are in line with the conclu- sions of Fang et al. http://dx.doi.org/10.21511/ppm.22(1).2024.55 4. DISCUSSION As a result, like other countries (Nassif, 2023), the Portuguese industry was forced into sudden technological in- Currently, financial efficiency levels cannot and should not be limited to economic agents’ ability to increase tax efficiency levels through local tax bene- fits (Soares & Pinheiro, 2023); efficiency must also be 711 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 be extended to other activities to understand the sit- uation of economic agents, since they are not isolated in the marketplace. measured in terms of investment decisions, wheth- er in assets or research and development (R&D). In line with Fahmy-Abdullah et al. (2018) and Du et al. (2022), the levels of R&D spending on financial efficiency are low and companies should promote a closer relationship between production and research. Policymakers need to encourage companies to invest in innovation in order to improve efficiency and in- crease the return on invested assets. A pertinent future research question involves analyz- ing the impact of efficiency on the circular economy and sustainability in line with current political agen- das. To this end, it is necessary to analyze the effi- ciency of circular models and strategies for extend- ing the lifespan of products. The assessment can cov- er the manufacturing, distribution, sales, consump- tion, and waste disposal phases, emphasizing the need for efficiency throughout the life cycle of textile and clothing products. In addition, it is possible to extend efficiency analysis to production methods us- ing renewable energies and ecological processes for the sustainable manufacture of textiles. As far as the hypotheses are concerned, the results mainly point to the validation of hypothesis 2, since all subsectors of the textile and clothing industry do not exhibit a relationship between the efficiency of R&D spending and return on assets for all years of technical efficiency equal to one. However, the rela- tionship between innovation and efficiency should 4. DISCUSSION (2013) and Li and Tsai (2018), there is a positive correlation between increased innovation and the financial efficiency of the pro- duction process. On the other hand, there is greater financial efficiency in the textile sector compared to the clothing sector. The higher the quality of re- search investments, the higher the levels of finan- cial efficiency produced. Research spending in the manufacturing industry has been the subject of much debate in Portugal in recent years. Academia recognizes the importance of the topic and the need to assess and discuss the levels of gains made by the various economic sectors (Rai, 2013). However, the results reveal the presence of low levels of investment in research and development, re- flected in low levels of efficiency and operational per- formance. The data are consistent with Rakhmawan et al. (2012), who investigated the effectiveness of performance indicators and how they affected pro- ductivity in the textile industry. In the Portuguese context, the level of financial efficiency of allocated assets varies between 14.36 and 74.81%, depending on the subsector of the textile and clothing industry, while the study indicated the efficiency of the textile industry was 40.0%. The technological modernization of the Portuguese textile and clothing industry requires new invest- ments in strategic assets, such as the purchase of pat- ents and intellectual property (WIPO, 2005) and ob- taining new competitive advantages (Proença, 2018). One of the challenges facing the textile and clothing subsectors is that it is difficult for companies to keep up with technology. Since some of the subsectors analyzed have zero efficiency levels, this underlines the economic imperative of increasing efficiency lev- els in allocating available resources, both financial- ly and in terms of sustainability, as highlighted by Sueyoshi and Goto (2019). The theory goes that Portuguese industrialization was late (Fortuna, 1993) and was mainly based on foreign models (Marques, 2023). However, although the adoption of foreign models led to the so-called “economic miracle,” the results were temporary, un- stable (Gonçalves & Caraça, 1986), and based on con- tinued energy dependence (Neto, 1991). CONCLUSION The aim of this study is to analyze the financial efficiency of the textile and clothing industry in Portugal, examining the performance from 2003 to 2022 and the correlation between spending on research and development (R&D) and return on assets. The results reveal a notable disparity in financial efficiency between industry subsectors (14.36% to 74.81%), emphasizing the need for innovative strategies. In Portugal’s textile and clothing sectors, inno- vation, research, and modernization are essential for increasing efficiency. The results show that there is a positive correlation between levels of investment in R&D and increased productivity. The study highlights the challenges the Portuguese textile sector faces, including low financial efficiency and obsolete assets. However, it identifies opportunities through the adoption of innovative production methods. Understanding the impact of financial efficiency on the circular economy is vital for sustain- ability. Increasing efficiency throughout the life cycles of textile products is crucial for sustainable prac- tices. The conclusions serve as a basis for formulating policies to promote innovation in the sector. The study reached several conclusions. The financial efficiency of the textile and clothing industry sectors varies, and the data point to different levels of financial efficiency. The industry subsectors have zero, mod- erate, and high levels of financial efficiency, and it is possible to define benchmarking periods. The clothing industry sector has higher levels of financial inefficiency compared to the textile sector. A low ratio of R&D expenditure to return on assets is observed in all sectors. The textile industry tends to be more technically efficient than the clothing industry. Finally, a more efficiency-centered view of the clothing industry sub- sectors is needed to achieve gains in the ratio of R&D expenditure to return on assets. The study has some limitations, including its reliance on historical data, which potentially does not fully rep- resent the current situation in the sector. The efficiency assessment was carried out based on specific indica- tors, and the use of alternative metrics could provide more information. In addition, the study only analyzes financial efficiency, neglecting crucial aspects such as operational efficiency and product quality. Despite its limitations, the study provides a broad overview of the financial efficiency levels of the textile and clothing industry in Portugal, highlighting the need to improve the industry’s performance. 712 http://dx.doi.org/10.21511/ppm.22(1).2024.55 Problems and Perspectives in Management, Volume 22, Issue 1, 2024 AUTHOR CONTRIBUTIONS Conceptualization: Ricardo de Moraes e Soares. Conceptualization: Ricardo de Moraes e Soares. p Formal analysis: Ricardo de Moraes e Soares. Formal analysis: Ricardo de Moraes e Soares. Investigation: Ricardo de Moraes e Soares. Investigation: Ricardo de Moraes e Soares. Investigation: Ricardo de Moraes e Soares. Investigation: Ricardo de Moraes e Soares. Methodology: Ricardo de Moraes e Soares, Alexandre Morais Nunes. oject administration: Ricardo de Moraes e Soares Project administration: Ricardo de Moraes e Soares. j Resources: Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro, Vanda Martins. S ft Ri d d M S j Resources: Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro, Vanda Martins. Software: Ricardo de Moraes e Soares. Resources: Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro, Vanda Martins. Software: Ricardo de Moraes e Soares. t Supervision: Ricardo de Moraes e Soares. Supervision: Ricardo de Moraes e Soares. Visualization: Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro, Vanda Martins. Writing – original draft: Ricardo de Moraes e Soares. Visualization: Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro, Vanda Martins. Writing – original draft: Ricardo de Moraes e Soares. Writing – review & editing: Ricardo de Moraes e Soares, Alexandre Morais Nunes, Paula Heliodoro, Vanda Martins. Vanda Martins. This article is financed by Instituto Politécnico de Setúbal [Polytechnic Institute of Setúbal]. This article is financed by Instituto Politécnico de Setúbal [Polytechnic Institute of Setúbal]. REFERENCES Retrieved from https://hdl.handle. net/11441/146165 158). Office for Strategy and Stud- ies of the Ministry of Economy and Digital Transition. Retrieved from https://www.gee.gov.pt/ RePEc/WorkingPapers/GEE_PA- PERS_158.pdf 1. Ar, I. M., & Baki, B. (2007). Mea- suring and evaluating efficiency of a glass company through data en- velopment analysis. Problems and Perspectives in Management, 5(1), 72-81. Retrieved from https:// www.businessperspectives.org/ images/pdf/applications/publish- ing/templates/article/assets/567/ PPM_EN_2007_01_Ar.pdf 8. Darji, I., & Dahiya, S. (2023). 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Economies, 11(11), Article 281. https://doi.org/10.3390/econo- mies11110281 REFERENCES Comércio internacional e crescimento a longo prazo: O contexto mundial, notas sobre Portugal [International trade and long-term growth: The global context, notes on Portugal]. Alme- dina. (In Portuguese). Retrieved from https://www.almedina. net/comercio-internacional-e- crescimento-a-longo-prazo-o- contexto-mundial-notas-sobre- portugal-1675161933.html 15. Fortuna, C. (1993). O fio da meada: A algodão de Moçam- bique, Portugal e a economia mundo, 1860–1960 [The thread: Mozambique cotton, Portugal and the world economy, 1860–1960]. Edições Afrontamento. (In Por- tuguese). Retrieved from https:// www.ces.uc.pt/iframe/publi- cacoes/csis/03.php 28. Neto, A. M. (1991). Industrial- ização de Angola: Reflexão sobre a experiência da administração portuguesa, 1961–1975 [Industri- alization in Angola: A reflection on the experience of the portu- guese administration, 1961–1975]. Escher. (In Portuguese). 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https://openalex.org/W1994145858	https://europepmc.org/articles/pmc4346795?pdf=render	English	null	Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases	Scientific reports	2,015	cc-by	8,222	OPEN Received 28 November 2014 Accepted 23 January 2015 Published 3 March 2015 1Institute of Plant Biology and Biotechnology, Westphalian Wilhelm’s-University Mu¨nster, Schlossplatz 8, 48143 Mu¨nster, Germany, 2Laboratory of Biochemistry, Institut Quı´mic de Sarria` (IQS), Universitat Ramon Llull (URL), Via Augusta 390, E-08017 Barcelona, Spain, 3Bio Base Europe Pilot Plant, Rodenhuizekaai 1, 9042 Ghent, Belgium. Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin d l h d f h h l d f h l h l d Correspondence and requests for materials should be addressed to S.K. (S.Kolkenbrock@ evocatal.com) y g g deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture. C C hitin, a b-(1, 4)-linked polymer composed of N-acetyl-D-glucosamine (GlcNAc, A) residues, is one of the most abundant biopolymers on earth, occurring e.g. in the exoskeleton of crustaceans and insects, and in the cell walls of fungi. Chitosan is the completely or partially deaceylated form of chitin, thus being composed of GlcNAc as well as D-glucosamine (GlcN, D) units. Chitosan polymers as well as oligomers of chitin and chitosan show diverse biological activities and, therefore, have a broad range of different potential applica- tions in various fields, including medicine, cosmetics, food, and agriculture1–4. * Current address: evocatal GmbH, Alfred-Nobel-Str. 10, 40789 Monheim am Rhein. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases Stefanie Nicole Hamer1, Stefan Cord-Landwehr1, Xevi Biarne´s2, Antoni Planas2, Hendrik Waegeman3, Bruno Maria Moerschbacher1 & Stephan Kolkenbrock1* Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases Stefanie Nicole Hamer1, Stefan Cord-Landwehr1, Xevi Biarne´s2, Antoni Planas2, Hendrik Waegeman3, Correspondence and requests for materials should be addressed to S.K. (S.Kolkenbrock@ evocatal.com) Results and Discussion Isolation and analysis of Rhizobium sp. GRH2 nodB. We designed consensus-degenerate hybrid oligonucleotide primers (CODEHOP)24 based on the sequences of multiple nodB genes from different Rhizobium strains, and used these to identify and subsequently clone the chitin deacetylase gene from Rhizobium sp. GRH2. The analysis of 16S rDNA sequences by BLASTn revealed that Rhizo- bium sp. GRH2 is most closely related to Rhizobium leguminosarum bv. trifolii (99% identity). Enzymatic sequencing23 in combination with UHPLC-ELSD-ESI- MS analysis showed that recombinant NodB deacetylated the first unit from the non-reducing end generating DA1–5 chitosan-oligo- mers (Figure 2), which is in agreement with previous reports on recombinant NodB, refolded from the pellet fraction20. Likewise, the same sequencing methodology showed that COD deacetylated the second unit from the non-reducing end generating ADA1–3 chit- osan-oligomers, in agreement with previous reports18. Production of enzymes in E. coli and characterization of purified enzymes. The GRH2 nodB gene was expressed in E. coli BL21 (DE3), whereas the cod gene was expressed in E. coli Rosetta 2 (DE3) [pLysSRARE2] essentially as previously reported23,25. Both genes contained a downstream located Strep-tag II encoding sequence for the purification by streptactin affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis revealed single bands for both enzymes, with apparent molecular masses close to the expected ones of 24.4 and 45.5 kDa for NodB and COD, respectively (Fig. 1). Production and analysis of defined chitosan oligomers. To test whether the chitin oligosaccharide deacetylases NodB and COD are also active on partially deacetylated chitosan oligomers – and whether the enzymes can be used in combination to broaden the spectrum of defined chitosan oligomers with a specific PA - we deacetylated GlcNAc5 with NodB, removed NodB, and used the mono-deacetylated chitosan pentamer as a substrate for COD. The same was done in reverse order, and in a combined reaction of NodB and COD together. In all cases, GlcNAc5 was completely converted into doubly deacetylated chitosan pentamers (Figure 3). The PA of the doubly deacetylated chitosan pentamer was determined using enzymatic/mass spectrometric sequencing which revealed that the first two units from the non-reducing end were deacetylated in the combined as well as in both sequential reactions of NodB and COD (Figure 4). In addition of GlcNAc5, also GlcNAc2–4 and GlcNAc6 were converted into the respective doubly deacetylated chitosan oligomers in a combined reaction of NodB and COD (Table 2). OPEN A fully controlled method potentially leading to a broad range of fully defined products is chemical syn- thesis of chitosan oligomers from monomeric building blocks. However, this attempt is time and labour intensive and the yields are rather low14,15. Alternatively, chitin oligomers can be efficiently converted into defined chitosan oligomers by the help of specific chitin deacetylases (EC 3.5.1.41) and chitin oligosaccharide deacety- lases (EC 3.5.1.-)9,16. A number of different chitin deacetylases from different sources have been described17–22. Among these, two highly interesting candidates for the production of fully defined chitosan oligomers are the highly specific chitin oligosaccharide deacetylases NodB from Rhizobium spp. and COD from Vibrio cholerae. NodB deacetylates exclusively the GlcNAc unit at the non-reducing end, whereas COD deacetylates the second unit from the non-reducing end18,20,23. The limitation of this technique lies in the rather low number of recombinant chitin deacetylases available today with a known and fully defined mode of action, leading to a very limited number of defined chitosan oligosaccharides that can be obtained in this way. Figure 1 \| SDS-PAGE and corresponding western blot of recombinant NodB from Rhizobium sp. GRH2 and COD from Vibrio cholerae purified from the crude extract of E. coli BL21 (DE3) [pET- 22b(1)::nodB_StrepIIC] and E. coli Rosetta 2 (DE3) [pLysSRARE2, pET- 22b(1)::cod_StrepIIC], respectively. The enzymes were purified by streptactin affinity chromatography and 3 mg of each enzyme was applied to the gel. Enzymes were visualized either by staining with ethyl violet/ zincon (a) or by enhanced chemiluminescence using HRP-coupled streptactin conjugate after western blotting (b). M: peqGOLD Protein- Marker II (Peqlab, Erlangen, Germany). Figure 1 \| SDS-PAGE and corresponding western blot of recombinant NodB from Rhizobium sp. GRH2 and COD from Vibrio cholerae To at least partially overcome this limitation, we have expressed cod from V. cholerae as well as nodB from Rhizobium sp. GRH2 in Escherichia coli and purified the recombinant enzymes. Starting with chitin oligomers of defined DP (An), we used the single enzymes for the in vitro generation of two different, fully defined mono-deacety- lated chitosan oligomers (DA(n-1) and ADA(n-2)). As a general agree- ment the first letter of the oligomers mentioned here represents the residue at the non-reducing end, while the last one represents that of the reducing end. OPEN g g Although chitin occurs in a wide variety of species, most of today’s commercially available chitin and its derivatives are produced from shrimp or crab shell waste through a combination of chemo-physical treatments5. These chitosans can be produced with different degrees of polymerization (DP) and different degrees of acetyla- tion (DA), but their pattern of acetylation (PA) is invariably random6. As chitin polymers are insoluble in aqueous solutions and chitosan polymers are soluble only at slightly acidic pH values, interest has shifted lately towards the more soluble chitin and chitosan oligomers2. Biological activities of chitosan oligomers have been shown or are hypothesized to be strongly dependent on the DP, DA, and PA7–9. To verify this hypothesis, defined chitosan oligomers with fully known architecture are needed. Production of chitosan oligomers by partial chemical or physical depolymerisation of the respective polymers, as typically done today, has severe disadvantages. Not only does the production of the oligomers typically involve harsh thermo-chemical treatments or strong physical forces, which may be environmentally unfriendly and/or partially destructive to the oligosaccharides produced, but also the production is highly difficult to control leading to broad heterogeneous mixtures, and the outcome is strongly dependent on the chemical and physical characteristics of the starting material10. Partial enzymatic hydrolysis of chitosan polymers using chitosan hydrolysing enzymes such as chitinases or chitosanases with well- defined cleavage specificities has been proposed as an alternative to chemical or physical depolymerisation11–13. But, this attempt is also strongly dependent on the starting material and it, too, leads to the production of SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 1 www.nature.com/scientificreports Figure 1 \| SDS-PAGE and corresponding western blot of recombinant NodB from Rhizobium sp. GRH2 and COD from Vibrio cholerae purified from the crude extract of E. coli BL21 (DE3) [pET- 22b(1)::nodB_StrepIIC] and E. coli Rosetta 2 (DE3) [pLysSRARE2, pET- 22b(1)::cod_StrepIIC], respectively. The enzymes were purified by streptactin affinity chromatography and 3 mg of each enzyme was applied to the gel. Enzymes were visualized either by staining with ethyl violet/ zincon (a) or by enhanced chemiluminescence using HRP-coupled streptactin conjugate after western blotting (b). M: peqGOLD Protein- Marker II (Peqlab, Erlangen, Germany). heterogeneous mixtures of chitosan oligomers. Still, due to the cleav- age specificities of the enzymes, the resulting mixture will be better defined than the chitosan oligomer mixtures obtained by chemical or physical depolymerisation2,12. OPEN As a proof-of-principle study, we then combined the two deacetylases in a single reaction and generated the expected doubly deacetylated oligomers (DDA(n-2)), showing that this approach opens a way to produce novel chitosan oligomers with hitherto unre- ported PA. tested COD with GlcNAc1–6 under the same conditions and analysed the hydrolysis products in the same way as we did for NodB. As expected, COD converted GlcNAc2–6 completely into mono-deace- tylated chitosan oligomers, but not GlcNAc1 (Table 1). The optimal pH for NodB was 9 and the optimal hydrolysis temperature was 37uC (Supplementary Fig. S1 online) as tested with GlcNAc5. According to literature, the optimal pH of COD is 8 and the optimal hydrolysis temperature is 45uC18. For COD no unspecific hydrolysis products were detected after prelonged incubation, as opposed to NodB, where negligible amounts of double deacetylated chitosan oligomers were detected after prolonged incubation at high enzyme concentrations. Results and Discussion y g The chitin deacetylase activity of NodB was tested against GlcNAc1–6. The hydrolysis products were characterized by ultra high performance liquid chromatography - evaporative light scattering detection - electrospray ionization - mass spectrometry (UHPLC- ELSD-ESI-MS), revealing that NodB was not active towards GlcNAc1, but converted GlcNAc2–6 completely into mono-deacety- lated chitosan oligomers (Table 1). To our knowledge, this is the first report of obtaining enzymatically active, recombinant NodB without the need of refolding it from insoluble inclusion bodies20. COD from V. cholerae is active on short chitin oligosaccharides18,25, and we SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 2 Table 1 \| Determination of substrate specificity of NodB and COD by UHPLC-ELSD-ESI-MS. GlcNAc1–6 (1 mM) was incubated with 2.5 mM NodB or COD in ammonium formate buffer (pH 9) or ammonium hydrogen carbonate buffer (pH 8), respectively, at 37uC overnight Table 1 \| Determination of substrate specificity of NodB and COD by UHPLC-ELSD-ESI-MS. GlcNAc1–6 (1 mM) was incubated with 2.5 mM NodB or COD in ammonium formate buffer (pH 9) or ammonium hydrogen carbonate buffer (pH 8), respectively, at 37uC overnight Subst. expected products determined products NodB determined products COD Mw A1–6 Mw A0–5D1 m/z Mw m/z Mw A1 221.09 179.08 A1 (465.14 [2M 1 Na]1) 221.07 A1 (465.14 [2M 1 Na]1) 221.07 A2 424.17 382.16 A1D1 (383.16 [M 1 H]1) 382.16 A1D1 (383.14 [M 1 H]1) 382.16 A3 627.25 585.24 A2D1 (586.24 [M 1 H]1) 585.24 A2D1 (586.23 [M 1 H]1) 585.23 A4 830.33 788.32 A3D1 (789.35 [M 1 H]1) 788.35 A3D1 (789.35 [M 1 H]1) 788.35 A5 1033.41 991.40 A4D1 (496.70 [M 1 H]21) 991.40 A4D1 (496.69 [M 1 H]21) 991.38 A6 1236.49 1194.48 A5D1 (598.24 [M 1 H]21) 1194.48 A5D1 (598.23 [M 1 H]21) 1194.48 trate specificity of NodB and COD by UHPLC-ELSD-ESI-MS. GlcNAc1–6 (1 mM) was incubated with 2.5 mM ormate buffer (pH 9) or ammonium hydrogen carbonate buffer (pH 8), respectively, at 37uC overnight complex with GlcNAc2, and GlcNAc3 25 were used as reference struc- tures (PDB accession codes: 4NZ1 and 4OUI respectively). The dimeric and trimeric substrates where manually converted to all In addition to the in vitro experiments, binding of partially dea- cetylated chitosan dimers and trimers to COD, was examined in silico. The recently solved three dimensional structures of COD in mination of the pattern of acetylation (PA) of GlcNAc5 (A5) after hydrolysis with NodB from Rhizobium sp. Results and Discussion The same was done vice versa: GlcNAc5 (A5) was deacetylated with COD (c) in the first step, and the enzyme was then replaced by NodB (d). Furthermore, NodB and COD were combined in a single reaction leading to a double-deacetylated chitosan pentamer in one reaction (e). All reactions were carried out in ammonium hydrogen carbonate buffer (pH 8) at 37uC for 2 h. possible deacetylated compounds (i.e., DA, AD, DD, DAA, ADA, AAD, DDA, DAD, ADD, DDD). Docking calculations were done with flexible ligands inside a search-space grid-box that covers the whole active site. Figure 5 shows the crystal structure of COD in complex with the disaccharide substrate AA, and the modelled structure of the complex with the NodB product DA, which properly binds to form a compet- ent complex. The closest amino acid residues to the 2-NAc or 2-NH2 in the glucosyl unit on the non-reducing end are Arg304 and Asn119. Removal of the acetyl group in the DA substrate does not alter sig- nificantly the interactions map where only one hydrogen-bond with Arg304 is lost. The binding affinity of all acetylated, partially deace- tylated, and fully deacetylated compounds to COD was calculated by means of the VINA scoring function26 (Table 3). Not surprisingly, the fully acetylated compounds are the ones with strongest binding affinity towards COD (29.4 kcal mol21 for AA, 210.6 kcal mol21 for AAA). On the other hand, the deacetylated compounds produced by COD (AD and ADA) loose affinity towards the enzyme (increase in energy of roughly 1 kcal mol21). Interestingly, the deacetylated com- pounds produced by NodB (DA and DAA) are able to bind COD with a stronger affinity than the COD originating products, but the inter- action energy to COD is still reduced by 0.8 to 0.4 kcal mol21 relative to the natural fully acetylated substrates. In other words, the contribution to the binding affinity of the N- acetyl substituent of the sugar ring at the non-reducing end is only 0.8 kcal mol21 for chitobiose in COD, in agreement with the lost of one hydrogen bond with Arg304 in the deacetylated substrate (Figure 5). This contribution is lower for chitotriose (just 0.5 kcal mol21). It is expected that this contribution will be even lower for longer oligosaccharides. Results and Discussion GRH2 by enzymatic mbination with UHPLC-ELSD-ESI-MS analysis. To determine the PA of the exclusively mono-deacetylated chitosan pentamer (A4D1) as dB (a), it was subsequently incubated with the GlmATK GlcNase from Thermococcus kodakaraensis KOD1. This enzyme exclusively s from the non-reducing end leading to GlcNAc4 (A4) and GlcN1 (D1) (b). In the next step GlmATK was replaced by the GlcNAcase illus subtilis 168, which exclusively removes terminal GlcNAc units, starting from the non-reducing end. The reaction yielded GlcNAc1 1) monomers (c). This approach revealed the pattern of acetylation of a chitin pentamer after incubation with NodB from Rhizobium sp. AA. RTS \| 5 : 8716 \| DOI: 10 1038/srep08716 3 Figure 2 \| Determination of the pattern of acetylation (PA) of GlcNAc5 (A5) after hydrolysis with NodB from Rhizobium sp. GRH2 by enzymatic sequencing in combination with UHPLC-ELSD-ESI-MS analysis. To determine the PA of the exclusively mono-deacetylated chitosan pentamer (A4D1) as generated by NodB (a), it was subsequently incubated with the GlmATK GlcNase from Thermococcus kodakaraensis KOD1. This enzyme exclusively cleaves GlcN units from the non-reducing end leading to GlcNAc4 (A4) and GlcN1 (D1) (b). In the next step GlmATK was replaced by the GlcNAcase BsNagZ from Bacillus subtilis 168, which exclusively removes terminal GlcNAc units, starting from the non-reducing end. The reaction yielded GlcNAc1 (A1) and GlcN1 (D1) monomers (c). This approach revealed the pattern of acetylation of a chitin pentamer after incubation with NodB from Rhizobium sp. GRH2 to be DAAAA. SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 3 www.nature.com/scientificreports Figure 3 \| UHPLC-ELSD-ESI-MS analysis of in vitro combinations of NodB and COD. The substrate GlcNAc5 (A5) was deacetylated with NodB (a). NodB was removed and the obtained mono-deacetylated chitosan pentamer (A4D1) was further deacetylated with COD (b). The same was done vice versa: GlcNAc5 (A5) was deacetylated with COD (c) in the first step, and the enzyme was then replaced by NodB (d). Furthermore, NodB and COD were combined in a single reaction leading to a double-deacetylated chitosan pentamer in one reaction (e). All reactions were carried out in ammonium hydrogen carbonate buffer (pH 8) at 37uC for 2 h. Figure 3 \| UHPLC-ELSD-ESI-MS analysis of in vitro combinations of NodB and COD. The substrate GlcNAc5 (A5) was deacetylated with NodB (a). NodB was removed and the obtained mono-deacetylated chitosan pentamer (A4D1) was further deacetylated with COD (b). Results and Discussion Thus, although COD is more active on AA and kcat decreases with increasing oligosaccharide length18,25, com- pounds such as chitotetraose or chitopentaose continue to be sub- strates of COD even if they are deacetylated at the non-reducing end because the loss of affinity due to the acetyl substituent of the sugar ring at the non-reducing end will presumably be negligible. possible deacetylated compounds (i.e., DA, AD, DD, DAA, ADA, AAD, DDA, DAD, ADD, DDD). Docking calculations were done with flexible ligands inside a search-space grid-box that covers the whole active site. Figure 5 shows the crystal structure of COD in complex with the disaccharide substrate AA, and the modelled structure of the complex with the NodB product DA, which properly binds to form a compet- ent complex. The closest amino acid residues to the 2-NAc or 2-NH2 in the glucosyl unit on the non-reducing end are Arg304 and Asn119. Removal of the acetyl group in the DA substrate does not alter sig- nificantly the interactions map where only one hydrogen-bond with Arg304 is lost. The binding affinity of all acetylated, partially deace- tylated, and fully deacetylated compounds to COD was calculated by means of the VINA scoring function26 (Table 3). Not surprisingly, the fully acetylated compounds are the ones with strongest binding affinity towards COD (29.4 kcal mol21 for AA, 210.6 kcal mol21 for AAA). On the other hand, the deacetylated compounds produced by COD (AD and ADA) loose affinity towards the enzyme (increase in energy of roughly 1 kcal mol21). Interestingly, the deacetylated com- pounds produced by NodB (DA and DAA) are able to bind COD with a stronger affinity than the COD originating products, but the inter- action energy to COD is still reduced by 0.8 to 0.4 kcal mol21 relative to the natural fully acetylated substrates. The fact that NodB and COD accept the products of each other offers new possibilities for the biotechnological production of defined chitosan oligomers. Assuming that also other chitin deace- tylases can accept partially deacetylated products as substrate, a rather large number of different fully defined chitosan oligomers with different PA could be produced by combining a rather limited number of different chitin deacetylases. Results and Discussion In order to produce sufficient amounts of specifically mono as well as doubly deacetylated chitosan oligomers for further down- stream bio-testing, we biotechnologically produced chitin pentamer SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 4 www.nature.com/scientificreports tic sequencing in combination with UHPLC-ELSD-ESI-MS analysis of double-deacetylated chitosan pentamer (A3D2). The product fter the incubation of GlcNAc5 (A5) with NodB and COD (a). To determine the PA, it was first hydrolysed with the GlcNase GlmATK, usively GlcN units from the non-reducing end. The reaction resulted in GlcN1 (D1) and GlcNAc3 (A3) (b). In the next step, GlmATK was NAcase BsNagZ, which exclusively removes GlcNAc units from the non-reducing end, resulting in GlcN and GlcNAc monomers equencing of the simultaneous hydrolysis product of NodB and COD revealed that the first two units starting from the non-reducing ted, giving a specific chitosan oligomer with the novel PA DDAAA. Figure 4 \| Enzymatic sequencing in combination with UHPLC-ELSD-ESI-MS analysis of double-deacetylated chitosan pentamer (A3D2). The product A3D2 was obtained after the incubation of GlcNAc5 (A5) with NodB and COD (a). To determine the PA, it was first hydrolysed with the GlcNase GlmATK, which removes exclusively GlcN units from the non-reducing end. The reaction resulted in GlcN1 (D1) and GlcNAc3 (A3) (b). In the next step, GlmATK was replaced by the GlcNAcase BsNagZ, which exclusively removes GlcNAc units from the non-reducing end, resulting in GlcN and GlcNAc monomers (c).The enzymatic sequencing of the simultaneous hydrolysis product of NodB and COD revealed that the first two units starting from the non-reducing end were deacetylated, giving a specific chitosan oligomer with the novel PA DDAAA. in E. coli, of which we then deacetylated 100 mg in a combined reaction using NodB and COD at mg-scale. The chitosan pentamers thus obtained were purified using size-exclusion chromatography (SEC), separating them from chitosan tetramers, which originated from a by-product of the recombinant production of chitin penta- mers in E. coli. The purified products were analysed using UHPLC- Table 2 \| Analysis of double deacetylation of GlcNAc2–6 by a combined action of NodB and COD, analysed using UHPLC-ELSD-ESI-MS. GlcNAc2–6 (1 mM) was incubated with 2.5 mM of both NodB and COD in ammonium hydrogen carbonate buffer (pH 8) at 37uC overnight Subst. Methods Bacterial strains, vectors and culture conditions. E. coli strains TOP10 (Invitrogen, Darmstadt, Germany) and DH5a27 were used for general cloning, whereas Rosetta 2 (DE3) [pLysSRARE2] and BL21 (DE3) (Novagen, Darmstadt, Germany) were used for protein expression. The vector pCRII-TOPO (Invitrogen) was used for cloning of PCR fragments, whereas pET-22b(1) (Novagen, Darmstadt, Germany) including a StrepII tag sequence upstream of the multiple cloning site (MCS)23 was used for cloning and expression. E. coli was grown on LB agar, in LB medium, or in auto- induction medium28. Media were supplemented with the appropriate antibiotics (34 mg ml21 chloramphenicol and/or 100 mg ml21 ampicillin). Plasmid construction. The nodB gene from Rhizobium sp. GRH2 was synthesized by GenScript (New York, USA) and inserted into the vector pET-22b(1)StrepIIC after digestion with NdeI and SacI. The construction of pET-22b(1)StrepIIC, pET- 22b(1)::cod_StrepIIC for overexpression of cod from V. cholerae (GenBank: AAF94439.1), and pET-22b(1)::glmATK_StrepIIC for overexpression of glmATK from T. kodakaraensis KOD1 (GenBank: BAD85943.1) was described earlier23. To improve the expression rate of nagZ from B. subtilis 168 (GenBank: CAB11942.1) in E. coli, the gene was codon optimized and synthesized by GenScript with a StrepII encoding sequence downstream of the gene and inserted into pET-22b(1)::StrepIIC after digestion with NdeI and SacI. Preparation of genomic DNA from Rhizobium sp. GRH2 and 16S rDNA analysis. Genomic DNA was extracted from Rhizobium sp. GRH2 as described by Rainey et al.29. The 16s rDNA was amplified with Phusion Hot Start II High Fidelity proof Table 3 \| Calculated binding affinities of partially and fully deace- tylated chitobiose and chitotriose to COD structure. * natural pro- ducts of COD reaction Chitobiose-like compound Binding Affinity (kcal mol21) Chitotriose-like compound Binding Affinity (kcal mol21) AA 29.4 AAA 210.6 DA 28.7 DAA 29.9 AD* 28.1 ADA* 29.5 DD 27.2 DDD 28.2 AAD 210.1 DDA 28.8 ADD 29.1 DAD 29.4 SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 Table 3 \| Calculated binding affinities of partially and fully deace- tylated chitobiose and chitotriose to COD structure. * natural pro- ducts of COD reaction Synthesis and purification of enzymes. E. coli BL21 (DE3) harbouring either pET- 22b(1)::nodB_StrepIIC or pET-22b(1)::nagZ_StrepIIN1C and E. coli Rosetta 2 (DE3) [pLysSRARE2] harbouring either pET-22b(1)::cod_StrepIIC or pET22- b(1)::glmATK_StrepIIC were cultivated in autoinduction medium. E. coli BL21 (DE3) [pET-22b(1)::nodB_StrepIIC] was cultivated at 37uC for 4 h and subsequently at 18uC for 40 h, whereas the other strains where incubated at 26uC for 48 h. Results and Discussion expected products determined products NodB 1 COD Mw A2–6 Mw A1–5D1 Mw A0–4D2 m/z Mw A2 424.17 382.16 340.15 A0D2 (341.16 [M 1 H]1) 340.15 A3 627.25 585.24 543.23 A1D2 (544.27 [M 1 H]1) 543.27 A4 830.33 788.32 746.31 A2D2 (747.35 [M 1 H]1) 746.35 A5 1033.41 991.40 949.39 A3D2 (475.73 [M 1 H]21) 949.46 A6 1236.49 1194.48 1152.47 A4D2 (577.28 [M 1 H]21) 1152.56 SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 5 Table 2 \| Analysis of double deacetylation of GlcNAc2–6 by a combined action of NodB and COD, analys GlcNAc2–6 (1 mM) was incubated with 2.5 mM of both NodB and COD in ammonium hydrogen carbonate acetylation of GlcNAc2–6 by a combined action of NodB and COD, analysed using UHPLC-ELSD-ESI-MS. ted with 2.5 mM of both NodB and COD in ammonium hydrogen carbonate buffer (pH 8) at 37uC overnight 5 Figure 5 \| Structures of AA and DA substrates in the active site of COD. A) X-ray structure of the complex COD?AA25. B) Modelled structure with the DA substrate. Ligands are shown as thick lines, metal ion as a green sphere. Only the amino acids interacting with the N-acetyl group at the non-reducing end are shown (as thick lines). The removal of this acetyl group does not alter significantly the interactions map at the active-site, where only one hydrogen-bond with R304 is lost. Figure 5 \| Structures of AA and DA substrates in the active site of COD. A) X-ray structure of the complex COD?AA25. B) Modelled structure with the DA substrate. Ligands are shown as thick lines, metal ion as a green sphere. Only the amino acids interacting with the N-acetyl group at the non-reducing end are shown (as thick lines). The removal of this acetyl group does not alter significantly the interactions map at the active-site, where only one hydrogen-bond with R304 is lost. reading DNA Polymerase (Fisher Scientific, Schwerte, Germany) using three different primer pair combinations (27F/1525R, 27F/926R and 16S-F/16S-Rev). All primer sequences are listed in Supplementary Table S1. Only 27F/926R yielded a product, which was purified, sequenced and analysed using BLASTn. ELSD-ESI-MS and proved to be highly pure (Figure 6). We obtained 27 mg of highly pure doubly deacetylated chitosan pentamer (DDAAA) and 4 mg of equally pure doubly deacetylated chitosan tetramer (DDAA). Identification of unknown nodB gene from GRH2 and plasmid construction. Results and Discussion Obtained PCR products were cloned in pCRII-TOPO for sequencing, followed by sequence analysis using BLASTx and alignment with CloneManager (Scientific & Educational Software, Cary, USA). Inverse PCR was used to isolate the upstream and downstream regions from genomic DNA. Total DNA was each digested with either SacI or HindIII and circularized with rapid T4 DNA ligase. The circulized DNA was used as template for a PCR with different inverse primer combinations. Amplified PCR products were ligated into pCRII-TOPO vector for sequencing. Promising sequences were aligned with CloneManager to generate a contig. The full-length nodB gene was isolated by standard PCR using a further CODEHOP primer (BC-for) designed for known nodB genes from different Rhizobium strains and specific primers that bind within the partially identified regions of the nodC gene from Rhizobium sp. GRH2, which is located upstream of nodB. In the last step the full length nodB gene was amplified by using specific primers and Phusion Hot Start II High Fidelity proof reading DNA Polymerase (Fisher Scientific) to exclude possible mutations, which may had occurred during the dif- ferent PCR amplification steps with the non-proof reading Mango-tag polymerase. Results and Discussion Consensus-degenerate hybrid oligonucleotide primers (CODEHOP) [24] were designed against known nodB genes from different Rhizobium strains as previously described. All primer sequences used during the study are listed in Supplementary Table S1. The genes were amplified using Mango-Taq DNA Polymerase (Bioline, Luckenwalde, Germany) and different combinations of CODEHOPs (B-for, D-rev, C-for, E-rev). Obtained PCR products were cloned in pCRII-TOPO for sequencing, followed by sequence analysis using BLASTx and alignment with CloneManager (Scientific & Educational Software, Cary, USA). Inverse PCR was used to isolate the upstream and downstream regions from genomic DNA. Total DNA was each digested with either SacI or HindIII and circularized with rapid T4 DNA ligase. The circulized DNA was used as template for a PCR with different inverse primer combinations. Amplified PCR products were ligated into pCRII-TOPO vector for sequencing. Promising sequences were aligned with CloneManager to generate a contig. The full-length nodB gene was isolated by standard PCR using a further CODEHOP primer (BC-for) designed for known nodB genes from different Rhizobium strains and specific primers that bind within the partially identified regions of the nodC gene from Rhizobium sp. GRH2, which is located upstream of nodB. In the last step the full length nodB gene was amplified by using specific primers and Phusion Hot Start II High Fidelity proof reading DNA Polymerase (Fisher Scientific) to exclude possible mutations, which may had occurred during the dif- ferent PCR amplification steps with the non-proof reading Mango-tag polymerase. As this method can be scaled up, it is feasible to produce fully defined chitosan oligomers in sufficient amounts for trial applica- tions in different fields such as cosmetics or bio-medicine. Evaluating the scientific and commercial potential of fully defined chitosan oligomers is not possible at the moment due to the lack of well- defined chitosan oligomers on the market. When the chitin oligo- mers used as a substrate are of biotechnological origin, as in this study, our method has the added advantage that the chitosan oligomers produced, can be guaranteed to be free of allergenic contaminants. Identification of unknown nodB gene from GRH2 and plasmid construction. Consensus-degenerate hybrid oligonucleotide primers (CODEHOP) [24] were designed against known nodB genes from different Rhizobium strains as previously described. All primer sequences used during the study are listed in Supplementary Table S1. The genes were amplified using Mango-Taq DNA Polymerase (Bioline, Luckenwalde, Germany) and different combinations of CODEHOPs (B-for, D-rev, C-for, E-rev). Methods Samples were eluted from the column with a gradient of A and B. Eluent A was composed of 80520 acetonitrile/water and eluent B of 20580 acetonitrile/water. Both eluents contained 10 mM NH4HCO2 and 0.1% (v/ v) formic acid. The separation of samples was done over 15 min using the following elution profile: 0–2.5 min, isocratic 100% A; 2.5–12.5 min, linear from 0% to 75% (v/v) B; followed by column re-equilibration: 12.5–13.5 min linear from 75% to 0% (v/v) B, isocratic 100% (v/v) A from 13.5–15 min. A 151 splitter (Accurate, Dionex Corporation, Sunnyvale, CA, USA) was used to split the eluent to the ELSD and to the ESI-MSn detector. The gas pressure of the ELSD HPLC nebulizer was set to 3.5 bar, the gas flow to 1.75 l min21, the evaporation temperature to 50uC and the gain to 12. MS- detection was performed in a positive mode with the capillary voltage set to 4 kV, the end plate offset voltage to 500 V, the pressure of the nebulizer to 15 psi, the flow rate of the dry gas to 8 l min21 and the dry temperature to 200uC. Mass spectra were The composition of the shake flask and minimal batch fermenter medium was the same as described by Waegeman et al.36. Fed-batch medium consisted of 500 g l21 glucose, 1 g l21 MgSO437H2O and 30 g l21 NH4Cl. All media were supplied with 0.1 g l21 ampicillin. The recombinant E. coli strain was pre-cultured in two shake flasks of 2 l total volume, filled with approximately 0.2 l shake flask medium and grown at 30uC while constantly shaking at 200 rpm. After 24 hours, 100 ml of broth was transferred into two shake flasks of 5 l total volume filled with 2 l shake flask medium at 30uC while constantly shaking at 200 rpm. After 24 hours the resulting fermentation broth (4 l in total) was inoculated into a 150 l fermenter (Sartorius, Go¨ttingen, Germany). Fermentation conditions were; tem- perature: 30uC, stirrer speed: minimum 500 rpm and if pO2 drops below 30% stepwise increased, aeration: 10 slpm, pH: 7, pressure: 500 mbar. Feeding was started when glucose was depleted in the batch phase and the feed rate applied was 4 l h21. The total fermentation time was 110 h. Methods coli enzyme, GlcNAc5 was in parallel incubated under the same conditions as describe above with 3 ml crude extract (4 mg ml21) of E. coli Rosetta2 (DE3) [pLysSRARE2] harbouring either pET22-b(1)::nodB_StrepIIC, pET22-b(1)::cod_StrepIIC or the empty vector control. The reaction volume was set to 20 ml. All products were analysed by UHPLC-ELSD-ESI-MS. Determination of pH and temperature optimum for NodB. The pH and temperature optima of NodB were determined indirectly, by measuring the amount of acetate released during the enzymatic reaction, using an acetic acid assay kit (R- bioharm AG, Darmstadt, Germany) adapted for microtiter plates. The pH optimum was determined over the pH range 4–12 at 37uC for 2 h, in buffer containing 100 mM NH4HCO3, 20 mM TEA, 20 mM KH2PO4 and 20 mM Na2HPO4. Teorell and Stenhagen buffer (100 mM citric acid, 100 mM phosphoric and 100 mM boric acid33) was used for high-pH conditions, overlapping at pH 10. The temperature optimum was determined at 4uC, 22uC, 37uC, 45uC and 55uC at pH 9 using the buffer and conditions described above. The reaction volume was set to 400 ml, comprising 50 mM buffer, 1 mM chitin pentamer (Megazyme, Bray, Ireland) and 0.5 mM purified NodB. The reactions were stopped and the amount of released acetic acid was measured directly. Determination of the PA of the generated chitosan oligomers. The PA of the different chitosan oligomers generated by NodB and COD, or by a combined action of both enzymes was determined by enzymatic sequencing23. Samples were analysed by UHPLC-ELSD-ESI-MS analysis instead of HPTLC. UHPLC-ELSD-ESI-MS analysis of chitin and chitosan oligomers. The applied UPLC-ELSD-ESI-MSn method was developed based on the methods described previously by Leijdekkers et al. and Remoroza et al.34,35. Analysis of chitin and chitosan oligomers were done using a Dionex Ultimate 3000RS UHPLC system (Thermo Scientific, Milford, USA) coupled to an evaporative light scattering detector (Model Sedex 90LT, Sedere, Alfortville Cedex, France) and an ESI-MSn-detector (amaZon speed, Bruker, Bremen, Germany). Chitin and chitosan oligomers were separated by hydrophilic interaction chromatography (HILIC) using an Acquity UPLC BEH Amide column (1.7 mm, 2.1 mm 3 150 mm; Waters Corporation, Milford, MA, USA) in combination with a VanGuard pre-column (1.7 mm, 2.1 mm 3 5 mm; Waters Corporation, Milford, USA). The flow rate was set to 0.4 ml min21 and the column oven temperature to 35uC. Aliquots of 1 ml of sample were injected into the system using an autosampler. Methods The enzymes were purified as described earlier23. The protein concentration was determined with PierceTM BCA Protein Assay Kit (Pierce Biotechnology, Rockford, USA). SDS-PAGE and western blot analysis. Protein samples were heated in SDS loading buffer (8% (w/v) SDS in 0.25 M Tris/HCl pH 6.8, 20% glycerol (v/v), 0.4% (w/v) bromophenol blue, 0.4% (v/v) b-mercaptoethanol) at 99uC for 10 min and separated SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 6 Figure 6 \| Production and UHPLC-ELSD-ESI-MS analysis of doubly deacetylated chitosan tetramers and pentamers at mg-scale. Doubly deacetylated chitosan tetramers (A2D2) and pentamers (A3D2) were produced by deacetylating biotechnologically produced chitin pentamer and tetramer with NodB and COD in a combined reaction overnight (a). The resulting doubly deacetylated chitosan oligomers A3D2 and A2D2 were purified and separated from each other using SEC (b). Fractions containing A3D2 (c) and A2D2 (d) were pooled separately and analysed using UHPLC-ELSD-ESI-MS. Figure 6 \| Production and UHPLC-ELSD-ESI-MS analysis of doubly deacetylated chitosan tetramers and pentamers at mg-scale. Doubly deacetylated chitosan tetramers (A2D2) and pentamers (A3D2) were produced by deacetylating biotechnologically produced chitin pentamer and tetramer with NodB and COD in a combined reaction overnight (a). The resulting doubly deacetylated chitosan oligomers A3D2 and A2D2 were purified and separated from each other using SEC (b). Fractions containing A3D2 (c) and A2D2 (d) were pooled separately and analysed using UHPLC-ELSD-ESI-MS. acquired over a scan range from m/z 50–2000 using enhanced resolution scan mode and were analysed using Data Analysis 4.1 software (Bruker, Bremen, Germany). Precision of the masses according to the instrument specifications are 60.15 m/z units. by SDS-PAGE in a 12% (w/v) gel30. Separated proteins were visualized with zincon/ ethyl violet staining31 or transferred to a nitrocellulose membrane (GE Healthcare Europe GmbH, Freiburg, Germany) using a semi-dry transfer procedure32. Strep-tag II fusion proteins were detected with Strep-Tactin-horseradish peroxidase (HRP) conjugate and chemiluminescent reaction was developed according to the instruction of the manufacturer (IBA GmbH, Go¨ttingen, Germany). Analysis of enzyme activity of NodB and COD. Substrate specificity of NodB and COD was tested towards GlcNAc1–6. GlcNAc1 was purchased from Sigma Aldrich (Mu¨nchen, Germany) whereas GlcNAc2–6 was purchased from Megazyme (Bray, Ireland). GlcNAc1–6 (1 mM) were incubated with either 2.5 mM NodB or 2.5 mM COD in 50 mM NH4HCO3 (pH 8) at 37uC overnight. To ensure that the hydrolysis activity was caused by the activity of NodB or COD and not by an endogenous E. Methods After fermentation, the broth was harvested and cells were separated from supernatant by ceramic tangential flow microfiltration (Tami Industries, Nyons, France, 0.45 mm). Both the cell and supernatant fraction contained the fully acetylated pentamer and were further purified. Cells were disrupted by a homogeniser (GEA Process Engineering, Mechelen, Belgium, 10 l h21) and cell debris was removed by tangential flow microfiltration (Kleenpak, Pall, Zaventem, Belgium, 0.45 mm). In both fractions, salts were removed by ion exchange (Amberlite, Dow, Tessenderlo, Belgium) and the solutions were further concentrated by wiped film evaporation (Carl Canzler, Germany, 150 l h21, 60uC, 70 mbar) and finally spray-dried (Xedev, Zelzate, Belgium). After purification, 371 g of product was obtained, which consisted for 85% of fully acetalyated pentamer (GlcNAc5). SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 7 www.nature.com/scientificreports Purified chitin pentamer (100 mg) was incubated with 0.2 mM NodB and 0.6 mM COD in 100 ml 50 mM NH4HCO3 buffer (pH 8) at 37uC overnight. The sample was freeze dried and solved in water before size exclusion chromatography (SEC). After SEC the sample was diluted with water and freeze dried. The obtained product was analysed using UHPLC-ELSD-ESI-MS. 18. Li, X., Wang, L.-X., Wang, X. & Roseman, S. The chitin catabolic cascade in the marine bacterium Vibrio cholerae: characterization of a unique chitin oligosaccharide deacetylase. Glycobiology 17, 1377–1387 (2007). 19. Zhao, Y., Park, R.-D. & Muzzarelli, R. A. A. Chitin deacetylases: properties and applications. Mar Drugs 8, 24–46 (2010). pp g 20. John, M., Ro¨hrig, H., Schmidt, J., Wieneke, U. & Schell, J. Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase. Proc Natl Acad Sci U S A 90, 625–629 (1993). Size-exclusion chromatography (SEC). Deacetylated chitosan oligomers were purified on three HiLoad 26/600 Superdex 30 pg columns (GE Healthcare Europe GmbH, Freiburg, Germany) in a row with an overall dimension of 2.60 3 180 cm. Ammonium acetate buffer (0.15 M, pH 4.5) was used as mobile phase and the flow rate was set to 0.8 ml min21. The effluent was monitored with an online refractive index detector (1260 Infinity Refractive Index Detector, Agilent Technologies Deutschland GmbH, Bo¨blingen, Germany) which was coupled to a datalogger13,37. Fractions containing oligomers were pooled and analysed by UHPLC-ELSD-ESI-MS. 21. Tokuyasu, K., Ono, H., Ohnishi-Kameyama, M., Hayashi, K. & Mori, Y. Deacetylation of chitin oligosaccharides of dp 2–4 by chitin deacetylase from Colletotrichum lindemuthianum. Methods Carbohydr Res 303, 353–358 (1997). 22. Tsigos, I., Zydowicz, N., Martinou, A., Domard, A. & Bouriotis, V. Mode of action of chitin deacetylase from Mucor rouxii on N-acetylchitooligosaccharides. Eur J Biochem 261, 698–705 (1999). 23. Hamer, S. N., Moerschbacher, B. M. & Kolkenbrock, S. Enzymatic sequencing of partially acetylated chitosan oligomers. Carbohydr Res 392, 16–20 (2014). Calculation of relative binding affinities of partially deacetylated chitobiose and chitotriose to COD structure. Three dimensional structures of COD in complex with chitobiose and chitotriose have been recently obtained25 and deposited in the Protein Data Bank with accession codes 4NZ1 and 4OUI respectively. Polar hydrogens were added to the receptor protein structure with AutoDockTools38. AutoDock4.2 atom typing was used. Gaisteger partial charges were computed for each atom with AutoDockTools. Ligand structures (chitobiose, AA and chitotriose, AAA) were extracted from the corresponding PDB files. For each ligand, a series of partially and fully deacetylated compounds were generated by removing the corresponding N-acetyl group with AutoDockTools while keeping the overall geometry of the molecules. Thus, three dimensional structures AA,AD,DA,DD,AAA,ADA, AAD, ADD, DAD, DDD were obtained. Every ligand was parametrized in the same way as the receptor. Each ligand was docked onto COD structure by means of AutoDock VINA algorithm26. A grid-box of 30 3 26 3 30 A3 centered at the active site was used as the search space for docking. Interaction energies between ligands and COD receptor were calculated with VINA scoring function26. Reported values in Table 3 are an estimation of the differences in free energy upon binding for those docking poses in which the ligand binds in a productive orientation at the active site. The uncertainty of these calculations is 0.6 kcal mol21. This was estimated as the average range of VINA score values obtained for a series of similar docking poses (structures within 1.5 A of the lowest score one). 24. Rose, T. M., Henikoff, J. G. & Henikoff, S. CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res 31, 3763–3766 (2003). 25. Andre´s, E. et al. Structural basis of chitin oligosaccharide deacetylation. Angew Chem Int Ed Engl 53, 6882–6887 (2014). 26. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comput Chem 31, 455–461 (2010). 27. Grant, S. G., Jessee, J., Bloom, F. R. & Hanahan, D. Methods FEBS J 273, 491–503 (2006). 7. El Gueddari, N. E. & Moerschbacher, B. Advances in Chitin Science VII [Boucher, I., Jamieson, K. & Retnakaran, A. (ed.)] [56–59], 2004. 38. Sanner, M. F. Python: A programming language for software integration and development. J Mol Graph Model 17, 57–61 (1999). 8. Vander, P., Va˚rum, K. M., Domard, A., El Gueddari, N. E. & Moerschbacher, B. M. Comparison of the ability of partially N-acetylated chitosans and 8. Vander, P., Va˚rum, K. M., Domard, A., El Gueddari, N. E. & Moerschbacher, B. M. Comparison of the ability of partially N-acetylated chitosans and chitooligosaccharides to elicit resistance reactions in wheat leaves. Plant Physiol 118, 1353–1359 (1998). chitooligosaccharides to elicit resistance reactions in wheat leaves. Plant Physiol 118, 1353–1359 (1998). Author contributions 12. Kohlhoff, M. et al. in Advances in Chitin Science XI [Rustichelli, F., Caramella, C., Senel, S. & Va˚rum, K. M. (ed.) [463–468], 2009. S.N.H., S.C.-L., X.B., A.P., H.W., B.M.M. and S.K. designed the experiments and discussed the results. S.N.H., S.C.-L., X.B. and H.W. performed the experiments. S.N.H. wrote the main manuscript text to which X.B. and H.W. contributed. S.N.H. prepared all figures and tables except for table 3 and figure 5 which were prepared by X.B. All authors reviewed and revised the manuscript. S.N.H., S.C.-L., X.B., A.P., H.W., B.M.M. and S.K. designed the experiments and discussed the results. S.N.H., S.C.-L., X.B. and H.W. performed the experiments. S.N.H. wrote the main manuscript text to which X.B. and H.W. contributed. S.N.H. prepared all figures and tables except for table 3 and figure 5 which were prepared by X.B. All authors reviewed and revised the manuscript. 13. Sørbotten, A., Horn, S. J., Eijsink, V. G. H. & Va˚rum, K. M. Degradation of chitosans with chitinase B from Serratia marcescens. Production of chito- oligosaccharides and insight into enzyme processivity. FEBS J 272, 538–549 (2005). 14. Trombotto, S., Ladavie`re, C., Delolme, F. & Domard, A. Chemical preparation and structural characterization of a homogeneous series of chitin/chitosan oligomers. Biomacromolecules 9, 1731–1738 (2008). Methods Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 87, 4645–4649 (1990). 28. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41, 207–234 (2005). 29. Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092 (1996). 30. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970). 31. Choi, J.-K. et al. Background-free, fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes, zincon and ethyl violet. Electrophoresis 23, 4053–4059 (2002). 32. Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354 (1979). 1. Shahidi, F., Arachchi, J. K. V. & Jeon, Y. J. Food applications of chitin and chitosans. Trends Food Sci Technol 10, 37–51 (1999). 33. Teorell, T. & Stenhagen, E. Ein Universalpuffer fu¨r den pH-Bereich 2.0 bis 12.0. Biochem Z 299, 416–419 (1938). 2. Aam, B. B. et al. Production of chitooligosaccharides and their potential applications in medicine. Mar Drugs 8, 1482–1517 (2010). 34. Leijdekkers, A. G. M., Sanders, M. G., Schols, H. A. & Gruppen, H. Characterizing plant cell wall derived oligosaccharides using hydrophilic interaction chromatography with mass spectrometry detection. J Chromatogr A 1218, 9227–9235 (2011). 3. Khoushab, F. & Yamabhai, M. Chitin research revisited. Mar Drugs 8, 1988–2012 (2010). 4. Rinaudo, M. Chitin and chitosan: properties and applications. Prog Polym Sci 31, 603–632 (2006). 35. Remoroza, C. et al. Combined HILIC-ELSD/ESI-MS(n) enables the separation, identification and quantification of sugar beet pectin derived oligomers. Carbohydr Polym 90, 41–48 (2012). 5. Synowiecki, J. & Al-Khateeb, N. A. Production, properties, and some new applications of chitin and its derivatives. Crit Rev Food Sci Nutr 43, 145–171 (2003). y y 36. Waegeman, H. et al. Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering. N Biotechnol 30, 255–261 (2013). 6. Weinhold, M. X., Sauvageau, J. C. M., Kumirska, J. & Tho¨ming, J. Studies on acetylation patterns of different chitosan preparations. Carbohydr Polym 78, 678–684 (2009). 37. Horn, S. J. et al. Endo/exo mechanism and processivity of family 18 chitinases produced by Serratia marcescens. Acknowledgments 9. El Gueddari, N. E. et al. Advances in Chitin Science X [Senel, S., Va˚rum, K. M., Sumnu, M. M. & Hincal, A. A. (ed.)] [119–126], 2007. We thank Prof. Dr. Lopez-Lara for supplying Rhizobium sp. GRH2 and Claudia Lu¨neberg for excellent technical assistance. The research leading to these results has received funding from the European Union Sixth Framework Programme ([FP6/2002–2006]) in the framework of the European Research Area – Industrial Biotechnology (ERA-IB) project ‘‘ChitoBioEngineering’’ under grant agreement EIB.10.042. We thank Prof. Dr. Lopez-Lara for supplying Rhizobium sp. GRH2 and Claudia Lu¨neberg for excellent technical assistance. The research leading to these results has received funding from the European Union Sixth Framework Programme ([FP6/2002–2006]) in the framework of the European Research Area – Industrial Biotechnology (ERA-IB) project ‘‘ChitoBioEngineering’’ under grant agreement EIB.10.042. 10. Chang, K. L. B., Tsai, G., Lee, J. & Fu, W.-R. Heterogeneous N-deacetylation of chitin in alkaline solution. Carbohydr Res 303, 327–332 (1997). 11. Va˚rum, K. M., Holme, H. K., Izume, M., Stokke, B. T. & Smidsrød, O. Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans. Biochim Biophys Acta 1291, 5–15 (1996). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if Additional information Supplementary information accompanies this paper at http://www.nature.com/ scientificreports Supplementary information accompanies this paper at http://www.nature.com/ scientificreports 15. Kuyama, H. et al. Stereocontrolled synthesis of chitosan dodecamer. Carbohydr Res 243, C1–C7 (1993). Competing financial interests: The authors declare no competing financial interests. 16. Tsigos, I., Martinou, A., Kafetzopoulos, D. & Bouriotis, V. Chitin deacetylases: new, versatile tools in biotechnology. Trends Biotechnol 18, 305–312 (2000). 17. Blair, D. E. et al. Structure and mechanism of chitin deacetylase from the fungal pathogen. Biochemistry 45, 9416–9426 (2006). 16. Tsigos, I., Martinou, A., Kafetzopoulos, D. & Bouriotis, V. Chitin deacetylases: new, versatile tools in biotechnology. Trends Biotechnol 18, 305–312 (2000). How to cite this article: Hamer, S.N. et al. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases. Sci. Rep. 5, 8716; DOI:10.1038/srep08716 (2015). 17. Blair, D. E. et al. Structure and mechanism of chitin deacetylase from the fungal pathogen. Biochemistry 45, 9416–9426 (2006). SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 8 www.nature.com/scientificreports the material is not included under the Creative Commons license, users will need to obtain permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ SCIENTIFIC REPORTS \| 5 : 8716 \| DOI: 10.1038/srep08716 9
https://openalex.org/W4207083675	https://thejournalofheadacheandpain.biomedcentral.com/track/pdf/10.1186/s10194-021-01234-6	English	null	Comparative Study of Magnesium, Sodium Valproate, and Concurrent Magnesium- Sodium Valproate Therapy in the Prevention of Migraine: A Randomized Controlled Double-Blind Trial.	Research Square (Research Square)	2,021	cc-by	7,897	RESEARCH ARTICLE Open Access (2021) 22:21 (2021) 22:21 The Journal of Headache and Pain Khani et al. The Journal of Headache and Pain https://doi.org/10.1186/s10194-021-01234-6 © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Objective: This study aimed to assess the efficacy of concurrent magnesium-sodium valproate therapy and compare it with either magnesium or sodium valproate alone in migraine prophylaxis. Materials and methods: This randomized single-center double-blind parallel-group controlled clinical trial study was conducted on migraine patients within the age range of 18–65 years. The subjects with at least four monthly attacks were randomly assigned to group A (n = 82) sodium valproate, group B (n = 70) magnesium with sodium valproate, and group C (n = 70) magnesium. The patients passed a one-month baseline without prophylactic therapy and then received a 3-month treatment. The characteristics of migraine, including frequency, severity, duration of the attacks, and the number of painkillers taken per month, were monthly recorded in each visit. The Migraine Disability Assessment (MIDAS) and Headache Impact Test-6 (HIT-6) scores were recorded at the baseline and after 3 months of treatment in each group. Within- and between-group analyses were performed in this study. Results: The obtained results revealed a significant reduction in all migraine characteristics in all groups compared to those reported for the baseline (P < 0.001). Intragroup data analysis indicated that there was no statistically significant difference in headache frequency between groups A and B in the third month (P = 0.525); nevertheless, three other parameters showed a significant reduction in group B, compared to those reported for group A in the third month (P < 0.05). On the other hand, group C could not effectively reduce measured parameters in the patients, compared to groups A and B after 3 months (P < 0.001). Furthermore, the MIDAS and HIT-6 scores significantly diminished in groups A, B, and C compared to those reported at the baseline (P < 0.001), and these changes were more significant in groups A and B than in group C (P < 0.001). Conclusion: The obtained results of this study revealed that magnesium could enhance the antimigraine properties of sodium valproate in combination therapy and reduce the required valproate dose for migraine prophylaxis. agnesium, Migraine, Prophylaxis, Sodium valproate Keywords: Magnesium, Migraine, Prophylaxis, Sodium valproate Keywords: Magnesium, Migraine, Prophylaxis, Sodium valproate * Correspondence: Sharifipour.e@gmail.com 1Department of Neurology, Neurosciences Research Center (NSRC), Qom University of Medical Sciences, Shahid-Beheshti hospital, Shahid-Beheshti street, Qom, Iran Full list of author information is available at the end of the article Comparative study of magnesium, sodium valproate, and concurrent magnesium- sodium valproate therapy in the prevention of migraine headaches: a randomized controlled double-blind trial Samira Khani1, Seyed Amir Hejazi1, Mehdi Yaghoubi2 and Ehsan Sharifipour1* Study design Beta-blockers, calcium-channel blockers, anticonvulsants, selective serotonin reuptake inhibitors, tricyclic antidepressants, and angiotensin blockers are some of the medications used for migraine prophylaxis. Furthermore, botulinum toxin, flunarizine, vitamins, minerals, and herbal agents were also sug- gested in many studies [14, 44]. p p A total of 260 patients entered into the present study with signed consent about the inclusion and exclusion criteria. The patients were recruited from those referred to an outpatient neurological clinic and also through ad- vertisements on social networks (i.e., Telegram and WhatsApp). The patients were randomly assigned into three groups, including the intervention groups A, B, and C. Intervention group A received a 200 mg sodium valproate tablet twice a day orally for 12 weeks and a placebo tablet twice a day orally for 12 weeks. Interven- tion group B received a 200 mg sodium valproate tablet twice a day orally for 12 weeks and a 250 mg magnesium oxide tablet twice a day orally for 12 weeks. Intervention group C received a 250 mg magnesium oxide tablet twice a day orally for 12 weeks and a placebo tablet twice a day orally for 12 weeks. Sodium valproate is one of the FDA-approved anti- epileptic drugs (AEDs) for the prevention of migraine [27]. Several functions have been proposed for the antimigraine action of sodium valproate in such patients; nevertheless, the exact mechanism is not completely perceived due to its various biochemical effects and multifactorial nature of migraine patho- physiology [6, 37, 38]. Despite the proven efficacy of valproate in the prevention of migraine, poor compli- ance with therapy has been observed due to its side effects, such as fatigue, dizziness, nausea, tremor, and weight gain [2, 29]. All the patients underwent a 1-month baseline period (without prophylactic medication) to assess the frequency of attacks, severity of attacks, and amount of collaboration with the physician. The study duration since the onset of drug prescription would be 3 months, and the patients were monthly followed. The concurrent administration of acute abortive treatment was not prohibited during this study; however, the patients should record the dose of pain killer in diaries. Headache diaries and headache ques- tionnaires (i.e., the Migraine Disability Assessment [MIDA S] and Headache Impact Test-6 [HIT]) were used to ob- tain clinical information. Study design y g This single-center placebo-controlled double-blind ran- domized trial study (registered in Iranian Registry of Clinical Trials with registry no. IRCT2015081923685N1) was conducted on migraine patients on October 11 in 2015. Ethical approval for this study was obtained from the Ethics Committee of Qom University of Medical Sciences in Qom, Iran (special approval ID: IR.MUQ.- REC.1394.73) on August 26 in 2015 in accordance with the Helsinki Declaration. This study was conducted at the Neurological Special Clinic of Shahid Beheshti Hos- pital in Qom. The research assistant evaluated eligibility obtained informed consent and enrolled the participants. A total of 260 patients entered into the present study with signed consent about the inclusion and exclusion criteria. The patients were recruited from those referred to an outpatient neurological clinic and also through ad- vertisements on social networks (i.e., Telegram and WhatsApp). The patients were randomly assigned into three groups, including the intervention groups A, B, and C. Intervention group A received a 200 mg sodium valproate tablet twice a day orally for 12 weeks and a placebo tablet twice a day orally for 12 weeks. Interven- tion group B received a 200 mg sodium valproate tablet twice a day orally for 12 weeks and a 250 mg magnesium oxide tablet twice a day orally for 12 weeks. Intervention group C received a 250 mg magnesium oxide tablet twice a day orally for 12 weeks and a placebo tablet twice a day orally for 12 weeks. This single-center placebo-controlled double-blind ran- domized trial study (registered in Iranian Registry of Clinical Trials with registry no. IRCT2015081923685N1) was conducted on migraine patients on October 11 in 2015. Ethical approval for this study was obtained from the Ethics Committee of Qom University of Medical Sciences in Qom, Iran (special approval ID: IR.MUQ.- REC.1394.73) on August 26 in 2015 in accordance with the Helsinki Declaration. This study was conducted at the Neurological Special Clinic of Shahid Beheshti Hos- pital in Qom. The research assistant evaluated eligibility obtained informed consent and enrolled the participants. A reduction in the frequency of headaches about 50% or more, reduced intensity, and improved response to symptomatic medication can be three outcomes of pre- ventive therapy [28]. However, a lack of effectiveness, adverse effects, and poor compliance are also common in these patients [16, 35]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 10 Khani et al. The Journal of Headache and Pain (2021) 22:21 Page 2 of 10 Khani et al. The Journal of Headache and Pain (2021) 22:21 Khani et al. The Journal of Headache and Pain Study design The paper diary which was used to record information about the attacks of migraine dur- ing the baseline and treatment period, included the Wong-Baker Faces Pain Rating Scale for severity assess- ment and data about the frequency, duration, days with migraine per month, and number of painkillers used per month. The patients should daily complete the diary, and the research assistant called the patients every week to en- sure patient compliance. Magnesium is a cofactor in more than 300 biochemical reactions and helps to maintain normal nerve and muscle functions [12]. In recent years, studies have focused on the clinical use of magnesium as a prophylac- tic regimen for migraine due to good efficacy and toler- ability in patients [9]. Oral magnesium supplementation has been reported with level B evidence for its efficacy in the prophylactic therapy of episodic migraine based on the American Academy of Neurology Guidelines [8]. Although there has been existing evidence on the ef- fectiveness of magnesium in migraine prophylaxis, no study has evaluated magnesium potency in increasing sodium valproate efficacy in combination therapy of mi- graine. The current study was the first attempt to com- pare the efficacy of combination therapy of magnesium and sodium valproate with each treatment alone in mi- graine prophylaxis. Magnesium is a cofactor in more than 300 biochemical reactions and helps to maintain normal nerve and muscle functions [12]. In recent years, studies have focused on the clinical use of magnesium as a prophylac- tic regimen for migraine due to good efficacy and toler- ability in patients [9]. Oral magnesium supplementation has been reported with level B evidence for its efficacy in the prophylactic therapy of episodic migraine based on the American Academy of Neurology Guidelines [8]. Although there has been existing evidence on the ef- fectiveness of magnesium in migraine prophylaxis, no study has evaluated magnesium potency in increasing sodium valproate efficacy in combination therapy of mi- graine. The current study was the first attempt to com- pare the efficacy of combination therapy of magnesium and sodium valproate with each treatment alone in mi- graine prophylaxis. Methods Study design Migraine as a primary headache disorder with substan- tial pain is included in the 20 most disabling diseases ac- cording to the World Health Organization [39]. Epidemiologic studies have indicated that the prevalence of migraine is about 14% in Iran similar to or even higher than that reported worldwide [11]. Lifestyle man- agement, acute treatment, and preventive treatment are three approaches to treat migraine [44]. Prophylactic therapy for migraine is recommended in patients with four or more attacks per month, eight or more headache days a month, debilitating headaches, and medication- overuse headaches [15]. Inclusion and exclusion criteria A total of 298 patients with recurrent migraine were en- rolled in the present study. After careful screening, 260 patients were randomized to different treatment groups (i.e., A, B, and C). Finally, 222 patients (including 125 fe- males and 97 males) completed the study. The consort diagram of the study is presented in Fig. 1. As shown in Table 1, there was no significant difference in demo- graphics, migraine type, and headache history at the baseline in various treatment groups. The inclusion criteria were the diagnosis of migraine ac- cording to the latest International Headache Society cri- teria, history of migraine with or without aura for at least 6 months, age range of 18–65 years, and experience of at least four monthly attacks. The exclusion criteria were non-migraine headaches, total number of headache days per month higher than 15, overuse of analgesics in migraine attacks (i.e., the use of ergots, nonsteroidal anti-inflammatory drugs, and triptans higher than 8 days a month), substance and alcohol dependence, illiteracy of patients and their family (unable to fill diaries), preg- nancy and nursing, history of magnesium or sodium valproate intolerance, history of renal, liver, and chronic diseases, elevated liver enzymes in the first sampling more than two times the normal, neurologic disorders other than migraine, having other comorbidities i.e. fibromyalgia or etc., use of supplements containing mag- nesium, use of herbal antimigraine, and use of anti- depressant and antipsychotic medications. The patients were evaluated in terms of headache fre- quency, headache severity, headache duration, and num- ber of painkillers taken per month after the onset of the treatment for 3 months. The intergroup and intragroup analyses of the data were performed. The obtained re- sults showed a significant reduction in the measured pa- rameters in the three arms of the study after 3-month of treatment, compared to those reported for the baseline (P < 0.001). However, a significant reduction in head- ache duration in group C was not demonstrated during the first month of the study (P = 0.153) (Figs. 2 and 3). Intragroup data analysis indicated that there was no statistically significant difference in headache frequency between groups A and B in the first (P = 0.972) and third (P = 0.525) months; nonetheless, group B revealed a significant reduction in the second month, compared to group A (P = 0.029). Primary efficacy measures The three groups were evaluated to compare the efficacy of the three treatment schedules on migraine frequency Page 3 of 10 Khani et al. The Journal of Headache and Pain (2021) 22:21 Khani et al. The Journal of Headache and Pain at the end of the baseline, and in the first, second, and third months after the treatment. 38.1%, based on the previous trials, respectively [22]. Therefore, 260 patients were divided into three groups via random divisions (A = 88, B = 86, C = 86) considering the dropout rate. Secondary efficacy measures Migraine severity, duration of attacks and number of painkillers used per month were compared among the three treatment groups. Also, the changes in the MIDAS and HIT-6 scores since the baseline were calculated after a 3-month treatment in each group. The HIT-6 was used to assess the severity of headache impact on the patient life, and the HIT-6 score is within the range of 36–78 [34]. The MIDAS questionnaire was also utilized to determine migraine-related disabilities over 3 months. The MIDAS scores include several ranges, namely 0–5 (MIDAS grade I: minor or no disability), 6–10 (MIDAS grade II: mild disability), 11–20 (MIDAS grade III: moderate disability), and 21 or higher (MIDAS grade IV: severe disability) [3, 18]. All the statistical analyses were performed using SPSS software (version 20). Firstly, the normal distribution of the data at the beginning of the study was investigated using the Kolmogorov-Smirnov test. Descriptive charac- teristics, such as mean and standard deviation, were used to explain the statistical results. Patient characteristics (e.g., gender, migraine type, and family history) were an- alyzed using the Pearson’s Chi-square test. Intergroup comparisons were performed using a paired student’s t- test. The analysis of variance and Tukey’s post-hoc test were used to compare the values among the three treat- ment groups. A p-value of less than 0.05 was considered statistically significant. Inclusion and exclusion criteria Furthermore, group C indicated a significant difference in headache frequency in compari- son to groups A and B over 3 months (P < 0.001) (Table 2). Randomization and blinding Block randomization sampling was used in the present study. Each subject received an ID code, and the medica- tions were delivered to the patients based on the order of the blocks in completely identical containers without the labels, marked as A, B, or C, to complete the sample size. The clinicians and patients were blinded to the re- ception of the drugs during the study period. No significant difference in headache severity was ob- served in the first month among the three groups; never- theless, severity reduction was significant in group B in comparison to those reported for group A (P = 0.01 and P = 0.002) and group C (P < 0.001) in the second and third month, respectively. Severity reduction was also Sample size and statistical methods The required sample size for the study was calculated at least 67 subjects in each group with a power of 80% and the significance level of 5%; nevertheless, the recovery rates in the two groups of the study were 65% and Page 4 of 10 Khani et al. The Journal of Headache and Pain (2021) 2 (2021) 22:21 Fig. 1 CONSORT flow diagram of participants through the study Fig. 1 CONSORT flow diagram of participants through the stu higher in group A, compared to that reported for group C in the second and third months (P < 0.001) (Table 2). The three groups had no significant difference in headache duration in the first month; however, groups A and B were both significantly better than group C re- garding headache duration with p-values of 0.006 and less than 0.001 in the second month and p-values of 0.004 and less than 0.001 in the third month, respectively. In addition, headache duration significantly differed between groups A and B in the second (P = 0.019) and third (P = 0.013) months (Table 2). respectively. In addition, headache duration significantly differed between groups A and B in the second (P = 0.019) and third (P = 0.013) months (Table 2). higher in group A, compared to that reported for group C in the second and third months (P < 0.001) (Table 2). The three groups had no significant difference in headache duration in the first month; however, groups A and B were both significantly better than group C re- garding headache duration with p-values of 0.006 and less than 0.001 in the second month and p-values of 0.004 and less than 0.001 in the third month, The number of painkillers had no statistically signifi- cant difference between groups A and B in the first month (P = 0.871); however, it was significantly lower in groups A (P = 0.031) and B (P = 0.010), compared to that reported for group C in the first month. Sample size and statistical methods The number of Table 1 Patient Demographic characteristics at baseline Characteristics A (n = 82) B (n = 70) C (n = 70) p-value Sex (Female), n 41 42 42 0.35 Age, y 35.16 ± 8.21 37.11 ± 6.56 34.41 ± 6.19 0.27 BMI (Kg/m2) 24.78 ± 8.34 24.76 ± 8.54 24.42 ± 7.70 0.17 Migraine type, n Without Aura 51 39 44 0.62 With Aura 31 31 26 Headache history, y 5.23 ± 2.49 5.73 ± 2.14 4.89 ± 3.14 0.25 Family history of migraine, n Yes 55 43 47 0.71 No 27 27 23 Khani et al. The Journal of Headache and Pain (2021) 22:21 Page 5 of 10 Fig. 2 Migraine characteristics of the three groups in different treatment periods; P values≤0.05 were considered statistically significant vs baseline [measured using sample (paired) t test]. A: Sodium Valproate; B: Sodium Valproate +Magnesium; C: Magnesium Fig. 2 Migraine characteristics of the three groups in different treatment periods; P values≤0.05 were considered statistically significant vs baseline [measured using sample (paired) t test]. A: Sodium Valproate; B: Sodium Valproate +Magnesium; C: Magnesium painkillers was significantly lower in group B in com- parison to those reported for group A in the second and third months (P < 0.001). Both groups A and B used sig- nificantly lower numbers of painkillers than group C in the second (P = 0.017 and P < 0.001, respectively) and third (P < 0.001) months (Table 2). were compared among the three treatment groups. The obtained results showed that the combination of magne- sium and sodium valproate had an appropriate efficacy in migraine prophylaxis, as headache severity, duration of headache, and amount of utilized painkillers were significantly lower in group B, compared to those re- ported for group A. Moreover, the MIDAS scores fur- ther reduced in group B than those reported for group A, and the changes in HIT scores were not significantly different between groups A and B. Furthermore, by the addition of magnesium to valproate (group B), the so- dium valproate dose significantly didn’t increase, com- pared to that reported for group A. The MIDAS and HIT-6 scores (Table 3) were signifi- cantly lower in all the treatment groups in comparison to those measured at the baseline (P < 0.001). Sample size and statistical methods The intragroup analysis of MIDAS and HIT-6 changes also indicated that groups A and B had significantly higher changes in scores than group C (P < 0.001). However, no significant difference was observed between groups A and B in HIT score change (P = 0.999). The changes in MIDAS scores were significantly different between the two groups (group A vs. group B; P = 0.023) (Fig. 4). Sodium valproate belongs to the class of AEDs with an important role in the treatment of migraine. Increasing GABA activity and inhibiting NMDA-evoked neuroexci- tatory signals are two main mechanisms of valproate in blocking cortical spreading depression during a migraine attack. It can inhibit GABA-degrading enzymes (i.e., aminotransferase and succinic semialdehyde) and in- crease the neuro-inhibitory activity of GABA. In addition, the active metabolites of valproate can activate the GABA-synthesizing enzyme (i.e., glutamic acid Discussion The Journal of Headache and Pain Page 7 of 10 Table 2 Intragroup monthly basis analysis of Migraine attack, Headache severity, Headache duration and Painkiller number (A: Sodium Valproate; B: Sodium Valproate +Magnesium; C: Magnesium); P values≤0.05 were considered statistically significant Group A B C A vs B A vs C B vs C p- value Mean Difference 95% CI p- value Mean Difference 95% CI p- value Mean Difference 95% CI Migraine attack (n), mean ± SD baseline 6.65 ± 1.65 6.89 ± 1.52 7.06 ± 1.54 0.621 −0.24 −0.85 to 0.37 0.248 −0.41 −1.02 to 0.20 0.797 −0.17 −0.80 to 0.46 After 1 month 4.09 ± 0.99 4.04 ± 0.93 5.49 ± 1.45 0.972 0.05 −0.40 to 0.48 < 0.001 −1.40 −1.84 to −0.96 < 0.001 −1.45 −1.90 to 0.99 After 2 month 2.83 ± 0.73 2.47 ± 0.71 4.21 ± 0.08 0.029 0.36 0.03 to 0.69 < 0.001 −1.38 −1.71 to −1.06 < 0.001 −1.74 −2.08 to −1.40 After 3 month 1.60 ± 0.76 1.40 ± 0.75 3.91 ± 0.86 0.525 0.20 −0.17 to 0.45 < 0.001 −2.31 −2.62 to −2.01 < 0.001 −2.51 −2.77 to −2.14 Headache severity, mean ± SD baseline 5.33 ± 0.67 5.27 ± 0.79 5.16 ± 1.02 0.885 0.06 −0.25 to 0.38 0.424 0.17 −0.15 to 0.49 0.732 0.11 −0.22 to 0.43 After 1 month 3.68 ± 0.81 3.69 ± 0.73 3.93 ± 1.24 0.999 −0.01 −0.37 to 0.35 0.243 −0.25 −0.61 to 0.11 0.287 −0.24 −0.62 to 0.13 After 2 month 2.51 ± 0.68 2.16 ± 0.67 3.32 ± 0.78 0.01 0.35 −0.06 to 0.61 < 0.001 −0.81 −1.08 to −0.54 < 0.001 −1.16 −1.44 to 0.87 After 3 month 1.71 ± 0.55 1.26 ± 0.59 2.41 ± 1.15 0.002 0.45 −0.13 to 0.75 < 0.001 −0.70 −1.00 to −0.39 < 0.001 −1.15 −1.46 to −0.82 Headache duration (h), mean ± SD baseline 11.56 ± 3.53 11.96 ± 1.73 10.99 ± 2.49 0.649 −0.40 −1.45 to 0.66 0.403 0.57 −0.48 to 1.63 0.093 0.97 −0.12 to 2.07 After 1 month 10.39 ± 2.87 9.95 ± 1.80 10.64 ± 1.96 0.481 0.44 −0.45 to 1.31 0.779 −0.25 −1.13 to 0.63 0.185 −0.69 −1.60 to 0.23 After 2 month 8.19 ± 2.76 7.22 ± 1.66 9.30 ± 1.84 0.019 0.97 0.12 to 1.80 0.006 −1.11 −1.94 to −0.26 < 0.001 −2.08 −2.94 to −1.20 After 3 month 7.06 ± 2.53 6.08 ± 1.75 8.15 ± 1.83 0.013 0.98 0.17 to 1.77 0.004 −1.09 −1.90 to −0.29 < 0.001 −2.07 −2.90 to −1.23 Painkiller (n),mean ± SD baseline 6.45 ± 0.95 6.27 ± 0.94 6.05 ± 1.48 0.601 0.18 −0.26 to 0.62 0.083 0.40 −0.04 to 0.84 0.489 0.22 −0.24 to 0.68 After 1 month 4.15 ± 0.93 4.07 ± 0.73 4.55 ± 1.13 0.871 0.08 −0.29 to 0.44 0.031 −0.40 −0.76 to −0.03 0.010 −0.48 −0.85 to −0.09 After 2 month 3.11 ± 0.68 2.21 ± 0.75 3.46 ± 0.89 < 0.001 0.90 0.60 to 1.19 0.017 −0.35 −0.64 to −0.05 < 0.001 −1.25 −1.55 to −0.93 After 3 month 1.72 ± 0.58 1.26 ± 0.59 2.37 ± 0.78 < 0.001 0.46 0.20 to 0.71 < 0.001 −0.65 −0.89 to −0.39 < 0.001 −1.11 −1.36 to −0.84 Table 2 Intragroup monthly basis analysis of Migraine attack, Headache severity, Headache duration and Painkiller number (A: Sodium Valproate; B: Sodium Valproate +Magnesium; C: Magnesium); P values≤0.05 were considered statistically significant Table 2 Intragroup monthly basis analysis of Migraine attack, Headache severity, Headache duration and Painkiller number (A: Sodium Valproate; B: Sodium Valproate +Magnesium; C: Magnesium); P values≤0.05 were considered statistically significant Table 2 Intragroup monthly basis analysis of Migraine attack, Headache severity, Headache duration and Painkiller number (A: Sodium Valproate; B: Sodium Valproate +Magnesium; C: Magnesium); P values≤0.05 were considered statistically significant neurogenic mechanisms of migraine [41]. Discussion The current study compared the efficacy of sodium val- proate (group A), sodium valproate plus magnesium oxide (group B), and magnesium oxide (group C) in the migraine prophylaxis of patients within the age range of 18–65 years. Furthermore, the MIDAS and HIT6 scores Khani et al. The Journal of Headache and Pain (2021) 22:21 Khani et al. The Journal of Headache and Pain Page 6 of 10 Fig. 3 Intra group comparison of duration, severity, number of attack and painkiller in different treatment periods Fig. 3 Intra group comparison of duration, severity, number of attack and painkiller in different treatment periods decarboxylase) and further increase GABA activity over time. Furthermore, valproate decreases neurogenic in- flammation by debilitating plasma extravasation of vaso- active neuropeptides, such as substance P, CGRP, and neurokinin A [27, 43]. therapeutic effect is achieved in migraine prophylaxis using a daily dose of sodium valproate within the range of 200–500 mg [4, 16, 19]. The effectiveness of magnesium in migraine prophy- laxis was firstly investigated by [10] [10]. Other trials also reported different results of magnesium efficacy in migraine patients [23, 40]. Slavin et al. demonstrated that the inadequate consumption of magnesium intake is associated with migraine in American adults within the age range of 20–50 years [36]. There is evidence that the concentrations of magnesium in the occipital lobes of patients with migraine and cluster headaches are lower than those reported for healthy controls [25]. There is abundant evidence that valproate is effective in the prevention of migraine attacks; however, different response rates were reported in various studies [26, 33]. Ichikawa et al. demonstrated that different factors, such as the history of hyperlipidemia, allergy, and psychiatric disorders, are involved in the clinical responses to val- proate [20]. Sodium valproate has been used in doses within the range of 500–1000 mg/day in migraine pre- vention trials [27]. Nevertheless, in the current study, a lower dose of 200 mg was prescribed twice a day for patients with an acceptable response. Other studies con- ducted on the Iranian population also revealed that the Therefore, further clinical trials were suggested to clar- ify the exact efficacy of magnesium in the prevention of migraine. It is proposed that magnesium is linked to mi- graine pathogenesis by counteracting both vascular and Khani et al. The Journal of Headache and Pain (2021) 22:21 Khani et al. Discussion Magnesium may be effective in migraine through the regulation of neuronal excitability because magnesium not only acts as a physiologic calcium-antagonist but also inhibits NMDA receptors and glutamate-dependent excitatory pathways [7, 17]. inflammatory intracellular signaling, such as the nu- clear factor kappa B pathway [24]. Other mechanisms, including the inhibition of voltage-gated calcium channels, connexin channels, and other ion channels, can be involved in the prevention of migraine [17]. Based on the evidence, magnesium was strongly rec- ommended by the Canadian Headache Society in mi- graine prophylaxis. The Swiss Headache Society also suggests magnesium to children and pregnant women with migraine [28]. In addition, magnesium can regulate neurotransmitter release and substance P release and reduce free radical accumulation within the cell and vasoconstriction [1, 10, 45]. Magnesium through modulating mitochondrial oxi- dative phosphorylation, 5-HT neurotransmission, and NO system, regulating the uptake of glutamate into as- trocytes, and blocking of NMDA receptor can be effect- ive in migraine-preventive therapy [38]. In a crossover study conducted by Karimi et al., the comparison of efficacy between magnesium oxide and sodium valproate was carried out for migraine prophy- laxis. The results showed that 500 mg/day magnesium has comparable efficacy to 400 mg/day valproate in mi- graine prophylaxis [21]. However, the obtained results of the present study indicated that valproate was signifi- cantly more effective than magnesium in the reduction Magnesium has numerous effects on the nervous system such as analgesic effects in neuropathic pain [30] and visceral inflammatory pain [42]. Magnesium can decrease inflammation by inhibiting pro- Table 3 Analysis of MIDAS and HIT score in a different group (A: Sodium Valproate; B: Sodium Valproate +Magnesium; C: Magnesium) compared to baseline A P-value B P-value C P-value Pre- intervention Post- intervention Pre- intervention Post- intervention Pre- intervention Post- intervention MIDAS 21.74 ± 4.44 17.11 ± 4.06 < 0.001 21.68 ± 3.72 16.11 ± 3.87 < 0.001 22.13 ± 1.88 18.81 ± 1.76 < 0.001 HIT-6 56.72 ± 4.59 49.91 ± 4.58 < 0.001 56.89 ± 3.84 50.50 ± 3.27 < 0.001 57.54 ± 2.13 53.03 ± 1.88 < 0.001 MIDAS migraine disability assessment score; HIT-6 Headache Impact Test-6 score Khani et al. The Journal of Headache and Pain (2021) 22:21 Page 8 of 10 Khani et al. The Journal of Headache and Pain Fig. 4 Comparison of MIDAS and HIT-6 score change between three treatment groups. Discussion MIDAS change, P value: (A Vs B = 0.023, A Vs C = 0.001, B Vs C < 0.001). HIT change, P value: (A Vs B = 0.999, A Vs C < 0.001, B Vs C < 0.001) Fig. 4 Comparison of MIDAS and HIT-6 score change between three treatment groups. MIDAS change, P value: (A Vs B = 0.023, A Vs C = 0.001, B Vs C < 0.001). HIT change, P value: (A Vs B = 0.999, A Vs C < 0.001, B Vs C < 0.001) assessment of adverse effects during the study was not completely carried out due to faulty reports; therefore, the precise analysis was impossible. of migraine frequency and severity, duration of attacks, painkiller number, and MIDAS and HIT scores. According to the literature, it was shown that the addition of magnesium to valproic acid in magnesium valproate can efficiently reduce calcium ion conductance, activate the Na+/K+ ion pump, and modulate the NMDA receptors of the neuronal membrane in epilepsy, compared to valproate alone [5]. Animal studies also demonstrated that magnesium enhances the anticonvulsant potential of a subprotective dose of valproic acid in pentylenetetrazol- treated rats by the improvement of redox balance and modulation of some brain excitatory amino acids, such as aspartate, asparagine, and glycine [31]. Study limitations Some participants in this study did not contribute to blood sampling. A lack of complete data about serum magnesium levels limits the analysis of the correlation between the serum levels of magnesium and efficacy of treatment in the three groups. Furthermore, the Authors’ contributions Ehsan Sharifipour and Samira Khani designed the study protocol. Samira Khani, Seyed Amir Hejazi, Mehdi Yaghubi, and Ehsan Sharifipour had an effective role in the implementation of this study and the data collection. Samira Khani and Ehsan Sharifipour were involved in the data extraction and analysis, and writing of the manuscript. All authors have contributed effectively to the final arrangement of this manuscript. The authors read and approved the final manuscript. Conclusion The results of the present study revealed that combin- ation treatment with a low dose of sodium valproate and magnesium has desired efficacy in the reduction of migraine frequency, severity, duration of attacks, and number of utilized painkillers. Considering these prom- ising results, it also indicated that by the addition of magnesium to valproate, sodium valproate unchanged low dose improves its efficacy. Consequently, the devel- opment of a new combination drug (Magnesium Valpro- ate) in this manner, can be a more potent and safe therapy for migraine prophylaxis. It should be noted that epilepsy and migraine have some similar pathophysiological mechanisms, including the imbalance between GABA-mediated inhibition, exci- tatory glutamate-mediated transmission, and abnormal function of voltage-gated sodium and calcium channels [32]. The results of in vitro studies demonstrated that magnesium improves valproic acid efficacy against 4- aminopyridine-induced ictal activity [13]. The findings of the present clinical study also confirmed this idea and indicated that a combination of magnesium and low- dose valproate (200 mg) have appropriate efficacy in mi- graine prophylaxis without any reported exacerbation of side effects. Acknowledgments g The authors would like to express their gratitude to Professor Abolfazl Mohammadbeigi for his valuable advices during the study analysis, and RAHA Pharmaceutical Company in Isfahan, Iran, for providing sodium valproate and placebo, and all the patients who participated in this study. References 24. Lingam, I. and N. J. J. D. n. Robertson (2018). "Magnesium as a neuroprotective agent: a review of its use in the fetus, term infant with neonatal encephalopathy, and the adult stroke patient." 40(1): 1–12 1. Altura BT, Altura BM (1980) Withdrawal of magnesium causes vasospasm while elevated magnesium produces relaxation of tone in cerebral arteries. Neurosci Lett 20(3):323–327 1. Altura BT, Altura BM (1980) Withdrawal of magnesium causes vasospasm while elevated magnesium produces relaxation of tone in cerebral arteries. Neurosci Lett 20(3):323–327 25. Maier, J. A., G. Pickering, E. Giacomoni, A. Cazzaniga and P. J. N. Pellegrino (2020). "Headaches and Magnesium: Mechanisms, Bioavailability, Therapeutic Efficacy and Potential Advantage of Magnesium Pidolate." 12(9): 2660 2. Assarzadegan, F., H. Tabesh, S.-M. Hosseini-Zijoud, A. D. Beale, A. Shoghli, M. G. Yazdi, B. Mansouri, O. Hesami, N. B. Moghadam and H. D. J. I. R. C. M. J. Kasmaei (2016). "Comparing zonisamide with sodium valproate in the management of migraine headaches: double-blind randomized clinical trial of efficacy and safety." 18(9) 2. Assarzadegan, F., H. Tabesh, S.-M. Hosseini-Zijoud, A. D. Beale, A. Shoghli, M. G. Yazdi, B. Mansouri, O. Hesami, N. B. Moghadam and H. D. J. I. R. C. M. J. Kasmaei (2016). "Comparing zonisamide with sodium valproate in the management of migraine headaches: double-blind randomized clinical trial of efficacy and safety." 18(9) 26. Mathew, N. T., J. R. Saper, S. D. Silberstein, L. Rankin, H. G. Markley, S. Solomon, A. M. Rapoport, C. J. Silber and R. L. J. A. o. N. Deaton (1995). "Migraine prophylaxis with divalproex." 52(3): 281–286 3. Bigal, M. E., A. M. Rapoport, R. B. Lipton, S. J. Tepper, F. D. J. H. T. J. o. H. Sheftell and F. Pain (2003). "Assessment of migraine disability using the migraine disability assessment (MIDAS) questionnaire: a comparison of chronic migraine with episodic migraine." 43(4): 336–342 27. Parikh, S. K. and S. D. J. C. t. o. i. n. Silberstein (2019). "Current Status of Antiepileptic Drugs as Preventive Migraine Therapy." 21(4): 16 28. Pringsheim, T., W. Davenport, G. Mackie, I. Worthington, M. Aubé, S. N. Christie, J. Gladstone and W. J. J. C. J. N. S. Becker (2012). "Canadian Headache Society guideline for migraine prophylaxis." 39(2 Suppl 2): S1–59 4. Bostani, A., A. Rajabi, N. Moradian, N. Razazian and M. J. I. J. o. N. Rezaei (2013). "The effects of cinnarizine versus sodium valproate in migraine prophylaxis." 123(7): 487–493 29. Declarations 16. Hesami, O., M. R. Shams, L. Ayazkhoo, F. Assarzadegan, B. S. Lima, H. D. Kasmaei and M. J. I. j. o. p. r. I. Sistanizad (2018). "Comparison of pregabalin and sodium valproate in migraine prophylaxis: a randomized double- blinded study." 17(2): 783 Availability of data and materials The supporting data is available. 15. Ha, H. and A. J. A. f. p. Gonzalez (2019). "Migraine Headache Prophylaxis." 99(1) Authors’ information Samira Khani: PHD of Pharmacology, Assistant Professor of Pharmacology; Seyed Amir Hejazi: M.D. Neurologist, Associate Professor of Neurology; Page 9 of 10 Khani et al. The Journal of Headache and Pain (2021) 22:21 Mehdi yaghubi: Master of Psychiatry. Ehsan Sharifipur: M.D. Neurologist, Associate Professor of Neurology; 12. Faryadi, Q. J. I. J. o. A. (2012). "The magnificent effect of magnesium to human health: a critical review." 2(3) 13. Fueta, Y., A. Siniscalchi, V. Tancredi and M. J. E. Avoli (1995). "Extracellular magnesium and anticonvulsant effects of valproate in young rat hippocampus." 36(4): 404–409 Competing interests Non 20. Ichikawa, M., H. Katoh, T. Kurihara and M. J. J. o. C. N. Ishii (2016). "Clinical response to valproate in patients with migraine." 12(4): 468–475 Ethical approval and consent to participate Ethical approval for this study was obtained from the Ethics Committee of Qom University of Medical Sciences in Qom, Iran (special approval ID: IR.MUQ.REC.1394.73) on August 26 in 2015 in accordance with the Helsinki Declaration. Also, written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Ethical approval for this study was obtained from the Ethics Committee of Qom University of Medical Sciences in Qom, Iran (special approval ID: IR.MUQ.REC.1394.73) on August 26 in 2015 in accordance with the Helsinki Declaration. Also, written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of 17. Hoffmann J, Charles A (2018) Glutamate and its receptors as therapeutic targets for migraine. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 15(2):361–370. https://doi.org/1 0.1007/s13311-018-0616-5 18. Holla, R., B. Darshan, Y. Z. Ying, T. Raymond, E. Ponniah, K. S. San, N. Y. J. I. J. o. P. H. R. Yen and Development (2019). 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https://openalex.org/W3094229657	https://ojrd.biomedcentral.com/track/pdf/10.1186/s13023-020-01584-5	English	null	Apparently benign craniocervical signs in achondroplasia: “neurologic leftovers” identified through a retrospective dataset	Orphanet journal of rare diseases	2,020	cc-by	5,487	© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Smid et al. Orphanet J Rare Dis (2020) 15:301 https://doi.org/10.1186/s13023-020-01584-5 Smid et al. Orphanet J Rare Dis (2020) 15:301 https://doi.org/10.1186/s13023-020-01584-5 Open Access Abstract Background: Achondroplasia is the most common dwarfing disorder. It can result in a variety of sequelae, includ- ing neurologic complications, among which high cervical myelopathy is one of particular concern. However, some individuals with achondroplasia appear to have persistent signs by physical examination that, while they might sug- gest the presence of high cervical myelopathy, remain isolated, non-progressive and apparently benign. To document and quantify these apparently benign craniocervical signs (ABCS) a cohort of 477 individuals with achondroplasia was retrospectively analyzed and information regarding persistent neurologic features suggestive of high cervical myelop- athy was recorded in a REDCap database. Results: Within this cohort, 151 individuals (31.7%) had neurologic examinations that were in some manner concern- ing.Of these, 46 (30.5% of the subpopulation) required cervicomedullary decompressive surgery. The remaining 105 had concerning signs by examination but no apparent evidence for clinically significant cervical myelopathy. Of those 105 individuals, 88 (83.8%; 18.4% of the entire population) remained neurologically intact throughout their follow-up, and without clinical sequelae. Conclusions: It appears that many individuals with achondroplasia, if carefully examined, may demonstrate isolated, initially concerning signs suggestive of cervical myelopathy, but in the vast majority these are benign and do not indicate need for aggressive neurosurgical intervention. Further investigations may help to identify ways to differenti- ate these benign features from the less common but more problematic true myelopathic ones. We postulate that the “neurologic leftovers” may arise from temporally remote, subtle damage to the spinal cord at the craniocervical junc- tion, which damage otherwise does not reach clinical relevance. Keywords: Achondroplasia, Cervical myelopathy, Cervicomedullary decompression, Craniocervical junction, Natural history, Neurologic disease Apparently benign craniocervical signs in achondroplasia: “neurologic leftovers” identified through a retrospective dataset Cory J. Smid1, Janet M. Legare1,2, Peggy Modaff1,2 and Richard M. Pauli1,2* Background abnormalities at the foramen magnum may result in com- pression of the upper cervical-spine and brainstem [2] that may be manifest as sudden unexpected death or cer- vical myelopathy [3]. Although the risk for sudden death is highest within the first year of life [4, 5], high cervical myelopathy can present at any age [6]. In young children, classic features suggestive of such cervical myelopathy include persistent hypotonia, asymmetric reflexes and/or strength, ankle clonus, or upgoing response to Babinski stimulation, while beyond infancy features may include Achondroplasia is characterized by disproportionate growth of the long bones, resulting in the most common form of short-limbed dwarfism [1]. Additionally, bony Correspondence: pauli@waisman.wisc.edu Correspondence: pauli@waisman.wisc.edu 1 Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, 1500 Highland Avenue, #359, Madison, WI 53705, USA USA Full list of author information is available at the end of the article USA Full list of author information is available at the end of the article *Correspondence: pauli@waisman.wisc.edu 1 Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, 1500 Highland Avenue, #359, Madison, WI 5370 USA © The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Smid et al. Orphanet J Rare Dis (2020) 15:301 Page 2 of 7 Page 2 of 7 increased fatigue, decreased walking endurance and a host of other features [3]. At least 8–17% of individu- als with achondroplasia will require cervicomedullary decompression surgery to minimize the effects of com- pression at the craniocervical junction [8–11]. signs” (ABCS) or “neurologic leftovers”. Isolated “neuro- logic leftovers” without further evidence for development of myelopathy have been reported previously only rarely in the literature concerning achondroplasia [12, 13]. However, it was our clinical impression that ABCS are common, but often without consequence on long-term outcome.h Due to risks of compression at the craniocervical junc- tion, all individuals with achondroplasia must undergo neurologic assessment including neurologic history and neurologic examination, imaging of the craniocervi- cal junction (by computerized tomography or magnetic resonance imaging), and polysomnography shortly after initial diagnosis [3]. Distinguishing those with need for urgent surgical intervention from infants who dis- play diagnosis-typical motor delays and axial hypotonia remains challenging. Criteria used in our clinics to deter- mine if craniocervical decompression is warranted have been published previously [3, 7]. Therefore, we conducted a retrospective study of 477 individuals with achondroplasia to assess the frequency, nature, and outcomes of patients with these “neurologic leftovers”. Frequency and character of ABCSh completed because of progressive syrinx development without abnormal neurologic findings), or there was no documentation of any neurologic evaluation, or, in one instance, decompression occurred subsequent to com- pletion of data analysis for this study. The most common abnormalities found by neurologic examination included bilateral ankle clonus, bilateral upgoing response to Babinski stimulation, and unilateral upgoing response to Babinski stimulation (Table 2). Sub- jects may have presented with multiple types of abnor- malities, and therefore the number of events shown in Table 2 exceeds the number of subjects. Thus, the remaining cohort of 105 individuals (22.0%) had abnormal findings by neurologic examination, but had not been judged to be in need of cervicomedullary decompression. Change in ABCS over time Demographic characteristics of the total cohort and the subpopulation of subjects with ABCS are shown in Table 1. A total of 53 males (50.5%) and 52 (49.5%) females had evidence for “neurologic lefto- vers” (Table 1). The median age of initial recognition of ABCS was 5.3 years. There is no significant difference by sex (p = 0.7424), or race/ethnicity (white/non-white p = 0.3041) between the total cohort and the subpopu- lation with ABCS. A large majority of subjects (91/105, 86.7%) was seen more than once, allowing for tracking of neurologic fea- tures over time. Age at which concerns were first iden- tified varied from 2 to 835 m with a mean of 126.0 m (and a median of 60 m). As summarized in Fig. 1, 35 of the 105 individuals (33.3%) had ABCS considered to be Early Transient—the abnormal neurologic feature was first recognized prior to 5 years of age but completely disappeared subsequently. Demographics Overall, 151 of 477 individuals (31.7%) had initially con- cerning neurologic examinations. Of these, 46 (30.5% of those with concerning examinations) were excluded from further consideration based on their need for cer- vicomedullary decompression surgery (Fig. 1). Note that those who were excluded because they underwent decompressive surgery does not include all patients within the entire population who underwent such sur- gery, since in some instances there was not an ante- cedent abnormal neurologic examination (e.g. surgery An abnormal neurologic examination raises concern. However, we began to recognize individuals with per- sistent but isolated neurologic findings by examination in whom there was no subsequent evidence for develop- ment of cervical myelopathy or further evidence for cer- vicomedullary compression; we refer to these apparently isolated findings as “apparently benign craniocervical Fig. 1 Compartmentalization of the cohort into four groups as defined in the text Smid et al. Orphanet J Rare Dis (2020) 15:301 Page 3 of 7 Page 3 of 7 Frequency and character of ABCSh Change in ABCS over time Nineteen individuals (18.1%) Table 1 Demographic characteristics Total cohort Percent (%) Those showing “leftovers” Percent (%) Sex Male 236 49.5 53 51 Female 241 50.5 52 49 Race/ethnicity White 415 87.0 94 90 Black 23 4.8 5 5 Hispanic 21 4.4 3 3 American Indian 2 0.4 1 1 Asian 35 7.3 5 5 Other 1 0.2 0 0 Total “white-alone” 398 83.4 92 88 Total “not-white-alone” 79 16.6 13 12 Table 1 Demographic characteristics Those showing “leftovers” Percent (%) Table 2 Frequency of neurologic signs of potential concern and proportion showing each sign who had normal or abnormal neurologic outcome a The number of events exceeds the total number of subjects since one patient could display more than one sign Type of concern Number of subjects N (% n/105)a Normal outcome, N Abnormal outcome, N Excessively brisk reflexes 24 (23%) 19 (19/24, 79%) 5 (5/24, 21%) Asymmetric reflexes, arms 3 (3%) 1 (1/3, 33%) 2 (2/3, 67%) Asymmetric reflexes, legs 15 (14%) 11 (11/15, 73%) 4 (4/15, 27%) Clonus, unilateral 27 (26%) 20 (20/28, 75%) 7 (7/27, 25%) Clonus, bilateral 39 (37%) 33 (33/39, 85%) 6 (6/39, 15%) Upgoing Babinski response, unilateral 35 (33%) 30 (30/35, 86%) 5 (5/35, 14%) Upgoing Babinski response, bilateral 39 (37%) 30 (30/39, 77%) 9 (9/39, 23%) Asymmetric strength, arms 3 (3%) 0 (0/3, 0%) 3 (3/3, 100%) Asymmetric strength, legs 6 (6%) 3 (3/6, 50%) 3 (3/6, 50%) Table 2 Frequency of neurologic signs of potential concern and proportion showing each s or abnormal neurologic outcome Table 2 Frequency of neurologic signs of potential concern and proportion showing each sign who had normal or abnormal neurologic outcome neurologic signs of potential concern and proportion showing each sign who had normal c outcome Smid et al. Orphanet J Rare Dis (2020) 15:301 Page 4 of 7 Page 4 of 7 clinically neurologically intact and had a benign outcome (Table 3). Of the 17 individuals who had abnormal out- comes, the most common abnormalities were partial bladder incontinence (n = 8), partial bowel incontinence (n = 6), and limitation of walking (n = 7), (with the num- ber of total events greater than the total number of sub- jects because an individual may have had more than one sequela). were considered to have Late Transient “leftovers”—the abnormal neurologic finding was initially recognized at or after 5 years of age and completely disappeared over time. Change in ABCS over time Combining the Early Transient and Late Transient groups, the interval between recognition and disappear- ance of “leftovers” averaged 26.8 m (median 18 m). A total of 42 individuals (40.0%) were classified as hav- ing Persistent ABCS—the initial abnormality on neuro- logic examination remained the same or diminished but did not disappear over time. However, note that within this Persistent “leftovers” group, 14 individuals were seen only once, and so, in fact, we cannot determine whether features did or did not subsequently remit in that sub- group. Nine individuals (8.6%) were considered to have Ongoing “leftovers”—in these the neurologic examina- tion was always abnormal, and over time the concerns became more prominent or additional features devel- oped. These latter groups, of persistent and ongoing abnormality by examination are those in which concern is likely to be the greatest. For those in the “Persistent” and “Ongoing” groups for whom there were assessments following recognition of the abnormal clinical features, length of follow-up ranged from 1 to 399 m, with a mean of 95.4 m and median of 72 m. Among the groups categorized as above, not surpris- ingly the Ongoing group was least likely to have a benign neurologic outcome (5/9, 55% normal). These are indi- viduals in whom abnormal neurologic features became more prominent, or additional features appeared over time. Compared with the remaining three groups com- bined, the Ongoing group was less likely to have a nor- mal outcome (p = 0.0351). There were no clear statistical differences for the proportion of neurologically intact outcomes among any of the remaining groups. Even in patients in whom one might have the greatest concern [groups Ongoing and Persistent having been seen more than once (together n = 37)], the outcome was completely benign regarding issues at the craniocervical junction in the large majority (28/37, 75.7%). In fact, of the 17 individuals subsequently defined as not neurologically normal, only three had craniocervi- cal junction concerns (alone) explanatory of their neu- rologic problems, one had both craniocervical junction issues and lumbosacral spinal stenosis, and one had craniocervical concerns plus an unrelated concurrent a The number of events exceeds the total number of subjects since one patient could be categorized as having more than one When should apparently isolated neurologic signs be cause for concern? When should apparently isolated neurologic signs be cause for concern? condition (congenital cerebellar hypoplasia). Most of those subsequently defined as being neurologically abnormal developed isolated features of either spi- nal claudication or lumbosacral spinal stenosis (12/17, 71%). While there are some individuals who present with une- quivocal progression of features of high cervical mye- lopathy in whom urgent neurosurgical intervention is appropriate, discovery of isolated, non-progressive and apparently benign neurologic features is considerably more common. Of those, 46 (30.5% of those with abnor- mal neurologic examinations) showed other features (by imaging, of polysomnography, onset of acute life-threat- ening events) that demonstrated that cervicomedullary decompression surgery was needed. The remaining 105 of 151 (69.5%) had neurologic examinations which ini- tially were concerning but did not develop further char- acteristics warranting neurosurgical intervention. In fact, in many (55/105, 52.4%) the unequivocally abnor- mal signs were transient. In many others (33/105, 31.4%) they persisted but without progression. In only a small minority (17/105, 16.2%) were any significant neurologi- cal concerns eventually present. Even in those, although categorized as having progressive issues, most had sub- sequent features unrelated to the upper cervical spine. Indeed, the vast majority of individuals with ABCS (83.8%) had a normal neurologic outcome. Individuals with initial presentations including asymmetric reflexes or asymmetric strength of the arms or legs appear to be the least likely to have a normal outcome (Table 2). Other predictors of a poor outcome include developing new features, or features becoming more prominent over time (Table 3). Outcomesh The outcomes of all subjects with ABCS are summa- rized in Table 3. Overall, 88 of the 105 (83.8%) subjects with such neurologic findings seemed to be otherwise Table 3 Outcomes by category as defined in the text a The number of events exceeds the total number of subjects since one patient could be categorized as having more than one Outcomesa Overall number of subjects N (%, N/105) “Early transient” number of subjects N (%, N/35) “Late transient” number of subjects N (%, N/19) “Persistent” number of subjects N (%, N/42) “Ongoing” number of subjects N (%, N/9) Normal function 89 (85%) 32 (91%) 17 (89%) 34 (81%) 5 (55%) Persistent hypotonia 2 (2%) 1 (3%) 0 0 1 (11%) Persistent disproportion- ate motor delay 3 (3%) 2 (6%) 0 0 1 (11%) Asymmetric strength 2 (2%) 0 1 (5%) 0 1 (11%) Hemiplegia 2 (2%) 0 0 1 (2%) 1 (11%) Marked limitation of walking 7 (7%) 0 0 4 (10%) 3 (33%) No independent walking 1 (1%) 0 0 1 (2%) 0 Partial bladder inconti- nence 8 (8%) 0 1 (5%) 5 (12%) 2 (22%) Complete bladder incontinence 0 0 0 0 0 Partial bowel inconti- nence 6 (6%) 0 1 (5%) 3 (7%) 2 (22%) Complete bowel incon- tinence 1 (1%) 0 0 0 1 (11%) Other abnormal 0 0 0 0 1 (11%) Table 3 Outcomes by category as defined in the text Smid et al. Orphanet J Rare Dis (2020) 15:301 Page 5 of 7 When should apparently isolated neurologic signs be cause for concern? Why might such “leftovers” arise? y g At this time there is no clear etiologic explanation for these ABCS. One attractive hypothesis is that these signs may reflect subtle, temporally remote damage to the upper cervical spinal cord [3]. Repetitive, minor com- pression from uncontrolled head movement, in combina- tion with infantile macrocephaly, marked hypotonia, and poor head control may more often result in subtle injury than has been previously recognized. Discussion P i b Persistent but apparently benign signs of possible cervi- cal myelopathy appear to be quite common in those with achondroplasia. If careful neurologic examinations are undertaken, 22% of all individuals with achondroplasia (excluding those with clearly actionable craniocervical junction compression in infancy) show evidence for what one might think are worrisome features referable to the upper cervical spinal cord. Yet in the vast majority, no progression arises and no clinically relevant or disabling characteristics develop. That is, most often these neuro- logic ABCS can be considered benign findings. i Previous descriptions similar to what we report here are rare. Reid et al. [12] may have been the first to rec- ognize that neurologic examination is not completely specific in assessment of possible cervicomedullary com- pression. More recently, this phenomenon may have been described in one instance by Sanders et al. [13]. In that retrospective study of 49 patients with achondroplasia seen between 1997 and 2017, one patient had a neuro- logic examination of concern but without evidence for cord compression by magnetic resonance imaging assess- ment [13]. Why more instances have not been noted in the literature is unclear. Pro forma examinations will fail to identify subtle neurologic abnormalities as were pre- sent in many of the individuals described here. Small numbers of patients seen within a particular clinic may mean uncertainty about what is and what is not within normal limits for a child with achondroplasia. The craniocervical junction in achondroplasia I di id l i h h d l i i The craniocervical junction in achondroplasia Individuals with achondroplasia are at an increased risk of neurological sequelae related to compression at the craniocervical junction [4, 5]. The foramen magnum is frequently small and “key-hole” shaped, constricting and compressing the upper cervical spine and brainstem. Within the first 4 years of life, children with achondropla- sia are at an increased risk for sudden death secondary to acute or chronic compression of the upper cervical spine and brainstem [5] with the greatest risk arising in the first year of life [4], while complications due to cervical mye- lopathy, however, may present at any age [6]. Individu- als presenting with unequivocal risk factors for clear-cut cervical myelopathy [6] or sudden infant death [7] need suboccipital decompression surgery. y g It is also uncertain how these clinical examination fea- tures relate to the imaging features described by Brouwer et al. [14] and van Dijk et al. [15]. This group demon- strated a remarkable frequency of cervical high-intensity intramedullary (CHII) lesions by magnetic resonance imaging (MRI) in adults with achondroplasia. This group initially examined MRIs of 25 adults with achondropla- sia and suspected neurologic lumbar spinal claudication. CHII lesions located between C1 and C2 were noted in 64% (16/25), without evidence for cervical spinal cord compression [15]. Since this initial cohort was ascer- tained because of neurologic concerns (although not of Smid et al. Orphanet J Rare Dis (2020) 15:301 Smid et al. Orphanet J Rare Dis (2020) 15:301 Page 6 of 7 These ABCS may reflect subtle, remote damage to the upper cervical spinal cord due to repetitive, minor com- pression from uncontrolled head movement in infancy.h the craniocervical junction), subsequently Brouwer et al. [14] analyzed the MRIs of 18 subjects with achondro- plasia who explicitly showed no clinical signs of cervical myelopathy. In this population, too, CHII lesions were common, observed in 39% (7/18), with only one of these individuals having anatomic evidence for compression at the craniocervical junction. We think it is conceivable that both the subtle clinical features described here and the imaging findings described in [14, 15] may have a common origin. The vast majority of individuals with these “neuro- logic leftovers” will have a normal outcome. Recommendations In order to minimize the risks associated with compres- sion at the craniocervical junction, all individuals with achondroplasia must undergo a neurologic assessment including: neurologic history and neurologic examina- tion, imaging of the craniocervical junction, and poly- somnography in early infancy [3]. However, if isolated abnormalities are noted by neurologic examination at this point or subsequently, without other evidence for cervical compression, then periodic clinical reassessment may be all that is warranted, with special attention paid to the type of abnormality, and the changes of neurologic features over time. If these remain isolated and non-pro- gressive, they should not precipitate a rush to neurosur- gical intervention. Those with asymmetries, those who develop new features, or whose features become more prominent over time should be more closely monitored, with additional neuroimaging to ensure that timely inter- vention, when necessary, is initiated. Data were collected from review of all available medi- cal records and entered into a supplement to a REDCap database [18] developed for a multi-center natural history study of achondroplasia [19]. Data regarding the pres- ence, characteristics, and changes of neurologic exami- nations of concern were entered, as were data related to overall outcome. Neurologic examinations were performed at each clini- cal visit. Concerning neurologic findings were defined as the following: excessively brisk reflexes; asymmetric reflexes in arms; asymmetric reflexes in legs; unilateral ankle clonus; bilateral ankle clonus; unilateral upgo- ing response to Babinski stimulation; bilateral upgoing response to Babinski stimulation; asymmetric strength in arms; and asymmetric strength in legs. In addition, note was made of hypotonia outside of the range expected for individuals with achondroplasia, and for disproportion- ate motor delays. Prior to 6 months of age, neurologic examinations were only considered abnormal if asym- metries were present since other neurologic abnormali- ties such as ankle clonus may persist for the first several months of life, but then disappear spontaneously around 6 months of age [20]. If patients were seen multiple times, changes in neurological findings could be documented Methodsh This study was limited to individuals with neurologic examinations of concern, and who ultimately were judged not to require cervicomedullary decompression. Indications for cervicomedullary decompression outside of neurologic examinations of concern, such as abnor- malities by polysomnography or MRI findings were not addressed. Additionally, patients presenting with neuro- logic examinations of concern only after cervicomedul- lary decompression are not be included in this study. This study was reviewed and approved by the Institu- tional Review Board of the University of Wisconsin-Mad- ison School of Medicine and Public Health, Madison, WI. All individuals with a diagnosis of achondroplasia who were seen in the Midwest Regional Bone Dyspla- sia Clinic between 1980 and 2017 were retrospectively analyzed. All individuals enrolled in this study had con- firmed molecular, radiographic, and/or clinical diagnoses of achondroplasia. All were comprehensively clinically assessed by a physician experienced in distinguishing achondroplasia from other skeletal dysplasias. Indi- viduals were excluded if they had confirmed molecular, radiographic, and/or clinical diagnoses of homozygous achondroplasia, compound heterozygosity (i.e. achon- droplasia + hypochondroplasia) or double heterozygosity (achondroplasia + another bone dysplasia [16, 17]. It is conceivable that certain patients with poor neuro- logic outcomes may have benefited from cervicomedul- lary decompression, yet surgery was never completed due to limited surveillance of the craniocervical junction. These patients were included in this study. The craniocervical junction in achondroplasia I di id l i h h d l i i Individuals with (1) asymmetric reflexes or asymmetric strength of the arms or legs or (2) development of new features or (3) features which become more prominent over time, appear to be less likely to have a normal outcome, and should be monitored closely. Author details 1 1 Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, 1500 Highland Avenue, #359, Madison, WI 53705, USA. 2 The Midwest Regional Bone Dysplasia Clinic, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA. Abbreviations 8. Ryken TC, Menezes AH. Cervicomedullary compression in achondroplasia. J Neurosurg. 1994;81:43–8. ABCS: Apparently benign craniocervical signs; CHII: Cervical high intensity intramedullary (lesions); MRI: Magnetic resonance imaging; REDCap: Research electronic data capture. 9. King JA, Vachhrajani S, Drake JM, Rutka JT. Neurosurgical implications of achondroplasia. J Neurosurg Pediatr. 2009;4:297–306. https://doi. org/10.3171/2009.3.PEDS08344. 9. King JA, Vachhrajani S, Drake JM, Rutka JT. Neurosurgical implications of achondroplasia. J Neurosurg Pediatr. 2009;4:297–306. https://doi. org/10.3171/2009.3.PEDS08344. Availability of data and materials 17. Flynn MA, Pauli RM. Double Heterozygosity in bone growth disorders: four new observations and review. Am J Med Genet A. 2003;121A:193–208. 17. Flynn MA, Pauli RM. Double Heterozygosity in bone growth disorders: four new observations and review. Am J Med Genet A. 2003;121A:193–208. HIPAA requirements preclude sharing of primary data upon which this study is based. HIPAA requirements preclude sharing of primary data upon which this study is based. 18. Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic data capture (REDCap)—a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform. 2009;42:377–81. https://doi.org/10.1016/j.jbi.2008.08.010. 18. Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic data capture (REDCap)—a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform. 2009;42:377–81. https://doi.org/10.1016/j.jbi.2008.08.010. Funding d f Funding for the publication costs of this study was, in part, from the Depart- ment of Pediatrics, University of Wisconsin—Madison School of Medicine and Public Health. 16. Pauli RM, Conroy MM, Langer LO, McLone DG, Nadich T, Franciosi R, Ratner IM, Copps SC. Homozygous achondroplasia with survival beyond infancy. Am J Med Genet. 1983;16:459–78. 16. Pauli RM, Conroy MM, Langer LO, McLone DG, Nadich T, Franciosi R, Ratner IM, Copps SC. Homozygous achondroplasia with survival beyond infancy. Am J Med Genet. 1983;16:459–78. Consent for publication Not applicable. 20. Sheller JM, Koch TK. Neurologic abnormalities of the newborn. In: Berg BO, editor. Child neurology: a clinical manual. 2nd ed. Philadelphia: Lipincott Williams & Wilkins; 1994. p. 219–29. 20. Sheller JM, Koch TK. Neurologic abnormalities of the newborn. In: Berg BO, editor. Child neurology: a clinical manual. 2nd ed. Philadelphia: Lipincott Williams & Wilkins; 1994. p. 219–29. Acknowledgements 10. Menezes AH. Achondroplasia. J Neurosurg Pediatr. 2009;4:295–6. https://doi. org/10.3171/2009.4.PEDS09148. 10. Menezes AH. Achondroplasia. J Neurosurg Pediatr. 2009;4:295–6. https://doi. org/10.3171/2009.4.PEDS09148. We extend gratitude to Adekemi Alade and Chengxin Liu for their guidance in REDCap navigation and data requests. BioMarin Pharmaceutical provided funding for the larger REDCap database upon which this study was grafted. We thank the PI and other co-PIs of that larger collaborative investigation, Julie Hoover-Fong, Johns Hopkins University; Jacqueline Hecht, University of Texas—Houston McGovern Medical School; and Michael Bober, Nemours/ Alfred I. DuPont Hospital for Children. g 11. Smid CJ, Legare JM, Modaff P, Pauli RM. Craniocervical junction issues after infancy in achondroplasia. Submitted for publication; 2020. 11. Smid CJ, Legare JM, Modaff P, Pauli RM. Craniocervical junction issues after infancy in achondroplasia. Submitted for publication; 2020. 12. Reid CS, Pyeritz RE, Kopits SE, Maria BL, Wang H, McPherson RW, Hurko O, Phillips JA III, Rosenbaum AE. Cervicomedullary compression in young patients with achondroplasia: value of comprehensive neurologic and respiratory evaluation. J Pediatr. 1986;110:522–30. y 13. Sanders VR, Sheldon SH, Charrow JC. Cervical spinal cord compression in infants with achondroplasia: should neuroimaging be routine? Genet Med. 2019;21:459–62. https://doi.org/10.1038/s41436-018-0070-0. 13. Sanders VR, Sheldon SH, Charrow JC. Cervical spinal cord compression in infants with achondroplasia: should neuroimaging be routine? Genet Med. 2019;21:459–62. https://doi.org/10.1038/s41436-018-0070-0. Authors’ contributions Study conception: RMP; Development of the REDCap database for this study: RMP; Patients evaluations: RMP, JML, PM; Access to patient records: PM; Data entry: CJS; Study design: RMP, CJS; Interpretation and analysis of data: CJS, RMP; Manuscript writing: CJS, RMP, JML; All authors read and approved the final manuscript. 14. Brouwer PA, Lubout CM, van Dijk JMC, Vleggeert-Lankamp CL. Cervical high- intensity intramedullary lesions in achondroplasia: aetiology, prevalence and clinical relevance. Eur Radiol. 2012;22:2264–72. https://doi.org/10.1007/ s00330-012-2488-0. 15. van Dijk JM, Lubout CM, Brouwer PA. Cervical high-intensity intramedullary lesions without spinal cord compression in achondroplasia. J Neurosurg Spine. 2007;6:304–8. Conclusions Isolated “neurologic leftovers” or ABCS are often a com- mon, benign finding in achondroplasia. Many individu- als may display such ABCS when carefully evaluated yet only a very small subset appear to have clinically relevant neurologic sequelae related to high cervical myelopathy. Smid et al. Orphanet J Rare Dis (2020) 15:301 Page 7 of 7 over time. Individuals were grouped in categories based on the change in neurological findings over time. 3. Pauli RM. Achondroplasia: a comprehensive clinical review. Orphanet J Rare Dis. 2019;14:1. https://doi.org/10.1186/s13023-018-0972-6. 3. Pauli RM. Achondroplasia: a comprehensive clinical review. Orphanet J Rare Dis. 2019;14:1. https://doi.org/10.1186/s13023-018-0972-6. 4. Pauli RM, Scott CI, Wassman RE, Gilbert EF, Leavitt LA, Ver Hoeve J, Hall JG, Partington MW, Jones KL, Sommer A, Feldman W, Langer LO, Rimoin DL, Hecht JT, Lebovitz R. Apnea and sudden unexpected death in infants with achondroplasia. J Pediatr. 1984;104:342–8. 5. Hecht JT, Francomano CA, Horton WA, Annegers JF. Mortality in Achondro- plasia. Am J Hum Genet. 1987;41:454–64. 6. Mukherjee D, Pressman BD, Krakow D, Rimoin DL, Danielpour M. Dynamic cervicomedullary cord compression and alterations in cerebrospinal fluid dynamics in children with achondroplasia: review of an 11-year surgical case series. J Neurosurg Pediatr. 2014;14:238–43. https://doi. org/10.3171/2014.5.PEDS12614. 4. Pauli RM, Scott CI, Wassman RE, Gilbert EF, Leavitt LA, Ver Hoeve J, Hall JG, Partington MW, Jones KL, Sommer A, Feldman W, Langer LO, Rimoin DL, Hecht JT, Lebovitz R. Apnea and sudden unexpected death in infants with achondroplasia. J Pediatr. 1984;104:342–8. i This study excludes all individuals without neuro- logic examinations of concern. In addition, individuals who underwent cervicomedullary decompression are excluded (Fig. 1), since those requiring cervicomedullary decompression had evidence for risk of sudden death, progressive cervical myelopathy and/or additional con- cerning features (e.g. abnormal imaging), and so did not demonstrate isolated, presumably benign ABCS. 5. Hecht JT, Francomano CA, Horton WA, Annegers JF. Mortality in Achondro- plasia. Am J Hum Genet. 1987;41:454–64. 6. Mukherjee D, Pressman BD, Krakow D, Rimoin DL, Danielpour M. Dynamic cervicomedullary cord compression and alterations in cerebrospinal fluid dynamics in children with achondroplasia: review of an 11-year surgical case series. J Neurosurg Pediatr. 2014;14:238–43. https://doi. org/10.3171/2014.5.PEDS12614. 7. Pauli RM, Horton VK, Glinski LP, Reiser CA. Prospective assessment of risks for cervicomedullary-junction compression in infants with achondroplasia. Am J Hum Genet. 1995;56:732–44. Publisher’s Note 1 Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, 1500 Highland Avenue, #359, Madison, WI 53705, USA. 2 The Midwest Regional Bone Dysplasia Clinic, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Received: 3 June 2020 Accepted: 13 October 2020 Received: 3 June 2020 Accepted: 13 October 2020 Ethics approval and consent to participate This study was reviewed and approved by the Institutional Review Board of the University of Wisconsin—Madison School of Medicine and Public Health. 19. Hoover-Fong JE, Alade AY, Bober MB, Hashmi S, Hecht J, Legare J, Little M, Liu K, McGready J, Modaff P, Pauli RM, Rodriguez-Buritica D, Schulze K, Serna E, Smid C. Achondroplasia natural history—a multi-center retrospective study in the US. Submitted for publication; 2020. 19. Hoover-Fong JE, Alade AY, Bober MB, Hashmi S, Hecht J, Legare J, Little M, Liu K, McGready J, Modaff P, Pauli RM, Rodriguez-Buritica D, Schulze K, Serna E, Smid C. Achondroplasia natural history—a multi-center retrospective study in the US. Submitted for publication; 2020. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References References 1. Oberklaid F, Danks DM, Jensen F, Stace L, Rosshandler S. Achondroplasia and hypochondroplasia. Comments on frequency, mutation rate, and radiologi- cal features in skull and spine. J Med Genet. 1979;16:140–6. 2. Yang SS, Corbett DP, Brough AJ, Heidelberger KP, Bernstein J. Upper cervical myelopathy in achondroplasia. Am J Clin Pathol. 1979;68:68–72. 1. Oberklaid F, Danks DM, Jensen F, Stace L, Rosshandler S. Achondroplasia and hypochondroplasia. Comments on frequency, mutation rate, and radiologi- cal features in skull and spine. J Med Genet. 1979;16:140–6. 2. Yang SS, Corbett DP, Brough AJ, Heidelberger KP, Bernstein J. Upper cervical myelopathy in achondroplasia. Am J Clin Pathol. 1979;68:68–72.
https://openalex.org/W1490445073	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-015-1612-3	English	null	A novel termini analysis theory using HTS data alone for the identification of Enterococcus phage EF4-like genome termini	BMC genomics	2,015	cc-by	6,147	METHODOLOGY ARTICLE Open Access Zhang et al. BMC Genomics (2015) 16:414 DOI 10.1186/s12864-015-1612-3 Zhang et al. BMC Genomics (2015) 16:414 DOI 10.1186/s12864-015-1612-3 © 2015 Zhang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Enterococcus faecalis and Enterococcus faecium are typical enterococcal bacterial pathogens. Antibiotic resistance means that the identification of novel E. faecalis and E. faecium phages against antibiotic-resistant Enterococcus have an important impact on public health. In this study, the E. faecalis phage IME-EF4, E. faecium phage IME-EFm1, and both their hosts were antibiotic resistant. To characterize the genome termini of these two phages, a termini analysis theory was developed to provide a wealth of terminal sequence information directly, using only high-throughput sequencing (HTS) read frequency statistics. Results: The complete genome sequences of phages IME-EF4 and IME-EFm1 were determined, and our termini analysis theory was used to determine the genome termini of these two phages. Results showed 9 bp 3′ protruding cohesive ends in both IME-EF4 and IME-EFm1 genomes by analyzing frequencies of HTS reads. For the positive strands of their genomes, the 9 nt 3′ protruding cohesive ends are 5′-TCATCACCG-3′ (IME-EF4) and 5′-GGGTCAGCG-3′ (IME-EFm1). Further experiments confirmed these results. These experiments included mega-primer polymerase chain reaction sequencing, terminal run-off sequencing, and adaptor ligation followed by run-off sequencing. Conclusion: Using this termini analysis theory, the termini of two newly isolated antibiotic-resistant Enterococcus phages, IME-EF4 and IME-EFm1, were identified as the byproduct of HTS. Molecular biology experiments confirmed the identification. Because it does not require time-consuming wet lab termini analysis experiments, the termini analysis theory is a fast and easy means of identifying phage DNA genome termini using HTS read frequency statistics alone. It may aid understanding of phage DNA packaging. Keywords: Termini analysis theory, Antibiotic-resistant Enterococcus, Phage, Genome termini Vancomycin was one of the last antibiotics to remain reliably effective against enterococci [12]. The continu- ous overuse and misuse of antibiotics has produced antibiotic-resistant Enterococcus. These, most notably vancomycin-resistant Enterococcus, have become a threat in nosocomial settings. However, phage therapy has great promise in antibiotic-resistant Enterococcus treatment. For this reason, the identification and study of phages for antibiotic-resistant Enterococcus may have a significant medical impact in the near future. A novel termini analysis theory using HTS data alone for the identification of Enterococcus phage EF4-like genome termini Xianglilan Zhang1†, Yahui Wang1,2†, Shasha Li1, Xiaoping An1, Guangqian Pei1, Yong Huang1, Hang Fan1, Zhiqiang Mi1, Zhiyi Zhang1, Wei Wang1, Yubao Chen3* and Yigang Tong1* * Correspondence: allexchen@126.com; tong.yigang@gmail.com †Equal contributors 3Beijing Computing Center, Beijing 100094, P.R. China 1State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P.R. China Full list of author information is available at the end of the article Background Enterococcus faecalis and Enterococcus faecium are the most common enterococcal bacteria cultured from humans, making up more than 90 % of clinical isolates [1]. Since the 1990s, Enterococcus strains have been found to be opportunistic Gram-positive pathogens, responsible for various diseases [2–8]. Recently, strains of enterococci resistant to vancomycin have emerged, with an increased incidence reported worldwide [9–11]. The most important information concerning a phage is its genome packaging, which affects its entire life cycle from initiation [13] to viral DNA replication [14], termin- ation, and regulation of transcription [15]. Identifying * Correspondence: allexchen@126.com; tong.yigang@gmail.com †Equal contributors 3Beijing Computing Center, Beijing 100094, P.R. China 1State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P.R. China Full list of author information is available at the end of the article Page 2 of 11 Zhang et al. BMC Genomics (2015) 16:414 2 mL of filtered (Millipore membranes, pore diameter 0.45 μM) sewage was mixed with 2 mL of 3*liquid LB medium and 100 μL E. faecalis or E. faecium that had been cultured overnight. To amplify the IME-EF4/IME- EFm1, the enrichment culture was incubated at 37 °C for at least 14 h with agitation and then centrifuged (10 min, 10,000 × g, 4 °C). The supernatant was filtered (Millipore membranes, pore diameter 0.45 μM) to re- move the residual bacterial cells. Then 100 μL of phage stock solution was mixed with 500 μL EF4/EFm1 cells in the exponential growth phase (OD600 = 0.2 to 0.5). The mixture was incubated at 37 °C for 5 min. Then 5 mL top agar (LB with 0.75 % agar) was added at 50 °C, and the mixture was poured into an LB plate that had been pre-warmed to 37 °C (double-layer method). This plate was then incubated overnight at 37 °C to produce phage plaques [21]. genome termini identification is crucial to the entire DNA packaging process. High-throughput sequencing (HTS) is an effective means of performing phage genome sequence analysis [16–19], including genome termini ana- lysis. HTS generates a large number of reads. Mining use- ful sequence information from these large datasets is a key problem in bioinformatics. Conventional methods first use these data to assemble the full sequence of a phage genome and then carry out molecular biology experiments to identify its termini. Background Unlike these conventional methods, our termini analysis theory can be used to study the phage’s genome termini and genome packaging directly using only the data from the HTS reads. This approach can prove a phage’s termini information without the sec- ondary molecular biological experiments required by con- ventional methods. It also reduces the time and expense of DNA packaging analysis. In this study, two newly introduced antibiotic-resistant Enterococcus phages, IME-EF4 and IME-EFm1, were an- alyzed using the current method. By using the termini analysis theory, we easily identified the IME-EF4/IME- EFm1’s termini and hypothesized that the IME-EF4 and IME-EFm1 both have special 9 bp 3′ protruding cohe- sive ends. Further molecular biological experiments con- firmed this hypothesis. This paper is the first to propose the termini analysis theory, which is a means of identify- ing a phage’s termini without any wet-lab termini con- formation experiment. IME-EF4/IME-EFm1 genome DNA extraction IME-EF4/IME-EFm1 DNA was extracted based on a method described in a previous work [22]. In brief, DNase I and RNase A (Thermo Scientific, America) were added to the phage IME-EF4 stock solution to a final concentra- tion of 1 μg/ml. The mixture was incubated overnight at 37 °C. Then samples were incubated at 80 °C for 15 min to deactivate the DNase I. Lysis buffer (final concentra- tion, 0.5 % sodium dodecyl sulfate, 20 mM EDTA, and 50 μg/ml proteinase K) was added to samples, which were then incubated at 56 °C for 1 h. An equal volume of phe- nol was added to extract the DNA. After centrifugation at 7000 × g for 5 min, the aqueous layer was removed to a fresh tube containing an equal volume of phenol- chloroform-isoamyl alcohol (25:24:1) and centrifuged at 7000 × g for 5 min. The aqueous layer was collected, mixed with an equal volume of isopropanol, and stored overnight at −20 °C. The mixture was centrifuged at 4 °C for 20 min at 10,000 × g, and the DNA pellet was washed with 75 % ethanol. The DNA was then air dried at room temperature, resuspended in deionized water, and stored at −20 °C. Methods IME-EF4, IME-EFm1, and their bacterial strains The lytic Enterococcus phages IME-EF4 and IME-EFm1 were isolated from sewage from PLA Hospital 307 (Beijing, China), and the host bacteria for the two phages were isolated from clinical samples in the same hospital. The collaboration between the PLA Hospital 307 and the present laboratory meant that no specific permits were required for these studies. The collected samples were neither privately owned nor protected and did not involve any endangered or protected species. IME-EF4/IME-EFm1 high-throughput sequencing IME-EF4/IME-EFm1 isolation IME-EF4/IME-EFm1 high-throughput sequencing After extraction of the phage IME-EF4 and IME-EFm1 genome DNA, the genomes were using the semiconductor Enrichment cultures were used to isolate IME-EF4 and IME-EFm1 from sewage [20]. Specifically, approximately Fig. 1 Generation of reads in dsDNA using high-throughput sequencing (HTS). F represents forward sequences and R represents reverse sequences. Ter. refers to termini. The sequence of Read F starts with base A. The sequence of Read R starts with base T. The starting bases are shown with red backgrounds. Reads Ter. F and Ter. R begin with natural termini Fig. 1 Generation of reads in dsDNA using high-throughput sequencing (HTS). F represents forward sequences and R represents reverse sequences. Ter. refers to termini. The sequence of Read F starts with base A. The sequence of Read R starts with base T. The starting bases are shown with red backgrounds. Reads Ter. F and Ter. R begin with natural termini Zhang et al. BMC Genomics (2015) 16:414 Page 3 of 11 Fig. 2 Adaptors and primers used in the experiments. Adaptor 1 includes C1 and C2, and adaptor 2 includes C3 and C4. P2 is highlighted in green, and P3 is highlighted in blue sequencer in the Life Technologies Ion Torrent Personal Genome Machine (PGM) IonTorrent sequencer (IonTor- rent). This technology uses emulsion polymerase chain re- action (PCR) and a sequencing-by-synthesis approach [23]. The library preparation, amplification, and sequen- cing were performed according to the Ion Torrent™ sequencing protocols. The IME-EF4 and IME-EFm1 gen- ome DNA samples were sheared using Ion Shear™Plus Reagents. These DNA fragments were then ligated to Ion Torrent adapters for subsequent nick repair and purifica- tion. Purified DNA fragments of about 300 bp were selected by using E-Gel® SizeSelect™agarose gel. After amplification and purification of the selected library, emulsion PCR was used to process it. PCR was performed in a water-in-oil microreactor containing a single DNA molecule on a bead [24]. The H+ Ion Torrent signal was detected during the sequencing-by- synthesis. In this process, four fluorescently labeled nucleotides were added to the flowcell channels during DNA synthesis. The florescent light signals were de- tected using a genome analyzer, which was used for base calling [25]. Theorem 1 R ¼ Freqter Freqave ¼ 2 Lreads ð1Þ ð1Þ ð1Þ Proof 1. There are m identical genomes. As illustrated in Fig. IME-EF4/IME-EFm1 isolation 1, the high-throughput sequencing (HTS) ma- chine reads each read from 5′ to 3′. In this way, each genome with dsDNA has two termini. The frequency of the reads starting with a natural terminus is as follows: Freqter ¼ m ð2Þ ð2Þ Freqter ¼ m There are a total of Nr reads in the m genomes. As shown in Fig. 1, two different reads, F and R, start with base A and base T. In this way, the average frequency of all reads is as follows: Freqave ¼ Nr 2 L ð3Þ ð3Þ The ratio of Freqter to Freqave is as follows: The ratio of Freqter to Freqave is as follows: R ¼ Freqter Freqave ¼ m Nr 2L ¼ 2 m L Nr ¼ 2 Lreads ð4Þ ð4Þ Termini analysis theory Our termini analysis theory provides a theoretical fre- quency ratio of terminal reads to general reads. In the experiment, we choose only 300 bp reads rather than the whole amount of reads for analysis. Such a library selection will decrease the practical frequency ratio. For- tunately, we can still practically distinguish the termini from the other reads (see results). A phage with a linear double-stranded DNA (dsDNA) has terminal repetitions. These repetitions are used for homologous recombination during the phage’s DNA replication process. The phage’s dsDNA can be circu- larized through the genome terminal repetitions. This makes the phage’s natural genome termini, which are cleaved by a terminase, difficult to identify. In this study, a termini analysis theory, which can be used to find natural termini using the read frequency, is described. Mega-primer PCR sequencing PCR amplification was performed on the IME-EF4 DNA genome to determine more about the IME-EF4 complete genome sequence acquired using the HTS data. The PCR involved mega-primer-guided polymerization through the protruding cohesive end. Suppose that there are m identical genomes and the length of each genome is L. All the genomes are divided into Nr short sequences. Each short sequence is called a read. The average length of the reads is Lreads. Table 1 Prepared adaptor list Adaptor Sequence (From 5′ to 3′) Adaptor C1-P P-GCCGGAGCTCTGCAGATATC 1 C2 GATATCTGCAGAGCTCCGGC-CGGTGATGA Adaptor C3 GCCGGAGCTCTGCAGATATC-TCATCACCG 2 C4-P P-GATATCTGCAGAGCTCCGGC Table 2 Prepared primer list Primer Sequence (From 5′ to 3′) P1 CTCTAGTTTGTTGCGTGCGTAAATC P2 GATATCTGCAGAGCTCCGGC P3 GCCGGAGCTCTGCAGATATC P4 AGGTACGGACCGCAATGGGTTGGGA Table 2 Prepared primer list Primer Sequence (From 5′ to 3′) P1 CTCTAGTTTGTTGCGTGCGTAAATC P2 GATATCTGCAGAGCTCCGGC P3 GCCGGAGCTCTGCAGATATC P4 AGGTACGGACCGCAATGGGTTGGGA Table 1 Prepared adaptor list Adaptor Sequence (From 5′ to 3′) Adaptor C1-P P-GCCGGAGCTCTGCAGATATC 1 C2 GATATCTGCAGAGCTCCGGC-CGGTGATGA Adaptor C3 GCCGGAGCTCTGCAGATATC-TCATCACCG 2 C4-P P-GATATCTGCAGAGCTCCGGC Zhang et al. BMC Genomics (2015) 16:414 Page 4 of 11 Fig. 3 Read occurrence distribution. Blue and red circles indicate the HTS reads of IME-EF4 and IME-EFm1 samples, respectively. The rank in the x-axis refers to the relative frequency of each read Fig. 3 Read occurrence distribution. Blue and red circles indicate the HTS reads of IME-EF4 and IME-EFm1 samples, respectively. The rank in the x-axis refers to the relative frequency of each read GACCGCAATGGGTTGGGA–3′). The Sangon Company synthesized the primers (Beijing, China). Terminal run-off sequencing The IME-EF4 complete genome was used as the template for terminal run-off sequencing, the process of which is described in [26]. The 3′ end of the IME-EF4 genome was marked using the primer P1 (5′–CTCTAGTTTGTTG CGTGCGTAAATC–3′). The 5′ end of the IME-EF4 gen- ome was marked using the primer P4 (5′–AGGTACG Ligation of the adaptors to the termini As shown in Table 3, the occurrence rates of terminal reads to gen- eral reads were 196 for the positive strand of IME-EF4, 344 for the negative strand of IME-EF4, 246 for the positive strand of IME-EFm1, and 341 for the negative strand of IME-EFm1. Biological experiments were used to verify our conclusion (see behind). Ligation of the adaptors to the termini To prove that IME-EF4 is linear dsDNA ending with a 3′ 9 nt protruding cohesive end, two pairs of adaptors ligated with the ends of IME-EF4 we created. They are Fig. 4 Occurrence rates of numbers of reads. Blue and red bars indicate the HTS reads of IME-EF4 and IME-EFm1 samples, respectively. The number on the x-axis represents the range from the last number to the next number Fig. 4 Occurrence rates of numbers of reads. Blue and red bars indicate the HTS reads of IME-EF4 and IME-EFm1 samples, respectively. The number on the x-axis represents the range from the last number to the next number Page 5 of 11 Zhang et al. BMC Genomics (2015) 16:414 Table 3 IME-EF4 and IME-EFm1 terminal sequence frequency statistics Phage Strand Ave. Freq. Ter. Freq. Ter:Freq: Ave:Freq: Terminal Sequence IME-EF4 Positive 6.73 1,322 196 ATTAGTTTCTTCAAAAAATT Negative 6.73 2,318 344 CTTTCGCTTAAACGAATCTC IME-EFm1 Positive 12.95 3,194 246 ATTAATTCGTTATAAAAAGG Negative 12.95 4,412 341 CTCTTCTTCGCACGAAATTC shown in Fig. 2. Adaptor 1 was created by combining C1 and C2 and adaptor 2 by combining C3 and C4. To ligate adaptors with IME-EF4 terminal sequences, phos- phoric acids were added to C1 and C4. The prepared adaptors are shown in Table 1. At the same time, primers were prepared for use in the next step of PCR amplification, as illustrated in Fig. 2. The primer se- quences are shown in Table 2. The Sangon Company prepared the oligonucleotides for these adaptors and primers. The adaptor oligonucleotide mixtures were hybridized by running the ligation program on a PCR machine. To ligate the IME-EF4 genome with the prepared adaptor, phosphoric acid was added for the IME-EF4 genome end repair. Specifically, 800 ng of the IME-EF4 genome was diluted to 16 μL using nuclease-free water, and prepared the end repair mix. Then the phosphate groups were added, also through PCR. The adaptor-ligated DNA was purified as listed in the NEBNext® Fast DNA Library Prep Set for Ion Torrent™instruction manual (version 3.1). The purified DNA samples were used as templates for PCR amplifica- tion and sequencing with the primer in two sets, P1 and P2, and P3 and P4. termini of phage IME-EF4 and IME-EFm1. IME-EF4/IME-EFm1 sequence assembly and identification of termini The IME-EF4 and IME-EFm1 sequence assembly results were acquired using Newbler (version 2.9, Roche). As shown in Table 4, the genome size of IME-EF4 is 40,713 bp and the read matches percentage was more than 97 %, while the genome size of IME-Efm1 is 42,599 bp and the read matches percentage was more than 95 %. The reads were then aligned to the reference assembly using a CLC Genomics Workbench (version 3.6.1). The CLC module was run with default parameters (mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.5, and similarity 0.8) using FASTQ files as read input and using FNA files as reference input. The results are shown in Fig. 5. The reference genomes are indexed as IME-EF4_Circled and IME-EFm1_Circled, respectively. For IME-EF4, the sequence TCATCACCG is the 9 nt tail that connects the 5′ and 3′ ends. For IME-EFm1, the sequence GGGTCAGCG is the 9 nt tail that connects 5′ and 3′ ends. Two 5′ ends start with ATTAGTTTCT TCAAAAAATT (IME-EF4) and ATTAATTCGTTATAA AAAGG (IME-EFm1). Two 3′ ends start with GAGAT TCGTTTAAGCGAAAG (IME-EF4) and CTCTTCTTC GCACGAAATTC (IME-EFm1). Fig. 6 shows the positions of terminal sequences in IME-EF4 and IME-EFm1, each with a 9 nt protruding cohesive end. The last bases of the 3′ termini were located at position 40,704 (IME-EF4, pos- ition = genome size (40,713) −protruding cohesive end size (9)) and 42,590 (IME-EFm1, position = genome size (42,590) −protruding cohesive end size (9)). IME-EF4/IME-EFm1 high-throughput sequencing (HTS) analysis IME-EF4/IME-EFm1 high-throughput sequencing (HTS) analysis IME-EF4/IME-EFm1 high-frequency read statistics g q y All HTS reads from the IME-EF4 and IME-EFm1 samples were statistically analyzed and then their frequencies were ranked in descending order. As shown in Fig. 3, IME-EF4 and IME-EFm1 HTS read data have two significant high- frequency reads, beginning with CTTTCGCTTAAACG AATCTC and ATTAGTTTCTTCAAAAAATT, respect- ively. The HTS data of IME-EF4 and IME-EFm1 share similar sequence occurrence frequency curves. Fig. 4 shows that more than 99 % of reads have frequencies below 420 for IME-EF4 and below 810 for IME-EFm1. Using the termini analysis theory, it was concluded that the two reads with the highest frequencies were the As shown in Fig. 5, there were fewer than 20 cases of short reads with the 9 nt protruding cohesive end. As Table 4 IME-EF4 and IME-EFm1 sample sequence assembly results Phage Length # Matched Reads # Total Reads Relative Matches Min Coverage Max Coverage IME-EF4 40,713 548,309 563,429 97.32% 11 7,215 IME-EFm1 42,599 1,103,439 1,154,818 95.55% 51 9,657 Zhang et al. BMC Genomics (2015) 16:414 Page 6 of 11 Fig. 5 (a) IME-EF4 and (b) IME-EFm1 mapping results. The mapped reads were acquired from the original HTS data. The 3′ terminal sequences are underlined in orange, the 5’ terminal sequences are underlined in dark red, and the 3′ protruding cohesive ends are underlined in blue Fig. 5 (a) IME-EF4 and (b) IME-EFm1 mapping results. The mapped reads were acquired from the original HTS data. The 3′ terminal sequences are underlined in orange, the 5’ terminal sequences are underlined in dark red, and the 3′ protruding cohesive ends are underlined in blue Fig. 5 (a) IME-EF4 and (b) IME-EFm1 mapping results. The mapped reads were acquired from the original HTS data. The 3′ terminal sequences are underlined in orange, the 5’ terminal sequences are underlined in dark red, and the 3′ protruding cohesive ends are underlined in blue Fig. 5 (a) IME-EF4 and (b) IME-EFm1 mapping results. The mapped reads were acquired from the original HTS data. The 3′ terminal sequences are underlined in orange, the 5’ terminal sequences are underlined in dark red, and the 3′ protruding cohesive ends are underlined in blue Zhang et al. BMC Genomics (2015) 16:414 Page 7 of 11 Fig. 6 Terminal sequences and their positions. Pos. refers to base positions. IME-EF4/IME-EFm1 high-frequency read statistics The dark orange arrows indicate the 5’ terminal sequences in HTS read data, and the light yellow arrows indicate the 3’ terminal sequences in HTS read data Fig. 6 Terminal sequences and their positions. Pos. refers to base positions. The dark orange arrows indicate the 5’ terminal ead data, and the light yellow arrows indicate the 3’ terminal sequences in HTS read data cohesive ends. The reason is explained as follows. If a terminase introduces a double-stranded break into the cos site of the IME-EF4/IME-EFm1, the 5′ and 3′ ter- minal sequences should ligate to each other without any internal sequence between them. However, this does not match the results of the present assembly experiments, shown in Table 3 and Fig. 3, the 9 nt protruding cohe- sive end was not detected in the most frequently occur- ring reads. Considering that HTS generates the dsDNA reads, it is possible that mature phage IME-EF4 and IME-EFm1 DNA molecules are the ones that have the 9 nt single-stranded 3′ ends, which are called protruding Fig. 7 (a) IME-EF4 and IME-EFm1 concatemer processing pathways and (b) terminal sequence details. The packaging of concatemeric dsDNA is initiated from the end generated by the initiation cleavage, which then proceeds unidirectionally before being terminated by the termination cleavage. IME-EF4/IME-EFm1 terminases introduce nicks staggered by 9 bp concatemer (highlighted in red) at cosN and melts the strands by its helicase activity after recognition of cosB (●)and cosQ (O) for initiation and termination cleavage, respectively Fig. 7 (a) IME-EF4 and IME-EFm1 concatemer processing pathways and (b) terminal sequence details. The packaging of concatemeric dsDNA is initiated from the end generated by the initiation cleavage, which then proceeds unidirectionally before being terminated by the termination cleavage. IME-EF4/IME-EFm1 terminases introduce nicks staggered by 9 bp concatemer (highlighted in red) at cosN and melts the strands by its helicase activity after recognition of cosB (●)and cosQ (O) for initiation and termination cleavage, respectively Fig. 7 (a) IME-EF4 and IME-EFm1 concatemer processing pathways and (b) terminal sequence details. The packaging of concatemeric dsDNA is initiated from the end generated by the initiation cleavage, which then proceeds unidirectionally before being terminated by the termination cleavage. IME-EF4/IME-EFm1 terminases introduce nicks staggered by 9 bp concatemer (highlighted in red) at cosN and melts the strands by its helicase activity after recognition of cosB (●)and cosQ (O) for initiation and termination cleavage, respectively Zhang et al. IME-EF4/IME-EFm1 high-frequency read statistics This means that a repeated sequence between the 5′ and 3′ terminal sequences should be detectable. However, there were few short reads including both 5′ and 3′ terminal sequences in the HTS data. Since the linear phage genome was circularized through the genome terminal repetitions during the replication of phage genome, the short reads including 5’ and 3’terminal se- quences found in the HTS data might be the inter- mediate form of the circularized linear phage genome. Therefore, IME-EF4 and IME-EFm1 may both have 9 bp 3′ protruding cohesive ends. As shown in Fig. 7, the terminase cleaves the IME-EF4/IME-EFm1 dsDNA from cosB and cosQ, which has a 9 nt protruding cohe- sive end between them. IME-EF4/IME-EFm1 high-frequency read statistics BMC Genomics (2015) 16:414 Page 8 of 11 hromatograms of the three molecular biology experiments, including (a) IME-EF4 complete genome sequencing, (b) IME-EF4 terminal equencing, and (c) IME-EF4 adaptor ligation to the terminal sequences Fig. 8 Chromatograms of the three molecular biology experiments, including (a) IME-EF4 complete genome sequencing, (b) IME-EF4 terminal run-off sequencing, and (c) IME-EF4 adaptor ligation to the terminal sequences Fig. 8 Chromatograms of the three molecular biology experiments, including (a) IME-EF4 complete genome sequencing, (b) IME-EF4 terminal run-off sequencing, and (c) IME-EF4 adaptor ligation to the terminal sequences Fig. 8 Chromatograms of the three molecular biology experiments, including (a) IME-EF4 complete genome sequencing, (b) IME-EF4 termina run-off sequencing, and (c) IME-EF4 adaptor ligation to the terminal sequences Page 9 of 11 Zhang et al. BMC Genomics (2015) 16:414 Fig. 9 Hypothetical genome dsDNA protruding cohesive end situations Fig. 9 Hypothetical genome dsDNA protruding cohesive end situations as shown in Fig. 5. In another assumption, the IME-EF4/ IME-EFm1 had a 5′ protruding cohesive end. Because the HTS experiment uses the T4 DNA polymerase and because T4 DNA polymerase catalyzes the synthesis of DNA in the 5′- > 3′ direction with 3′- > 5′ exonuclease activity rather than with 5′- > 3′ exonuclease function, the 5′ protruding cohesive end must be repaired. This means that a repeated sequence between the 5′ and 3′ terminal sequences should be detectable. However, there were few short reads including both 5′ and 3′ terminal sequences in the HTS data. Since the linear phage genome was circularized through the genome terminal repetitions during the replication of phage genome, the short reads including 5’ and 3’terminal se- quences found in the HTS data might be the inter- mediate form of the circularized linear phage genome. Therefore, IME-EF4 and IME-EFm1 may both have 9 bp 3′ protruding cohesive ends. As shown in Fig. 7, the terminase cleaves the IME-EF4/IME-EFm1 dsDNA from cosB and cosQ, which has a 9 nt protruding cohe- sive end between them. as shown in Fig. 5. In another assumption, the IME-EF4/ IME-EFm1 had a 5′ protruding cohesive end. Because the HTS experiment uses the T4 DNA polymerase and because T4 DNA polymerase catalyzes the synthesis of DNA in the 5′- > 3′ direction with 3′- > 5′ exonuclease activity rather than with 5′- > 3′ exonuclease function, the 5′ protruding cohesive end must be repaired. Experimental verification To confirm the conclusions drawn from the HTS data analysis, three molecular biology experiments were per- formed on IME-EF4. These experiments showed that the high-frequency reads were indeed the termini, which means that this termini analysis theory can be used to locate the phage’s termini and their positions using only HTS data even if the procedure is performed quickly. Mega-primer PCR sequencing g p q g To identify the IME-EF4 complete genome sequence acquired by the HTS data, PCR amplification was performed on the IME-EF4 DNA genome. The PCR involved mega-primer-guided polymerization through the protruding cohesive end. The genome sequence snapshot including the 9base is shown in Fig. 8(a), where the upstream sequence was …GAGATTCGTTT AAGCGAAAG and the downstream sequence was AT TAGTTTCTTCAAAAAATT… The results of PCR were consistent with those of HTS data statistical analysis (Fig. 5(a)). It proves that both the IME-EF4 Fig. 10 Agarose gel electrophoresis to confirm adaptor ligation. “M” represents marker, “1” is the PCR result of the 3′ IME-EF4 genome sequence ligated with adaptor 1 (primer P1 and P2), “3” is the PCR result of the 5′ IME-EF4 genome sequence ligated with adaptor 2 (primer P3 and P4), and “2” and “4” are the negative control PCR results of the 3′ and 5′ IME-EF4 genome sequences without any adaptor Fig. 10 Agarose gel electrophoresis to confirm adaptor ligation. “M” represents marker, “1” is the PCR result of the 3′ IME-EF4 genome sequence ligated with adaptor 1 (primer P1 and P2), “3” is the PCR result of the 5′ IME-EF4 genome sequence ligated with adaptor 2 (primer P3 and P4), and “2” and “4” are the negative control PCR results of the 3′ and 5′ IME-EF4 genome sequences without any adaptor Fig. 10 Agarose gel electrophoresis to confirm adaptor ligation. “M” represents marker, “1” is the PCR result of the 3′ IME-EF4 genome sequence ligated with adaptor 1 (primer P1 and P2), “3” is the PCR result of the 5′ IME-EF4 genome sequence ligated with adaptor 2 (primer P3 and P4), and “2” and “4” are the negative control PCR results of the 3′ and 5′ IME-EF4 genome sequences without any adaptor Page 10 of 11 Zhang et al. BMC Genomics (2015) 16:414 complete genome and the protruding cohesive ends ac- quired from the HTS data statistics are correct. IME-EFm1, have enriched the knowledge of antibiotic- resistant Enterococcus phages, which is essential to future antibiotic-resistant Enterococcus phage therapy. Acknowledgments This research was supported by a grant from the National Hi-Tech Research and Development (863) Program of China (No. 2012AA022003 and No. 2014AA021402), the China Mega-Project on Infectious Disease Prevention (No. 2013ZX10004605, No. 2011ZX10004001, No. 2013ZX10004607-004 and No. 2013ZX10004217-002-003), and the State Key Laboratory of Pathogen and BioSecurity Program (No. SKLPBS1113). Adaptor ligation to the termini As shown in Fig. 10, the 3′ IME-EF4 genome sequence ligated to adaptor 1 was about 280 bp in size, and 5′ IME-EF4 genome sequence ligated to adaptor 2 was about 250 bp in size. These findings were consistent with experimental design (P1 and P2 illustrations in Fig. 2). The sequencing results are shown in Fig. 8(c). This further proves that the two adaptors (adaptor 1 and adaptor 2) had successfully ligated to the IME-EF4 gen- ome, which means the IME-EF4 genome has a 9 bp 3′ protruding cohesive end. Authors’ contributions XZ carried out the data analysis and drafted and revised the manuscript. YW performed the molecular biology experiments. SL, XA, and GP participated in the HTS implementation. YH and HF contributed to the proof of the termini analysis theory. ZM and ZZ adjusted the experimental design. WW contributed to the preparation of the figures. YC and YT designed and managed the experiments and revised the manuscript. All authors have read and approved the final manuscript. XZ carried out the data analysis and drafted and revised the manuscript. YW performed the molecular biology experiments. SL, XA, and GP participated in the HTS implementation. YH and HF contributed to the proof of the termini analysis theory. ZM and ZZ adjusted the experimental design. WW contributed to the preparation of the figures. YC and YT designed and managed the experiments and revised the manuscript. All authors have read and approved the final manuscript. Terminal Run-off sequencing As shown in Fig. 9, two different cases of dsDNA pro- truding cohesive ends hypothetically exist. Case 1 shows the 5′ protruding cohesive end situation, and case 2 describes the putative IME-EF4 situation of the 3′ pro- truding cohesive end. If the IME-EF4 has the protruding cohesive end situation according to cases 1, then there would be signals after either the terminal sequence GAGATTCGTTTAAGCGAAAG, ATTTTTTGAAGAA ACTAATA, or both of them in the terminal run-off sequencing result. However, as shown in Fig. 8(b), no signal was detected after the termini GAGATTCGT TTAAGCGAAAG in the positive strand or after the termini ATTTTTTGAAGAAACTAATA in the nega- tive strand. These results confirmed the conclusion that IME-EF4 has a linear, double-stranded DNA genome with a 9 nt 3′ protruding cohesive end, represented by case 2–the 3′ protruding cohesive end situation. Author details 1 1State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P.R. China. 2School of Life Science & Technology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P.R. China. 3Beijing Computing Center, Beijing 100094, P.R. China. Received: 4 July 2014 Accepted: 4 May 2015 Availability of supporting data The data sets supporting the results of this article are available in the NCBI GenBank repository, [IME-EF4 complete genome http://www.ncbi.nlm.nih.gov/nuccore/ 589892984?report=genbankand http://www.ncbi.nlm.nih.- gov/nuccore/641468964]. References Page 11 of 11 Page 11 of 11 Zhang et al. BMC Genomics (2015) 16:414 11. Hoshuyama T, Moriguchi H, Muratani T, Matsumoto T. 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Analysis of the IME-EF4/ IME-EFm1’s HTS data indicated that both IME-EF4 and IME-EFm1 have 9 bp 3′ protruding cohesive ends (TCATCACCG in IME-EF4 positive strand, and GGGTCAGCG in IME-EFm1 positive strand). Further molecular biological experiments, including mega- primer PCR sequencing, terminal run-off sequencing, and adaptor ligation to the termini, fully supported these conclusions. Results indicated that this termini analysis theory facilitated acquisition of important phage genome termini information through analysis of HTS data alone, with no further molecular biology experiments required. In addition, the current analysis of the newly isolated antibiotic-resistant E. faecalis and antibiotic-resistant E. faecium phages, IME-EF4 and 1. Fraser SL, Lim J, Donskey J, Salata R. Enterococcal Infections. Retrieved June. 2008;12:2009. 1. Fraser SL, Lim J, Donskey J, Salata R. Enterococcal Infections. Retrieved June. 2008;12:2009. 2. Moellering Jr RC. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W4239706210	http://journal.ipb.ac.id/index.php/bulagron/article/download/15799/11704	Indonesian	null	Pengaruh Pemberian Auksin (NAA) dengan Sitokinin (BAP, Kinetin dan 2ip) terhadap Daya Proliferasi Tanaman Kantong Semar (Nepenthes mirabilis) Secara In Vitro	Buletin Agrohorti/Buletin Agrohorti : Agrohorti Bulletin	2,015	cc-by-sa	4,749	ABSTRACT Auxin and sitokinin are usually used as in vitro plant growth regulators. Combination of auxin (NAA) with sitokinin (BAP, kinetin, and 2ip) on in vitro bud proliferation of Nepenthes mirabilis was find out. This research were designed using completely randomized design with two factors, auxin as a first factor and cytokinin as second factor. The combination of auxin and cytokinin used was NAA: 1 and 2 mg/l while BAP, kinetin, and 2ip was respectively as much as 0, 2.5, and 5 mg/l. The combination of growth regulator that added to Murashige and Skoog (MS) medium. The best result for bud proliferation was no sitokinin and additional NAA 1 mg/l as much as 5.2 buds in 10 weeks. Keywords: 2ip, BAP, in vitro, kinetin, NAA, Nepenthes mirabilis, shoot proliferation Agus Setyo Yudhanto dan Ni Made Armini Wiendi* Departemen Agronomi dan Hortikultura, Fakultas Pertanian, Institut Pertanian Bogor (Bogor Agricultural University), Jl. Meranti, Kampus IPB Darmaga, Bogor 16680, Indonesia Telp. & Faks. 62-251-8629353 e-mail agronipb@indo.net.id Penulis Korespondensi : nmarmini@gmail.com Kata kunci: 2ip, BAP, in vitro, kinetin, NAA, Nepenthes mirabilis, proliferasi tunas Pengaruh Pemberian Auksin (NAA) dengan Sitokinin (BAP, Kinetin dan 2ip) terhadap Daya Proliferasi Tanaman Kantong Semar (Nepenthes mirabilis) Secara In Vitro ngaruh Pemberian Auksin (NAA) dengan Sitokinin (BAP, Kinetin dan 2ip) terhadap Daya Proliferasi Tanaman Kantong Semar (Nepenthes mirabilis) Secara In Vitro Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) PENDAHULUAN Penelitian tentang pengaruh media terhadap pertumbuhan dan perkembangan nepenthes mirabilis secara in vitro telah dilakukan sebelumnya oleh Sayekti (2007) dan Alitalia (2008). Menurut Sayekti (2007) media perkecambahan nepenthes terbaik adalah ¼ KC dan ½ MS. Pada penelitian ini juga diperoleh tanaman yang membentuk kalus dan multiplikasi tunas pada minggu ke-8 setelah berkecambah. Media yang mampu memberikan respon pertumbuhan tersebut adalah ¼ KC + Thidiazuron (TDZ) dan ½ MS + TDZ. Pertumbuhan tanaman pada media ini berbeda dengan yang lain. Tanaman menjadi kerdil (abnormal), daun tidak berkantong, roset, berukuran kecil dengan jumlah yang banyak. Hal ini disebabkan oleh aktivitas TDZ sebagai sitokonin yang sangat aktif walaupun dalam konsentrasi yang sangat rendah. Penelitian bertujuan untuk mempelajari pengaruh kombinasi auksin (NAA) dengan sitokinin (BAP, kinetin dan 2iP) terhadap pertumbuhan tunas dan akar (ploriferasi) Nepenthes mirabilis secara in vitro. Indonesia adalah negara kepulauan yang berada pada garis khatulistiwa yang merupakan salah satu negara tropis di dunia. Sebagai negara tropis, Indonesia memiliki berbagai jenis flora dan fauna endemik yang tidak dimiliki oleh negara atau tempat lain di bagian dunia ini. Salah satu flora endemik yang hanya tumbuh di Indonesia adalah tanaman kantong semar (Nepenthes sp.). Keberadaan Nepenthes sp. termasuk spesies langka dan dilindungi oleh negara maupun dunia. Sebagai tanaman yang dilindungi maka perdagangan internasional tanaman yang berasal dari habitat alam harus dikontrol dengan ketat dan hanya diperkenankan untuk kepentingan non komersial tertentu dengan izin khusus. Menurut Phillip dan Lamb (1996) Nepenthes merupakan satu-satunya genus dari famili Nepenthaceae. Kurang lebih terdapat 83 spesies yang saat ini telah teridentifikasi tersebar di dunia dan 53 spesies diantaranya (>60%) terdapat di Indonesia. Kalimantan menempati urutan pertama dengan jumlah Nepenthes lebih dari 29 jenis. Berdasarkan hasil penelusuran spesimen di Herbarium Bogor, ditemukan bahwa di Sulawesi terdapat minimum 10 jenis, New Guinea 9 jenis, Maluku 4 jenis, dan Jawa hanya terdapat 2 jenis Nepenthes saja. ABSTRAK Auksin dan sitokinin umumnya digunakan sebagai zat pengatur tumbuh secara in vitro. Sudah ditemukan bahwa kombinasi auksin (NAA) dan sitokinin (BAP, kinetin, dan 2ip) untuk proliferasi tunas Nepenthes mirabilis secara in vitro. Penelitian ini dirancang menggunakan rancangan acak lengkap dua faktor, auksin sebagai faktor pertama dan sitokinin sebagai faktor kedua. Kombinasi auksin dan sitokinin yang digunakan adalah NAA: 1 dan 2 mg / l sedangkan BAP, kinetin, dan 2ip masing-masing sebanyak 0, 2,5, dan 5 mg / l. Kombinasi zat pengatur tumbuh yang ditambahkan ke media Murashige dan Skoog (MS). Hasil terbaik untuk proliferasi tunas yaitu perlakuan tanpa sitokinin dan tambahan NAA 1 mg / l sebanyak 5,2 tunas dalam 10 minggu. 276 Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) NAA, sedangkan faktor kedua yaitu jenis dan konsentrasi sitokinin berupa BAP, kinetin dan 2 iP masing-masing 0, 2.5 dan 5 ppm. Pelaksanaan NAA, sedangkan faktor kedua yaitu jenis dan konsentrasi sitokinin berupa BAP, kinetin dan 2 iP masing-masing 0, 2.5 dan 5 ppm. Pelaksanaan NAA, sedangkan faktor kedua yaitu jenis dan konsentrasi sitokinin berupa BAP, kinetin dan 2 iP masing-masing 0, 2.5 dan 5 ppm. Pelaksanaan kultur yang telah disediakan. Botol-botol yang telah diisi dengan larutan media agar sama rata ditutup rapat dengan plastik transparan. Botol- botol yang telah tertutup plastik dengan baik disterilkan dengan autoklaf pada suhu 121ᴼC dan tekanan 17.5 psi selama 20 menit. Media steril disimpan dalam ruang kultur. Media ini dapat digunakan setelah inkubasi selama 4 hari dan bebas dari kontaminasi. Sterilisasi Alat dan Botol. Sterilisasi merupakan faktor terpenting dari keberhasilan pelaksanaan kultur jaringan. Alat-alat dan botol yang akan digunakan dalam pembuatan media dan penanaman dicuci hingga bersih kemudian disterilkan dengan menggunakan autoklaf selama satu jam pada suhu 121 dan tekanan 17.5 psi. Perhitungan lama waktu dimulai ketika tekanan telah berada pada 17.5 psi selama satu jam. Alat yang perlu disterilkan antara lain pinset, botol kultur, kertas saring, scalpel, cawan petri, dan pengaduk. Persiapan Ruang Tanam. Ruang tanam yang digunakan sebagai tempat menanam eksplan atau yang biasa disebut laminar air flow cabinet. Sebelum digunakan terlebih dahulu dibersihkan dengan menggunakan alkohol 96% dan disterilkan dengan sinar ultra violet selama 1 jam. Alat tanam dan bahan-bahan yang akan digunakan disemprot alkohol 70% sebelum di masukkan ke dalam laminar air flow cabinet. Hal ini dilakukan untuk menghindari resiko bahan penelitian terkontaminasi. Pembuatan Larutan Stok. Pembuatan larutan stok dilakukan sebelum pembuatan media tanam bertujuan untuk memudahkan dalam pencampuran media kultur. Media yang dibuat terdiri dari media Murashige Skoog (MS). Larutan stok dibuat sesuai dengan komposisi media MS yang disimpan dalam erlenmeyer dengan konsentrasi yang lebih pekat. Beberapa jenis larutan stok media MS tahan disimpan di ruangan dengan suhu ruang tetapi adapula yang harus disimpan dalam lemari es. Larutan stok F, myo- inositol dan vitamin disimpan dalam lemari es, sedangkan larutan stok A,B,C,D dan E dapat disimpan dalam suhu ruang. Penanaman. Eksplan tunas Nepenthes mirabilis diperoleh dari biji yang telah ditanam ± 1.5 tahun sejak dikecambahkan. Pengaruh Pemberian Auksin… BAHAN DAN METODE Penelitian dilaksanakan di Laboraturium Bioteknologi Tanaman, Departemen Agronomi dan Hortikultura, Fakultas Pertanian, Institut Pertanian Bogor, pada bulan Juni - Oktober 2010. Keunikan tanaman ini dapat dilihat dari bentuk dan struktur khas yang menjadikan daya tarik tersendiri. Morfologi kantong beraneka ragam, merupakan modifikasi daun, berfungsi sebagai perangkap serangga terutama semut dan dedaunan. Dalam kantong terdapat cairan berupa enzim yang dapat menghancurkan bagian-bagian tubuh serangga dan dedaunan sehingga menjadi sumber nutrisi untuk pertumbuhan tanaman ini. Selain bentuk yang beraneka ragam, ukuran dan warna kantong yang bervariasi sesuai dengan tempat tumbuh dan jenisnya menambah daya tarik tanaman (Mansur, 2006). Bahan tanaman digunakan eksplan Nepenthes mirabilis steril yang telah ditanam pada media ½ MS dan telah berumur ± 1.5 tahun sejak dikecambahkan.. Bahan untuk media tanam meliputi Zat Pengatur Tumbuh NAA 1 dan 2 mg/l (auksin), BAP, Kinetin dan 2iP masing-masing 0, 2.5, dan 5 mg/l (sitokinin), larutan stok MS, agar- agar 8 g/l, gula pasir 30 g/l dan aquades. Pengaturan pH media dengan menambahkan KOH (1 N) atau HCl (1 N). Bahan yang digunakan untuk sterilisasi berupa alkohol 70%. Bahan lain yang digunakan antara lain aquadest, karet gelang, plastik, tissue, spirtus dan label. Salah satu metode perbanyakan tanaman yang dapat ditempuh yaitu melalui kultur in vitro. Metode ini diharapkan mampu menghasilkan tanaman dalam skala besar dengan waktu yang relatif cepat serta kualitas tanaman yang dihasilkan menjadi lebih baik. Menurut Gunawan (1992), melalui kultur jaringan kebutuhan ketersediaan bibit tanaman dalam skala besar dapat terpenuhi. Sundorowati et al. (2002) juga menambahkan bahwa perbanyakan tanaman dengan teknik kultur jaringan (in vitro) telah banyak dilakukan untuk tanaman yang bernilai ekonomi tinggi atau tergolong langka dan sulit dipropagasi dengan cara konvensional. Alat-alat yang digunakan antara lain botol kultur, pipet, cawan petri, labu takar, bunsen, erlenmeyer, sprayer, mata pisau, scalpel, timbangan, gunting, pinset, autoclaf dan laminar air flow cabinet. Rak penyimpanan dilengkapi dengan lampu fluorescence dengan intensitas cahaya antara 500-750 lux sebagai sumber penyinaran dengan suhu ruangan diatur antara 20 -22 . Agus Setyo Yudhanto dan Ni Made Armini Wiendi Penelitian ini menggunakan metode Rancangan Acak Lengkap (RAL), dengan dua faktor yaitu perbandingan konsentrasi auksin dengan konsentrasi sitokinin. Faktor pertama yaitu konsentrasi auksin berupa 1 dan 2 ppm 277 Agus Setyo Yudhanto dan Ni Made Armini Wiendi Bul. Agrohorti 3 (3) : 276 – 284 (2015) Eksplan dipilih dari dalam botol yang memiliki penampilan baik, seperti pertumbuhan optimum, daun hijau tua, tidak berwarna kuning, tidak vitrus, tidak kekurangan hara dan tidak berkalus. Eksplan diambil dari tunas yang dipotong dari pangkal batang tanpa akar, tiap potongan berukuran 1-1.5 cm dan tanpa pemotongan tunas apikal. Potongan kecil ini kemudian ditanam di media perlakuan. Setiap botol kultur terdiri dari 3 eksplan. Botol kultur yang telah ditanami diletakkan di rak dalam ruang kultur di bawah cahaya penuh. Pembuatan Media Kultur. Media kultur yang digunakan dalam penelitian ini adalah media MS. Media MS dibuat dengan memipet 20 ml larutan stok A, B dan vitamin, 5 ml larutan stok C, D dan E, 10 ml larutan F dan Myoinositol ke dalam labu takar satu liter (Tabel Lampiran 1). Setelah itu ditambahkan NAA sebanyak 1 ml untuk mendapatkan media dengan konsentrasi 1mg/l dari stok 1g/l NAA dan BAP sebanyak 1 ml untuk mendapatkan media dengan konsentrasi 1mg/l dari stok 1g/l BAP. Kemudian gula dilarutkan sebanyak 30 g kedalam aquades dan dicampurkan ke dalam larutan media. Masukkan aquadest hingga tanda terra 1 liter. Untuk mengukur kemasaman media digunakan pH meter 5,8 diatur dengan menambahkan KOH atau HCl. Pemeliharaan. Pemeliharaan dilakukan dengan pengontrolan harian maupun mingguan. Botol kultur harus tetap dalam keadaan bersih dan dihindarkan dari media yang terkontaminasi. Penyemprotan alcohol 70% dilakukan tiap minggu dan penggantian tutup botol jika tutup botol yang lama telah rusak. Kondisi ruang kultur dipertahankan antara 20-22 ºC. Intensitas cahaya dipertahankan pada 500-750 lux dengan pencahayaan selama 16 jam penuh. Pengamatan. Pengamatan dilakukan selama 10 minggu. Pengamatan dilakukan setiap minggu. Peubah yang diamati antara lain: Pengamatan. Pengamatan dilakukan selama 10 minggu. Pengamatan dilakukan setiap minggu. Peubah yang diamati antara lain: Larutan media yang telah siap dipindahkan ke dalam panci pemanas lalu ditambahkan agar-agar sebanyak 8 gram. Larutan media tersebut dipanaskan sambil diaduk-aduk hingga mendidih. Botol-botol kultur yang telah steril disiapkan untuk diisi dengan larutan media yang telah dipanaskan. Larutan media agar yang telah mendidih dituangkan ke dalam botol-botol 1. Persentase kontaminasi 2. Presentase hidup 3. Warna tanaman 4. Jumlah tunas 5. Jumlah daun 6. Jumlah akar 278 Bul. Agrohorti 3 (3) : 276 – 284 (2015) 7. Jumlah kantong 8. Waktu inisiasi akar 9 Waktu inisiasi tunas 10. Waktu inisiasi daun 11. Waktu inisiasi kantong 10. Waktu inisiasi daun 11. Waktu inisiasi kantong Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) dikecambahkan. Kontaminasi pada kultur terjadi pada minggu kedua sebesar 0.8%, meningkat pada minggu keempat sebesar 4.0 %. Tingkat kontaminasi tertinggi terjadi pada minggu ketujuh sebesar 6.4%. Rekapitulasi sidik ragam menunjukkan pengaruh NAA nyata terhadap jumlah daun dan jumlah tunas. NAA memberikan pengaruh yang nyata pada 10 MST terhadap jumlah daun yang terbentuk. NAA juga memberikan pengaruh yang nyata pada 7-10 MST terhadap jumlah tunas. Sitokinin memberikan pengaruh yang nyata terhadap waktu inisiasi akar, waktu inisiasi daun dan jumlah akar pada 5-6 MST, 8 MST dan 10 MST. Sitokinin memberikan pengaruh sangat nyata pada jumlah akar pada 7 dan 9 MST. Waktu Inisiasi Ploriferasi Tunas. Berdasarkan hasil sidik ragam, kombinasi NAA dan sitokinin tidak memberikan pengaruh nyata pada waktu inisiasi ploriferasi tunas Nepenthes mirabilis baik secara tunggal maupun kombinasi. Waktu inisiasi ploriferasi tunas tercepat pada media tanpa penambahan zat pengatur tumbuh yaitu 5.2 HST, sedangkan waktu inisiasi ploriferasi tunas terlama pada media dengan pemberian BAP sebesar 2.5 mg/l yaitu 13.0 HST. Pemberian zat pengatur tumbuh menyebabkan waktu ploriferasi tunas menjadi lebih lama terlihat dari Tabel 2, dimana pemberian zat pengatur tumbuh berupa sitokinin maupun auksin tidak memberikan pengaruh waktu inisiasi tunas lebih cepat dibandingkan dengan tanpa pemberian zat pengatur tumbuh. Kontaminasi yang ditemukan selama pengamatan yaitu kontaminasi cendawan dan bakteri. Kultur yang terkontaminasi cendawan ditandai dengan adanya benang-benang hifa maupun spora cendawan pada eksplan, botol kultur ataupun media, sedangkan eksplan yang terkontaminasi bakteri ditandai dengan munculnya lendir pada eksplan maupun pada media. Menurut Gunawan (1992), inisiasi kultur yang bebas kontaminasi merupakan langkah yang sangat penting. g p g Kontaminan akan tumbuh dengan cepat pada media yang mengandung gula, vitamin dan mineral bila faktor kontaminasi tidak dihilangkan. Eksplan dapat mati akibat langsung dari serangan cendawan atau bakteri, atau secara tidak langsung akibat persenyawaan toksik yang diproduksi cendawan atau bakteri. Terjadinya kontaminasi pada eksplan diduga karena beberapa hal, antara lain kurang sterilnya ruang tanam maupun laminar air flow cabinet saat penanaman, peralatan yang digunakan tidak steril dan kurangnya perlakuan sterilisasi pada ruang kultur. Waktu inisiasi ploriferasi tunas dapat dilihat saat pengamatan harian beberapa hari setelah tanam (HST) yang ditandai dengan munculnya daun baru pada ruas atau ketiak daun. Tunas ini akan tumbuh dan mengeluarkan daun dan kantong baru setelah daun pertama. HASIL DAN PEMBAHASAN Kondisi Umum Tabel 1. Rekapitulasi sidik ragam respon peubah yang diamati pada kultur Nepenthes mirabilis secara in vitro Peubah Perlakuan KK (%) NAA Sitokinin NAA Sitokinin Inisiasi Akar tn * tn 30.539 Inisiasi Daun tn * tn 13.931 Inisiasi Kantong tn tn tn 21.607 Inisiasi Poriferasi Tunas tn tn tn 19.789 Jumlah Akar 2 MST tn tn tn 2.697 4 MST tn tn tn 8.320 5 MST tn * tn 11.298 6 MST tn * tn 13.841 7 MST tn ** tn 16.389 8 MST tn * tn 19.475 9 MST tn ** tn 20.986 10 MST tn * tn 22.538 Jumlah Daun 2 MST tn tn tn 8.030 4 MST tn tn tn 13.590 5 MST tn tn tn 13.046 6 MST tn tn tn 15.913 7 MST tn tn tn 17.171 8 MST tn tn tn 17.161 9 MST tn tn tn 17.160 10 MST tn tn tn 17.211 Jumlah Kantong 2 MST tn tn tn 7.577 4 MST tn tn tn 13.128 5 MST tn tn tn 12.089 6 MST tn tn tn 14.608 7 MST tn tn tn 16.525 8 MST tn tn tn 17.028 9 MST tn tn tn 17.464 10 MST tn tn tn 18.214 Jumlah Tunas 2 MST tn tn tn 14.337 3 MST tn tn tn 13.757 4 MST tn tn tn 12.900 5 MST tn tn tn 13.229 6 MST tn tn tn 13.174 7 MST * tn tn 13.194 8 MST * tn tn 13.663 9 MST * tn tn 14.713 10 MST * tn tn 15.686 Keterangan: tn : Tidak nyata pada uji F 5%; * : Nyata pada uji F 5%, ** : Sangat nyata pada uji F 5%; MST : Minggu Setelah Tanam; Semua data merupakan hasil transformasi (x+1) Eksplan tunas in vitro nepenthes yang digunakan dalam penelitian ini berasal dari biji yang dikecambahkan di media ½ MS, eksplan tersebut telah berumur ± 1.5 tahun sejak Keterangan: tn : Tidak nyata pada uji F 5%; * : Nyata pada uji F 5% Tanam; Semua data merupakan hasil transformasi (x+1) Eksplan tunas in vitro nepenthes yang digunakan dalam penelitian ini berasal dari biji yang dikecambahkan di media ½ MS, eksplan tersebut telah berumur ± 1.5 tahun sejak yang dikecambahkan di media ½ MS, eksplan tersebut telah berumur ± 1.5 tahun sejak 279 Bul. Agrohorti 3 (3) : 276 – 284 (2015) Dalam satu tanaman, tunas dapat tumbuh lebih dari satu sehingga pengamatan jumlah tunas dilakukan tiap minggunya setelah tunas pertama tumbuh. Tabel 2. Pengaruh pemberian BAP dan NAA terhadap wInisiasi ploriferasi tunas dan waktu inisiasi kantong Nepenthes mirabilis secara in vitro Perlakuan Waktu Ploriferasi Inisiasi Tunas (HST) Waktu Inisiasi Kantong (HST) Sitokinin (mg/l) 0 5.2 11.8 BAP 2.5 13.0 19.3 5.0 11.6 21.7 2iP 2.5 5.3 17.4 5.0 8.3 13.8 Kinetin 2.5 11.0 17.4 5.0 11.1 13.6 NAA (mg/l) 1.0 10.4 18.7 2.0 8.3 14.2 KK (%) 21.607 19.789 Tabel 2. Pengaruh pemberian BAP dan NAA terhadap wInisiasi ploriferasi tunas dan waktu inisiasi kantong Nepenthes mirabilis secara in vitro Perlakuan Waktu Ploriferasi Inisiasi Tunas (HST) Waktu Inisiasi Kantong (HST) Sitokinin (mg/l) 0 5.2 11.8 BAP 2.5 13.0 19.3 5.0 11.6 21.7 2iP 2.5 5.3 17.4 5.0 8.3 13.8 Kinetin 2.5 11.0 17.4 5.0 11.1 13.6 NAA (mg/l) 1.0 10.4 18.7 2.0 8.3 14.2 KK (%) 21.607 19.789 ruh pemberian BAP dan NAA terhadap wInisiasi ploriferasi tunas dan waktu inisiasi kantong nthes mirabilis secara in vitro Pengaruh Pemberian Auksin… Pemberian zat pengatur tumbuh tidak memberikan pengaruh terhadap waktu inisiasi ploriferasi tunas diduga adanya sitokinin endogen yang cukup tinggi di dalam tanaman induk 280 Bul. Agrohorti 3 (3) : 276 – 284 (2015) Nepenthes mirabilis yang dijadikan eksplan, sehingga pemberian sitokinin akan menghambat inisiasi ploriferasi tunas. Nepenthes mirabilis yang dijadikan eksplan, sehingga pemberian sitokinin akan menghambat inisiasi ploriferasi tunas. menyebabkan waktu inisiasi kantong menjadi lebih lama pula. Hal ini terlihat dari Tabel 2, dimana pemberian zat pengatur tumbuh berupa sitokinin maupun auksin tidak berpengaruh nyata waktu inisiasi kantong lebih cepat dibandingkan dengan tanpa pemberian zat pengatur tumbuh. Hal ini terjadi sejalan dengan pembentukan tunas, dimana waktu pembentukan tunas yang lebih cepat mempengaruhi waktu pembentukan kantong yang lebih cepat pula. Menurut Mufa’adi (2003) bahwa tanaman terpacu untuk lebih cepat melakukan multiplikasi tunas disebabkan oleh pemberian sitokinin BAP. Pada penelitian ini BAP dan hormon sitokinin yang lain tidak menyebabkan perbedaan yang nyata untuk menginisiasi ploreferasi tunas sehingga tanpa pemberian sitokinin pun sudah dapat menginisiasi tunas nepenthes mirabilis lebih cepat. Menurut Sudarmonowati et al. (2002), apabila tanpa hormon tidak berpengaruh nyata maka tidak diperlukan penggunaan BAP sehingga biaya produksi akan jauh lebih murah. Waktu Inisiasi Daun. Berdasarkan hasil sidik ragam, pemberian kombinasi NAA dan sitokinin tidak memberikan pengaruh yang nyata terhadap waktu inisiasi akar Nepenthes mirabilis. Pengaruh nyata diperoleh dari pemberian sitokinin secara tunggal. Bul. Agrohorti 3 (3) : 276 – 284 (2015) Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) memberikan pengaruh nyata terhadap jumlah tunas. Pengaruh yang nyata diperoleh dari pemberian NAA secara tunggal pada minggu 7-10 MST. Pemberian NAA sebesar 1 ppm menghasilkan jumlah tunas terbanyak yaitu sebesar 5.2 tunas/eksplan pada pengamatan 10 MST. Waktu inisiasi akar dapat dilihat beberapa hari setelah tanam (HST). Ditandai dengan terbentuknya bulu-bulu akar dan terlihat ujung akar berwarna putih. Setelah diketahui waktu inisiasi akar maka dilanjutkan dengan pengamatan jumlah akar yang dilakukan tiap minggunya. Jumlah Tunas. Berdasarkan sidik ragam, pemberian kombinasi NAA dan sitokinin tidak aruh pemberian NAA terhadap rata-rata jumlah tunas Nepenthes mirabilis pada 2-10 MST a in vitro Tabel 4. Pengaruh pemberian NAA terhadap rata-rata jumlah tunas Nepenthes mirabilis pada 2-10 MST secara in vitro NAA (mg/l) Jumlah Tunas Waktu (MST) 2 4 6 8 10 1.0 1.7 3.0 3.6 4.1 a 5.2 a 2.0 1.1 1.8 2.1 2.2 b 2.5 b KK (%) 14.3 12.9 13.2 13.7 15.7 Keterangan: Nilai yang diikuti oleh huruf yang sama pada kolom yang sama tidak berbeda nyata pada uji DMRT 5%, data merupakan hasil transformasi (x+1) Tabel 4. Pengaruh pemberian NAA terhadap rata-rata jumlah tunas Nepenthes mirabilis secara in vitro Jumlah Daun. Berdasarkan hasil sidik ragam, kombinasi NAA dan sitokinin tidak memberikan pengaruh nyata pada jumlah daun Nepenthes mirabilis baik secara tunggal maupun kombinasi. Pemberian media BAP sebesar 2.5 ppm mampu menghasilkan jumlah daun terbesar sebanyak 22.8 selama 10 MST. nepenthes. Hal ini dapat dilihat pada tabel dimana rata-rata jumlah daun nepenthes pada media dengan sitokinin 2.5 ppm lebih besar dibandingkan dengan media dengan sitokinin 5 ppm, begitu juga dengan pemberian NAA 1 ppm menunjukkan jumlah daun yang lebih besar dibandingkan dengan pemberian NAA 2 ppm. Peningkatan pemberian sitokinin maupun NAA terbukti menurunkan jumlah daun Peningkatan pemberian sitokinin maupun NAA terbukti menurunkan jumlah daun Tabel 5. Pengaruh Pemberian Auksin… Bul. Agrohorti 3 (3) : 276 – 284 (2015) Pemberian sitokinin berupa 2iP 5 mg/l mampu menghasilkan waktu inisiasi daun tercepat yaitu 4.0 HST, sedangkan pemberian sitokinin berupa BAP 5 ppm menghasilkan waktu inisiasi terlama yaitu 13.0 HST. Waktu Inisiasi Kantong. Waktu inisiasi kantong dapat dilihat beberapa hari setelah tanam (HST). Pengamatan waktu inisiasi kantong diawali dengan tumbuhnya daun baru yang disusul pembentukan kantong, kantong yang telah terbuka pada daun baru merupakan awal dari pengamatan waktu inisiasi kantong. Pengamatan jumlah kantong selanjutnya dilakukan setiap minggu setelah kantong pertama terbuka di setiap tanaman. Waktu inisiasi daun diamati beberapa hari setelah tanam (HST). Pengamatan waktu inisiasi daun dapat dilihat dengan munculnya daun baru melalui ujung tunas apikal maupun tunas lateral. Setelah waktu inisiasi daun diketahui maka dilanjutkan dengan pengamatan jumlah daun setiap minggunya. Berdasarkan hasil sidik ragam, kombinasi NAA dan sitokinin tidak memberikan pengaruh nyata pada waktu inisiasi kantong Nepenthes mirabilis baik secara tunggal maupun kombinasi. Waktu inisiasi kantong tercepat pada media tanpa penambahan zat pengatur tumbuh yaitu pada 11.8 HST, sedangkan waktu inisiasi kantong terlama pada media dengan pemberian BAP 5 mg/l yaitu pada 21.7 HST. Waktu Inisiasi Akar. Berdasarkan hasil sidik ragam, pemberian kombinasi NAA dan Sitokinin tidak berpengaruh nyata terhadap waktu inisiasi akar Nepenthes mirabilis. Pengaruh yang nyata diperoleh dari pemberian sitokinin secara tunggal. Pemberian zat pengatur tumbuh selain menyebabkan waktu inisiasi tunas lebih lama juga Tabel 3. Pengaruh pemberian sitokinin terhadap waktu inisiasi akar dan waktu inisiasi daun Nepenthes mirabilis secara in vitro Sitokinin (mg/l) Waktu Inisiasi Akar (HST) Waktu Inisiasi Daun (HST) 0 24.9 a 5.7 abc BAP 2.5 6.2 bc 11.1 ab 5.0 0.0 c 13.5 a 2iP 2.5 14.2 abc 5.8 abc 5.0 0.0 c 4.0 c Kinetin 2.5 13.5 abc 10.9 abc 5.0 19.0 ab 5.4 bc KK (%) 30.539 13.931 Keterangan: Nilai yang diikuti oleh huruf yang sama pada kolom yang sama tidak berbeda nyata pada uji DMRT 5% Data merupakan hasil transformasi (x+1) emberian sitokinin terhadap waktu inisiasi akar dan waktu inisiasi daun Nepenthes i i Tabel 3. Pengaruh pemberian sitokinin terhadap waktu inisiasi akar dan waktu inisiasi da mirabilis secara in vitro inisiasi akar.Pada pengamatan selama 10 minggu inisiasi akar tidak terjadi pada media dengan penambahan BAP dan 2iP sebesar 5 ppm. Pemberian sitokinin berupa BAP 2.5 ppm mampu menghasilkan waktu inisiasi akar tercepat. Media tanpa sitokinin justru memperlambat waktu Agus Setyo Yudhanto dan Ni Made Armini Wiendi Agus Setyo Yudhanto dan Ni Made Armini Wiendi Bul. Bul. Agrohorti 3 (3) : 276 – 284 (2015) Pengaruh pemberian NAA dan sitokinin terhadap rata-rata jumlah daun Nepenthes mirabilis pada 2- 10 MST secara in vitro Perlakuan Jumlah daun Waktu (MST) 2 4 6 8 10 Sitokinin (mg/l) 0 1.0 3.3 5.6 7.9 11.8 BAP 2.5 1.1 3.7 11.6 17.8 22.8 5.0 1.3 4.7 10.7 15.3 20.0 2iP 2.5 1.8 4.7 7.1 12.4 14.6 5.0 1.4 2.7 8.0 11.0 13.5 Kinetin 2.5 1.3 5.8 11.8 15.0 17.7 5 1.2 4.0 7.8 11.0 13.4 NAA (mg/l) 1.0 1.3 4.7 10.4 15.2 19.6 2.0 1.3 3.5 7.4 10.6 12.8 KK (%) 8.1 13.6 15.9 17.2 17.2 uh pemberian NAA dan sitokinin terhadap rata-rata jumlah daun Nepenthes mirabilis pada 2- ST secara in vitro l 5. Pengaruh pemberian NAA dan sitokinin terhadap rata-rata jumlah daun Nepenthes mi 10 MST secara in vitro Jumlah Kantong. Berdasarkan hasil sidik ragam, kombinasi NAA dan sitokinin tidak memberikan pengaruh nyata pada jumlah kantong Nepenthes mirabilis baik secara tunggal maupun kombinasi. Pemberian kinetin sebesar 2.5 ppm mampu menghasilkan jumlah kantong sebanyak 12.6 selama 10 MST. Peningkatan pemberian sitokinin maupun NAA terlihat menurunkan jumlah kantong yang diduga menghambat pertumbuhan tanaman. 282 Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) Pertumbuhan terhambat dengan semakin kecilnya jumlah kantong yang terbentuk sejalan dengan penambahan zat pengatur tumbuh di dalam media penambahan zat pengatur tumbuh di dalam media . Tabel 6. Pengaruh Pemberian NAA dan Sitokinin Terhadap Rata-rata Jumlah Kantong Nepenthes mirabilis pada 2-10 MST secara in vitro Perlakuan Jumlah Kantong Waktu (MST) 2 4 6 8 10 Sitokinin (mg/l) 0 0.4 2.8 4.5 6.3 8.6 BAP 2.5 0.0 1.5 4.5 6.8 9.5 5.0 0.4 1.6 4.2 7.0 10.1 2iP 2.5 0.3 2.4 5.2 8.9 11.4 5.0 0.6 1.6 3.8 7.0 8.8 Kinetin 2.5 0.5 2.4 6.9 10.5 12.6 5.0 0.7 1.8 4.5 7.1 8.8 NAA(mg/l) 1.0 0.4 2.2 5.5 9.1 12.0 2.0 0.4 1.8 4.1 6.2 7.9 KK (%) 7.6 13.1 14.6 17.0 18.2 aruh Pemberian NAA dan Sitokinin Terhadap Rata-rata Jumlah Kantong Nepenthes mirabilis 2-10 MST secara in vitro Jumlah Akar. Berdasarkan hasil sidik ragam, pemberian kombinasi NAA dan sitokinin tidak memberikan pengaruh yang nyata terhadap jumlah akar. KESIMPULAN Gunawan, L. W. 1992. Teknik Kultur Jaringan Tanaman. Departemen Pendidikan Tinggi. Pusat Antar Universitas Bioteknologi. Bogor (ID) : Institut Pertanian Bogor. Interaksi antara NAA dengan sitokinin tidak memberikan pengaruh nyata pada semua pengamatan. Pengaruh nyata didapat dari pemberian NAA atau sitokinin secara tunggal. Pemberian NAA dengan konsentrasi 1 mg/l terbukti mampu memberikan jumlah tunas terbanyak (5.2 tunas) selama 10 minggu, sedangkan pemberian BAP dengan konsentrasi 2.5 mg/l terbukti mampu memberikan waktu inisiasi akar tercepat (6.2 HST) selama 10 minggu. Mansur, M. 2006. Nepenthes Kantong Semar yang Unik. Jakarta (ID) : Penebar Swadaya. Mufa’adi, A. 2003. pengaruh kombinasi zat pengatur tumbuh bap dan iaa terhadap pertumbuhan dan perkembangan tanaman daun dewa (Gynura procumbens (Back.)) dalam kultur in vitro. [skripsi]. Bogor (ID): Institut Pertanian Bogor. Pemberian NAA berpengaruh nyata pada pengamatan jumlah tunas 7-10 MST, sedangkan pemberian sitokinin berpengaruh sangat nyata pada pengamatan jumlah akar pada 7 dan 9 MST. Pemberian 2iP dengan konsentrasi 5 mg/l terbukti mampu memberikan waktu inisiasi daun tercepat (4.0 HST) selama 10 minggu, sedangkan pemberian kinetin dengan konsentrasi 5 mg/l terbukti mampu memberikan jumlah akar terbanyak (14.1) selama 10 minggu pengamatan. Phillipps, A., Lamb, A.1996. Pitcer Plant of Borneo. Kuala Lumpur (MAL) : United Selangor Press. Rasco Jr., Maquilan, M.A.D. 2005. Initial studies on in vitro germination and early seedling growth of nepenthes truncata macf. Carnivorous Plant Newslatter. (34):51. Bul. Agrohorti 3 (3) : 276 – 284 (2015) Bul. Agrohorti 3 (3) : 276 – 284 (2015) DAFTAR PUSTAKA Sundorowati, E., Hartati, R., Taryana, T. 2002. Produksi tunas, regenerasi dan evaluasi hasil ubi kayu (manihot esculenta) indonesia asal kultur jaringan di lapang [Internet]. [Diunduh 2007 Nov 14]. Tersedia pada : http://www.unri.ac.id/jurnal/jurnal_natur/ vol4(2)/enny.pdf Cheek, M., Jebb, M. 2001. Flora Malesiana Series I. National Herbarium Netherland, Universiteit Leiden Brach: Leiden Brach. Cheek, M., Jebb, M. 2001. Flora Malesiana Series I. National Herbarium Netherland, Universiteit Leiden Brach: Leiden Brach. Cheek, M., Jebb, M. 2001. Flora Malesiana Series I. National Herbarium Netherland, Universiteit Leiden Brach: Leiden Brach. 284 Bul. Agrohorti 3 (3) : 276 – 284 (2015) Pengaruh yang nyata diperoleh dari pemberian sitokinin secara tunggal pada 5, 6, 8 dan 10 MST serta berpengaruh sangat nyata pada 7 dan 9 MST. Pemberian sitokinin berupa kinetin sebesar 5 ppm menghasilkan jumlah akar terbanyak yaitu sebanyak 14.1 akar/eksplan pada waktu 10 MST, sedangkan pemberian sitokinin berupa 2ip dan BAP sebesar 5 ppm tidak menghasikan akar sampai pengamatan 10 MST. Pemberian sitokinin berupa BAP dan 2iP pada tanaman nepenthes tidak menunjukkan penambahan jumlah akar yang signifikan selama 10 minggu pengamatan. Pemberian BAP dan 2iP lebih dari 2.5 ppm terlihat menghambat pertumbuhan akar. Berbeda dengan pemberian BAP dan 2 iP pemberian kinetin justru memberikan jumlah akar yang lebih banyak. Peningkatan pemberian kinetin hingga 5 ppm mampu meningkatkan jumlah akar hingga 14.1 akar/eksplan. 283 Agus Setyo Yudhanto dan Ni Made Armini Wiendi Tabel 7. Pengaruh Pemberian Sitokinin Terhadap Rata-rata Jumlah Akar Nepenthes mirabilis pada 2-10 MST secara in vitro Sitokinin (mg/l) Jumlah akar Waktu (MST 4 6 8 9 10 0 0.7ab 3.2ab 6.6a 8.2ab 9.8ab BAP 2.5 0.0b 0.2c 0.8b 1.0c 1.2c 5.0 0.0b 0.0c 0.0b 0.0c 0.0c 2iP 2.5 0.8ab 0.8abc 2.8ab 4.1abc 5.3ab 5.0 0.0b 0.0c 0.0c 0.0c 0.0c Kinetin 2.5 0.2ab 0.4bc 0.4b 1.3bc 2.1bc 5.0 1.5a 3.6a 8.8a 12.1a 14.1a KK (%) 8.320 13.841 19.475 20.986 22.538 Keterangan: Nilai yang diikuti oleh huruf yang sama pada kolom yang sama tidak berbeda nyata pada uji DMRT 5% Data merupakan hasil transformasi (x+1) emberian Sitokinin Terhadap Rata-rata Jumlah Akar Nepenthes mirabilis pada 2-10 in vitro h Pemberian Sitokinin Terhadap Rata-rata Jumlah Akar Nepenthes mirabilis pada 2-10 i i Tabel 7. Pengaruh Pemberian Sitokinin Terhadap Rata-rata Jumlah Akar Nepenthes mirab MST secara in vitro Agus Setyo Yudhanto dan Ni Made Armini Wiendi 283
https://openalex.org/W4205165260	https://journals.ufs.ac.za/index.php/pie/article/download/5564/4218	English	null	Pre-service Accounting teachers’ confidence and motivation in doing mathematics	Perspectives in education	2,021	cc-by	9,378	Research Article Research Article AUTHOR: Prof Msizi Mkhize1 ABSTRACT The purpose of the study was to determine pre-service accounting teachers’ perceptions of the confidence and motivation in doing mathematics in Bachelor of Education Accounting courses at a higher education institution, with the aim of informing higher education heutagogy as it relates to teaching and learning in accounting. This study included a convenience sample of 255 pre- service accounting teachers from a higher education institution in South Africa and a purposive sample of 18 students was drawn. Data collection was done through questionnaires and an interview schedule. A sequential explanatory design and sampling were employed. Data were analysed using SPSS for quantitative data and the interviews were transcribed and analysed qualitatively. The mean scores are above average (positive) for the cohort, however, there were numerous students with low scores in the effectance motivation and confidence scales. A more positive attitude towards effectance motivation in doing mathematics came from Indian, English and suburban respondents (p<.050). These students were not discouraged by difficult mathematical problems and are thus motivated by the challenges of mathematics. The interviews showed that parents positively influenced these students to persevere when faced with challenging mathematics problems. The results show no significant differences in the confidence in doing mathematics subscale (p >.050) in relation to gender, age, race group, mother tongue, Grade 12 mathematics, accounting module or location of schools. This finding runs counter to interview responses where 10 of the participants did not believe that they had a lot of self- confidence when it comes to mathematical accounting calculations and could not do advanced work without seeking help from others, compared with 6 participants who agreed that they had a lot of self- confidence when it comes to mathematical accounting calculations and could do advanced work without seeking help from others. In third-year accounting, strong significant connections were found between motivation in doing mathematics, confidence in doing mathematics and achievement in Accounting 420. Keywords: Pre-service teachers; accounting; mathematics; confidence; motivation; teaching. Published by the UFS http://journals.ufs.ac.za/index.php/pie © Creative Commons With Attribution (CC-BY) PRE-SERVICE ACCOUNTING TEACHERS’ CONFIDENCE AND MOTIVATION IN DOING MATHEMATICS Prof Msizi Mkhize1 AFFILIATION: 1University of KwaZulu-Natal AFFILIATION: 1University of KwaZulu-Natal DOI: http://dx.doi. org/10.18820/2519593X/pie.v39. i4.10 e-ISSN 2519-593X Perspectives in Education 2021 39(4): 135-155 PUBLISHED: 6 December 2021 RECEIVED: 14 August 2021 ACCEPTED: 07 October 2021 1. INTRODUCTION There is a limited understanding of the nature of the relationship between mathematics and accounting (Yunker et al., 2009, Mkhize, 2019) and the extent to which 135 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021 39(4): 135-155 2021: 39(4) Perspectives in Education 2021: 39(4) mathematics influences the teaching and learning of accounting. Accounting students perform a variety of calculations in accounting courses. This makes mathematics integral to the successful study of accounting. The majority of accounting definitions assert that accounting requires numerical and analytical skills, which are also integral elements of mathematics (Shaftel & Shaftel, 2005, Fedoryshyn et al., 2010, Heck & van Gastel, 2006). The transition from basic education to higher education mathematics modules, which involve advanced mathematics, is a challenge for many students (Rach & Ufer, 2020, Heck & van Gastel, 2006). The new entry students who fail to master basic skills, such as solving arithmetic and algebra problems, will most likely face difficulties in their further modules (Nortvedt & Siqveland, 2018). Undergraduate accounting students with majors in mathematics from pre- university institutes perform better than those with arts or social sciences backgrounds (Zandi, Shahabi & Bagheri, 2012). Number and operations, patterns and algebra, and data handling and probabilities skills that can be acquired in the subject of mathematics are found to be useful in accounting courses. It is vital to have mathematics skills to complete accounting tasks or activities as they are full of basic and advanced calculations and report accurate financial information (Mkhize, 2019, Villamar et al., 2020). This study was driven by the need to investigate pre-service teachers’ perceptions of the confidence and motivation in doing mathematics in Bachelor of Education Accounting courses at the higher education institution (HEI), with the aim of informing higher education heutagogy as it relates to teaching and learning in accounting. The research questions of this study were formulated as follows: • What are the pre-service teachers’ perceptions of the confidence and effectance motivation in doing mathematics in BEd Accounting courses? • What are the pre-service teachers’ perceptions of the confidence and effectance motivation in doing mathematics in BEd Accounting courses? 1. INTRODUCTION This question is answered in two ways: firstly, by looking at the confidence and motivation in doing mathematics domains and reviewing questions in the interview instrument and secondly, by examining groupings and determining if one can draw some significant differences in the perceptions of students across demographic variables as they relate to mathematics confidence and effectance motivation. • How do confidence and effectance motivation in doing mathematics relate to learning and achievement in accounting? The question is answered by looking at the correlation between dimensions of motivation and confidence in doing mathematics and achievement in accounting modules. 2.1 Confidence in learning mathematics 2.1 Confidence in learning mathematics i Hurst (2012) points out that confidence and positive self-efficacy can be a positive step towards success in any discipline. In his study, Hurst found that one method to increase student confidence and interest in mathematics was to “humanise” it, make the history of mathematics a key aspect of the module – for example, asking the students to undertake research on the history of a concept they are learning. Another study, by Hoffman and Schraw (2009), reported that students with higher mathematics self-efficacy persist longer with difficult mathematics problems and are more accurate in mathematics calculations than those lower in mathematics self-efficacy. In addition, Wenner (2001) found that the self-efficacy beliefs of in-service teachers – in particular, science and mathematics teachers – tend to be higher than those of pre-service teachers in other non-science or mathematics disciplines and that pre-service teachers should be the targeted population for intervention. Pajares and Schunk 136 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021 39(4): 136-155 2021 39(4): 136-155 Pre-service Accounting teachers doing mathematics Mkhize (2001) emphasise that students with low self-efficacy beliefs tend to be less confident and consequently become more anxious and stressed when attempting tasks. Canada (2011), who examined the mathematics attitudes of African-American elementary students, found that African American students in this sample enjoyed mathematics significantly more than White students and perceived themselves in mathematics at the same level, regardless of teachers underrating them and overrating their White counterparts on both measures. A study by Curtis (2006) found a significant difference in enjoyment of mathematics on college students’ race. No significant differences were found in confidence in doing mathematics, motivation in doing mathematics and value of mathematics. The qualitative data showed that a change occurred in confidence in doing mathematics, motivation in doing mathematics and value of mathematics. The study also identified that cooperative learning, problem solving, discourse and graphing calculators increased students’ confidence in doing mathematics, and that students felt more competent in performing problems during examinations. Similarly, Farooq and Shah (2008), investigating high school students’ attitudes towards mathematics, found no significant difference in confidence in doing mathematics between male and female students (t-value = 1.276; p > 0.05). 2.2 Effectance motivation in learning mathematics Incorporating stimulating questions, computer analysis (internet search included), and classical famous problems are important motivating resources in mathematics, which are particularly beneficial in the framework of action learning (Abramovich et al., 2019). Vandecandelaere et al. (2012) posit that the learning environment is linked with the motivation that comes with enjoying the challenges in mathematics. is effective. Incorporating stimulating questions, computer analysis (internet search included), and classical famous problems are important motivating resources in mathematics, which are particularly beneficial in the framework of action learning (Abramovich et al., 2019). Vandecandelaere et al. (2012) posit that the learning environment is linked with the motivation that comes with enjoying the challenges in mathematics. A study by Noble (2011) investigated the personal stories of African American male students who excelled in mathematics to understand the impact of their self-efficacy beliefs on their motivation and subsequent academic achievement in mathematics at the post- secondary level, and reported that enactive attainment and vicarious experience were influential sources for these students’ beliefs and were supported by family, friends and peers. The vicarious experiences seemed to be more influential than enactive attainment. This finding challenges Bandura (1986), who found that enactive attainment has the most significant influence on self-efficacy. Instead, Noble’s finding supports claims that friends play a major role in the development of attitudes to academics for African American male students. A study by Aruwa (2011) on influences on attitudes towards mathematics found that participants’ own motivational orientations, their belief about their mathematical ability, and the reasons they gave for success and failure appeared to have influenced their achievement and attitudes towards mathematics. The presence of an extrinsic achievement goal, especially in the later stages of secondary school, appeared to have motivated participants to strive for mathematical achievement. Anthony (2000) found that self-motivation influenced success in mathematics subjects and that lack of effort was the most likely factor to influence failure in mathematics subjects. Finally, a study by Frazier-Kouassi (1999) found a significant difference between high- achieving and low-achieving groups (t=-3.02; p=.003). High-achieving students had a more positive mean score than low-achieving students, meaning that they agreed more strongly on the statement “challenges of mathematics motivate” me than did low-achieving students. Additionally, these students may be intrinsically motivated or have a history of positive reinforcement from parents or teachers for persistence in the face of difficulty. 2.2 Effectance motivation in learning mathematics f According to self-determination theory (Deci & Ryan, 2000; Deci & Ryan, 1985), motivation can be divided into three broad categories: amotivation, extrinsic motivation and intrinsic motivation. The three categories exist on a continuum according to the level of self- determination underlying the motives behind behaviours. Amotivation occurs when individuals feel that an activity has no value, when individuals do not feel competent to complete a task or do not expect any desirable outcome from the activity. Extrinsic motivation is the desire or drive to engage in an activity because it leads to the attainment of an unrelated outcome (Eccles & Wigfield, 2002; Ames, 1992; Deci, 1972). External regulation is caused by external imposed rewards or punishments. Introjection occurs when individuals internalise the reasons for their behaviours and impose their own rewards or constraints. Identification occurs when an individual identifies with reason for behaving in a particular manner and behaviour is valued by the individual and occurs because the individual chooses to do so (Deci & Ryan, 1985; Deci & Ryan, 2000). Extrinsically motivated students engage in academic tasks because of rewards, such as recognition or pressures and failure (O’Keefe, 2010). Intrinsic motivation refers to an inner desire to accomplish a task, and pleasure is derived in the process. Intrinsically motivated students engage in academic tasks because they have an interest in studying and enjoy tasks or activities. Students have an intrinsic motivation to know, to accomplish tasks and to experience stimulation (Deci & Ryan, 2000). Instruction that supports developmental students to develop a positive self-concept, self- efficacy, independence and engagement will be beneficial in increasing student motivation (Hannula, 2002). The application of sound teaching and learning principles fosters an environment where students are motivated to reach for their potential (Tsanwani, 2009). Motivation to achieve in mathematics is not solely a product of mathematics ability nor is it so stable that intervention programmes cannot be designed to improve it (Middleton & Spanias, 1999). Instead, achievement motivation in mathematics is strongly influenced by instructional practices, and, if appropriate practices are implemented consistently over a long period of time, students can and do learn to enjoy and value mathematics (Middleton & Spanias, 1999, Posamentier, 2017). Teaching mathematics based on action learning and natural motivation 137 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021 39(4): 137-155 Perspectives in Education 2021: 39(4) 2021: 39(4) is effective. 2.3 Integration of mathematics in accounting Integration is the act, process or an instance of integrating. For example, the integration of mathematics and/with accounting (Merriam-Webster, 1999). An integrated study is one in which students broadly explore knowledge in various subjects related to certain aspects of their environment (Humphreys et al., 1981). Interdisciplinary integration is when students learn concepts and skills from two or more disciplines that are tightly linked to deepen knowledge and skills. Teachers arrange the curriculum around common knowledge or learnings throughout disciplines and break apart or chunk together the common knowledge or learnings embedded in the disciplines to emphasise interdisciplinary skills and concepts (Drake & Burns, 2004; Kaufman et al., 2003), as illustrated in Figure 1 below However, Mwakapenda and Dhlamini (2010) state that some tensions that students may face is where opportunities for making connections between mathematics and other learning areas are available but are either neglected or inappropriately used by the teachers. 2021 39(4): 138-155 2021 39(4): 138-155 138 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Pre-service Accounting teachers doing mathematics her learning areas are available but hers Mkhize d or ina Figure 1. The interdisciplinary integrated approach (Drake & Burns, 2014). . The interdisciplinary integrated approach (Drake & Burns, 2014). Mathematics Accounting Economics English Topics Concepts Interdisciplinary skills (e.g. numeracy skills, reading skills, critical thinking skills) Figure 1. The interdisciplinary integrated approach (Drake & Burns, 2014). The interdisciplinary integrated approach (Drake & Burns 201 Accounting activities require the use of mathematics or mathematical principles to successfully complete any introductory accounting courses or modules (Babalola & Abiola, 2013). The rules of debits and credits are entirely based on mathematical logic. There is a need for learning accounting from the perspective of mathematics (Warsono, Darmawan & Ridha, 2009). Selesho (2000) concurs that mathematics plays an important role in learning accounting. Studies by Yu (2011) and Zandi, Shahabi and Bagheri (2012) found that mathematics proficiency is the factor that has the biggest impact on accounting students’ output performance. A study by Stenberg, Varua and Yong (2010) suggested that tertiary institutions should recognise that high failure rates are due to inadequate mathematics exposure in secondary schooling, and corrective actions such as remedial lessons might not be sufficient. Specifying a minimum mathematics entry requirement and/or providing foundation programmes to ensure students have the essential basic mathematical skills, would increase student success in quantitative courses or modules. 2.3 Integration of mathematics in accounting ng activities require the use of mathematics or mathematical principles to su any introductory accounting courses or modules (Babalola & Abiola, 2013) and credits are entirely based on mathematical logic. There is a need fo ng from the perspective of mathematics (Warsono, Darmawan & Ridha, 2009 oncurs that mathematics plays an important role in learning accounting. Stu nd Zandi, Shahabi and Bagheri (2012) found that mathematics proficiency is the biggest impact on accounting students’ output performance. A study by nd Yong (2010) suggested that tertiary institutions should recognise that h 3.1 Research design and sampling A pragmatic, sequential explanatory mixed methods research design and sampling were adopted for this study and included a sequential collection of quantitative and qualitative data to provide answers to the research questions. For the first phase, convenience sampling was used for the surveys, because the pre-service accounting teachers are based at the HEI 6 139 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021 39(4): 139-155 2021: 39(4) Perspectives in Education 2021: 39(4) and are easy to reach and easy to contact. The randomly selected quantitative sample size consisted of 225 pre-service teachers. For the second phase, a purposeful choice sample for interviews was randomly selected from the quantitative sample. This sampling method was relevant for the study since pre-service accounting teachers elaborated on their quantitative responses. The qualitative sample size consisted of 18 pre-service accounting teachers (6 students from each first-, second- and third-year level of study). 3.2 Data collection methods The two data collection methods used to source the information were questionnaires and interviews. 3.2.1 Questionnaires The questionnaires were used to collect quantitative data. The composite statement for the confidence in doing mathematics scale is “I am confident in doing mathematics”. The composite statement for the motivation in doing mathematics scale is “The challenges of mathematics motivate me”. The scales consisted of 12 statements each. The first six statements measure positive attitudes, and six measure negative attitudes, with the following possible responses: strongly agree, agree, neutral, disagree, and strongly disagree. Each of the Likert responses was given a value of 5 to 1 respectively, for the positively stated questions, and 1 to 5 respectively, for the negatively stated questions. A minimum possible score was 12 and the maximum possible score was 60. A higher score indicates a more positive attitude towards the confidence of learning mathematics and effectance motivation. A lower score indicates a more negative attitude towards the confidence of learning mathematics and effectance motivation. The questionnaires adapted from the Fennema-Sherman Confidence and Motivation of doing Mathematics Scales were pilot tested. The reliability test was confirmed by determining the Cronbach’s Alpha for the scales. The confidence in learning mathematics scale reliability coefficient of r = 0.922 was found and Effectance motivation scale reliability coefficient of r = 0.847 was found. Figure 2: Example of a Likert-scale question used in a survey via interview Figure 2: Example of a Likert-scale question used in a survey via interview The interviewer pre-tested the survey via interviews with two pre-service accounting teachers at the HEI School of Education to confirm whether the interviewees understood the questions. The interviewer pre-tested the survey via interviews with two pre-service accounting teachers at the HEI School of Education to confirm whether the interviewees understood the questions. 3.2.2 Interviews The interview schedule was constructed from Fennema-Sherman Confidence and Effectance Motivation in doing Mathematics Scales and from literature identified and developed in the literature review. A number of the interview questions were posed as follows: “I have a lot of confidence when it comes to mathematical accounting calculations and could do advanced work in mathematics without seeking help from others”, “Watching and listening to a teacher in an accounting class performing an algebraic equation or applying a formula on the chalkboard makes me feel happy”. Do you agree with these statements? The 5-point Likert scale was used and students were required to explain during the interview. Open-ended questions were asked so that participants could express their views (Figures 1 and 2). I have a lot of confidence when it comes to mathematical accounting calculations and could do advanced work in mathematics without seeking help from others. 2021 39(4): 140-155 140 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Pre-service Accounting teachers doing mathematics w. Open-ended questions were asked so that d 2) Mkhize were ti i Figure 1: Example of a Likert-scale question used in a survey via interview I have a lot of confidence when it comes to mathematical accounting calculations and could do advanced work in mathematics without seeking help from others. Do you agree with the statement? Strongly agree Agree Undecided Disagree Strongly disagree If agree, why? _________________Explain: _______________________________________ If disagree, why not? __________________________________________________________ Figure 1: Example of a Likert-scale question used in a survey via interview Pre-service Accounting teachers doing mathematics I have a lot of confidence when it comes to mathematical accounting calculations and could do advanced work in mathematics without seeking help from others. I have a lot of confidence when it comes to mathematical accounting calculations and could do advanced work in mathematics without seeking help from others. Figure 1: Example of a Likert-scale question used in a survey via interview Figure 1: Example of a Likert-scale question used in a survey via interview Pre-service Accounting teachers doing mathematics Figure 1: Example of a Likert-scale question used in a survey via interview Figure 1: Example of a Likert-scale question used in a survey via interview Pre-service Accounting teachers doing mathematics Figure 2: Example of a Likert-scale question used in a survey via interview 8 Watching and listening to a teacher in an accounting class performing an algebraic equation or applying a formula on the chalkboard makes me feel happy. 3.2.2 Interviews Do you agree with the statement? Strongly Agree Undecided Disagree Strongly Watching and listening to a teacher in an accounting class performing an algebraic equation or applying a formula on the chalkboard makes me feel happy. Do you agree with the statement? Strongly agree Agree Undecided Disagree Strongly disagree Explain: ______________________________________________________________ Figure 2: Example of a Likert scale question used in a survey via interview Watching and listening to a teacher in an accounting class performing an algebraic equation or applying a formula on the chalkboard makes me feel happy. Do you agree with the statement? Watching and listening to a teacher in an accounting class performing an algebraic equation or applying a formula on the chalkboard makes me feel happy. 3.2.3 Procedures 3.2.3 Procedures Survey: The questionnaire was administered during the accounting class. The instructions in English were read to the participants. The participants were assured that the data was for academic research only. No identifying information was to be disclosed on the questionnaire, and for this reason the questionnaire was conducted anonymously. Participants were told that pseudonyms would be used. Participants were also given assurance that their participation or non-participation would not affect their marks or disadvantage them in any way, even though the researcher was the accounting lecturer. This gave respondents the assurance of confidentiality and anonymity. A consent form was distributed for the participants to complete. Questionnaires were also distributed, and participants were given 45 minutes to fill it in. Two research assistants assisted in collecting the questionnaires. Survey: The questionnaire was administered during the accounting class. The instructions in English were read to the participants. The participants were assured that the data was for academic research only. No identifying information was to be disclosed on the questionnaire, and for this reason the questionnaire was conducted anonymously. Participants were told that pseudonyms would be used. Participants were also given assurance that their participation or non-participation would not affect their marks or disadvantage them in any way, even though the researcher was the accounting lecturer. This gave respondents the assurance of confidentiality and anonymity. A consent form was distributed for the participants to complete. Questionnaires were also distributed, and participants were given 45 minutes to fill it in. Two research Interviews: The office was used because it is situated in a quiet area. No interruptions occurred. The individual face-to-face open-ended interviews with 18 pre-service accounting teachers were conducted. The instructions in English were read to the participants. The 18 participants were asked to indicate their perceptions of confidence in learning mathematics and effectance motivation in Bachelor of Education accounting courses at the higher education institution. The interviews were recorded and notes taken. Permission was obtained from participants before commencing. assistants assisted in collecting the questionnaires. Interviews: The office was used because it is situated in a quiet area. No interruptions occurred. The individual face-to-face open-ended interviews with 18 pre-service accounting teachers were conducted. The instructions in English were read to the participants. 3.2.3 Procedures 3.2.3 Procedures The 18 participants were asked to indicate their perceptions of confidence in learning mathematics and effectance motivation in Bachelor of Education accounting courses at the higher education institution The 2021 39(4): 141-155 141 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021: 39(4) Perspectives in Education 3.3 Data analysis strategy The quantitative data were analysed using SPSS. Descriptive and inferential statistics were computed. Mean scores of students falling in each category were calculated. Box and whisker plotting showed the overall means to conceal the key differences between individual student responses. The t-test and analysis of variance were used to test whether there was a significant difference between demographic variables and students’ confidence in doing mathematics and effectance motivation, significance level, p < 0.05. Pearson correlations computed for confidence in doing mathematics and effectance motivation and accounting mark sheets in order to investigate which of the affectance factors correlate, whether positively or negatively. Statistical significance was found to be at p < 0.01 and p < 0.05. The interview data were transcribed to produce a written word document. The interview participants were emailed data to verify their responses. The responses were coded and salient items categorised into themes. To enhance the credibility of the analyses, member checks were performed by supervisors, academics with doctorate degrees at a workshop and two retired education professors. 4. RESULTS Quantitative and qualitative results are explained below. 3.4 Ethical considerations The research complied with ethical considerations for dealing with human subjects. Ethical clearance was obtained from Human Research Ethics Committee of the relevant higher education institution. I – First year students: The large number of outliers indicated the diversity in the class. Most of the students in the first year agreed with statement, “the challenges of mathematics motivate me” and “I am confident in doing mathematics”. Despite the mean score being positive (above average) for the cohort, there were numerous students who had achieved low scores in the factors. II – Second year students: These students also had low scores in the motivation and confidence factors despite the mean being positive. 4.1.1 Descriptive statistics Cronbach’s Alpha coefficient for confidence in doing mathematics was .922 and effectance motivation in doing mathematics was .847. The mean score showed the overall effectance and motivation in doing mathematics of all 255 students. A mean equal to 36 indicated that effectance and motivation in doing mathematics is neutral, mean less than 36 (below average) indicated that students were in disagreement with the composite statement and the mean greater than 36 (above average) indicated that students agreed with the composite statement. Table 3 indicates that the mean values were above average for both the factors: Confidence in doing mathematics – the mean score for all 255 respondents was 41.57 (SD = 11.01) which represents moderate agreement with the composite statement, “I am confident in doing mathematics” and effectance motivation in doing mathematics – the mean score for all 255 respondents was 40.13 (SD = 9.28), which represented moderate agreement with the composite statement, “The challenges of mathematics motivate me”. Table 1. Mean scores and standard deviations for the personal affect factors Factor (N=255) Mean Std. Deviation Cronbach Alpha Confidence in doing mathematics 41.57 11.013 .922 Effectance motivation in doing mathematics 40.13 9.275 .847 Table 1. Mean scores and standard deviations for the personal affect factors 142 4): 142-155 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 142 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021 39(4): 142-155 Pre-service Accounting teachers doing mathematics Mkhize The overall means cited in the previous paragraph conceal the key differences between the individual student responses. As shown in Figure 3, the spread of factor and mean scores for students are depicted. 143 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 able 3 shows the data disaggregated by gen ing background year of study and location of III – Third year students: Pre ser ice Acco ntin It was noted that from the means for third-year accounting students scored lower than the means for first- and second-year accounting students. Pre-service Accounting teachers doing mathematics Figure 3: Spread of factor and mean scores for accounting students, Fennema-Sherman Motivation and Confidence in doing Mathematics Scales Figure 3: Spread of factor and mean scores for accounting students, Fen Motivation and Confidence in doing Mathematics Scales I – First year students II – Second year students III – Third year students II – Second year students Figure 3: Spread of factor and mean scores for accounting students, Fennema-Sherman Motivation and Confidence in doing Mathematics Scales Figure 3: Spread of factor and mean scores for accounting students, Fen M ti ti d C fid i d i M th ti S l 2021 39(4): 143-155 motivation in d mother tongue 143 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 able 3 shows the data disaggregated by gen ing background year of study and location of 2021 39(4): 143-155 motivation in mother tongue 2021: 39(4) 2021: 39(4) Perspectives in Education Inferential statistics Inferential statistics ance motivation in doing mathematics Effectance motivation in doing mathematics Effectance motivation in doing mathematics Table 2 shows the number of students selecting each level of agreement on effectance motivation in doing mathematics. Table 3 shows the data disaggregated by gender, age, race, mother tongue, mathematics schooling background, year of study and location of schooling. Number of students selecting each level of agreement on effectance motivation in doing mathematics Table 2. Number of students selecting each level of agreement on effectance motivation in doing mathematics Effectance motivation in doing mathematics subscale (N = 255) Strongly agree Agree Undecided Disagree Strongly disagree n n n n n I like maths puzzles. 47 100 46 40 22 Maths is enjoyable to me. 43 84 42 61 25 When a maths problem comes up that I cannot solve right away, I stick with it until I find the solution. 57 99 30 43 26 Once I start working on a maths puzzle, it is hard to stop. 35 66 39 70 45 When I have a question that doesn’t get answered in maths class, I keep thinking about it. 58 100 28 43 26 I am challenged by maths problems I cannot understand right away. 54 93 31 59 18 Figuring out maths problems is not something I like to do. 30 48 42 97 38 The challenge of maths problems does not appeal to me. 15 41 74 85 40 Maths puzzles are boring. 14 24 61 92 64 I do not understand how some people can spend so much time on maths and seem to like it. 33 42 40 85 55 I would rather have someone else figure out a tough maths problem than have to work it out myself. 32 48 44 65 66 I do as little work in maths as possible. 28 68 29 72 58 (N = 255) Table 3. Comparison: effectance motivation in doing mathematics with demographic variables using means, T-test and ANOVA Variables Sub-groups N Mean T-test ANOVA T Df P f p Gender Male Female 105 150 39.52 40.55 -.872 253 .384 Age (years) 18 – 20 years 21 years and above 165 90 40.50 39.44 .871 253 .385 Table 3. 145 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Effectance motivation in doing mathematics Comparison: effectance motivation in doing mathematics with demographic variables using means, T-test and ANOVA 144 2021 39(4): 144-155 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 144 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Pre-service Accounting teachers doing mathematics Mkhize Mkhize Variables Sub-groups N Mean T-test ANOVA T Df P f p Race group African Indian 225 30 39.56 44.40 -2.718 253 .007* Mother tongue English Zulu 32 223 44.50 39.50 2.891 253 .004* Grade 12 mathematics Mathematics Mathematical Literacy 146 109 40.86 39.16 1.451 253 .148 Accounting module First year Second year Third year 143 77 35 40.97 39.35 38.43 1.445 .238 Areas/ location of school Rural area Township area Suburban area 134 72 49 39.75 38.32 43.84 5.595 .004* p<0.05 The significant differences were between African and Indian, English and Zulu, and rural, township and suburban students (p < 0.050). The significant differences were between African and Indian, English and Zulu, and rural, township and suburban students (p < 0.050). Table 4. Number of students selecting each level of agreement on confidence in doing mathematics Confidence in doing mathematics domain (N = 255) Strongly agree Agree Undecided Disagree Strongly disagree n n n n n I feel confident trying maths. 95 92 27 27 14 I am sure that I could do advanced work in maths. 73 83 54 31 14 I am sure that I can learn maths. 89 113 34 10 9 I think I could handle more difficult maths. 55 77 67 26 30 I can get good marks (scores) in maths. 57 121 39 25 13 I have a lot of self-confidence when it comes to maths. 52 75 52 59 17 I am no good at maths. 31 47 35 91 51 I do not think I could do advanced maths. 21 46 42 97 49 I am not the type to do well in maths. 22 37 30 110 56 For some reason, even though I study, maths is really hard for me. 39 72 34 66 43 I do fine in most subjects, but when it comes to maths I really mess up. 62 53 21 72 47 Maths is my worst subject. 47 39 36 59 73 . Number of students selecting each level of agreement on confidence in doing mathematics 145 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Perspectives in Education 2021: 39(4) 2021: 39(4) Table 4 shows the number of students selecting each level of agreement on confidence in doing mathematics. Effectance motivation in doing mathematics Table 5 shows the data is disaggregated by gender, age, race, mother tongue, mathematics schooling background, year of study and location of schooling. Table 5. Comparison: confidence in doing mathematics with demographic variables using means, T-test & ANOVA Variables Sub-groups N Mean t-test ANOVA T Df P f P Gender Male Female 105 150 41.86 41.37 .349 253 .727 Age (years) 18 -20 years 21 years and above 165 90 42.07 40.66 .978 253 .329 Race group African Indian 225 30 41.40 42.87 -.687 253 .493 Mother tongue English Zulu 32 223 43.47 41.30 1.044 253 .298 Grade 12 mathematics Mathematics Mathematical Literacy 146 109 42.38 40.49 1.358 253 .176 Accounting module First year Second year Third year 143 77 35 42.91 40.31 38.86 2.656 .072 Areas/ location of school Rural area Township area Suburban area 134 72 49 40.86 41.08 44.22 1.785 .170 p<0.05 The results of the study show no significant differences between any of the grouping variables (p > 0.050). Correlation between effectance motivation and confidence in doing mathematics scores and achievement in accounting modules This section answers the question: How do personal factors (confidence in doing mathematics and motivation in doing mathematics) relate to learning and achievement in accounting? Comparison: confidence in doing mathematics with demographic variables using means, T-test & ANOVA The results of the study show no significant differences between any of the grouping variables (p > 0.050). The results of the study show no significant differences between any of the grouping variables (p > 0.050). 146 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Correlation between effectance motivation and confidence in doing mathematics scores and achievement in accounting modules 2021 39(4): 147-155 147 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021: 39(4) 2021: 39(4) Perspectives in Education Interviews on confidence in doing mathematics Figure 4 shows the responses of participants to the question “I have a lot of confidence when it comes to mathematical accounting calculations and could do advanced work in mathematics without seeking help from others. Do you agree with this statement?”. The results indicated that the majority of participants disagreed with the statement. Figure 4. Confidence in mathematical accounting calculations. Figure 4. Confidence in mathematical accounting calculations. Correlation between effectance motivation and confidence in doing mathematics scores and achievement in accounting modules Correlation between effectance motivation and confidence in doing mathematics scores and achievement in accounting modules This section answers the question: How do personal factors (confidence in doing mathematics and motivation in doing mathematics) relate to learning and achievement in accounting? 2021 39(4): 146-155 146 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Pre-service Accounting teachers doing mathematics Mkhize Table 6. Correlation between dimensions of motivation and confidence in doing mathematics and achievement in accounting modules Motivation subscale Confidence subscale First year Accounting 210 Pearson correlation Sig. (2-tailed) N .248** .005 128 .227* .010 128 Accounting 220 Pearson correlation Sig. (2-tailed) N .241* .010 112 .229* .015 112 Second year Accounting 310 Pearson correlation Sig. (2-tailed) N .178 .167 62 .098 .449 62 Accounting 320 Pearson correlation Sig. (2-tailed) N .178 .203 53 .075 .591 53 Third year Accounting 410 Pearson correlation Sig. (2-tailed) N .327 .072 31 .339 .062 31 Accounting 420 Pearson correlation Sig. (2-tailed) N .506** .007 27 .437* .023 27 ** Correlation is significant at the 0.01 level (2-tailed), * Correlation is significant at the 0.05 level (2-tailed). Table 6. Correlation between dimensions of motivation and confidence in doing mathematics and achievement in accounting modules Table 6. Correlation between dimensions of motivation and confidence in doing mathematics and achievement in accounting modules ** Correlation is significant at the 0.01 level (2-tailed), * Correlation is significant at the 0.05 level (2-tailed). The correlations between factors of motivation and confidence in doing mathematics and achievement in accounting modules studied in the year of the research have been calculated. The results were tabulated in Table 6 and it should be noted that six different modules of accounting were used to measure accounting achievement. This affects the comparability of the results across the year groups, the two modules in the current year of study; nevertheless, certain trends could be observed. The study revealed the following aspects: In first-year accounting, there were low positive and significant correlations between motivation in doing mathematics and confidence in doing mathematics. This indicated a marginal connection between accounting 210 and 220 achievement and motivation in doing mathematics as well as confidence in doing mathematics. In second-year accounting, no significant correlations were found between motivation and confidence in doing mathematics and achievement in Accounting 310 and 320. In third-year accounting, strong significant connections were found between motivation in doing mathematics, confidence in doing mathematics and achievement in Accounting 420. 4.2 Qualitative results Interviews on confidence in doing mathematics Interviews on confidence in doing mathematics 148 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Explanations given by the participants: Interviews on effectance motivation in doing mathematics Interviews on effectance motivation in doing mathematics Most of the participants (15) agreed with the statement, “I 149 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 18 Explanations given by the participants: I have self-confidence when it comes to doing calculations and I like to do advanced work so that when we are doing it in class I am already prepared. Although when I find challenges I often seek help but not always (Sibo). I have confidence I enjoy mathematics and challenges. I sometimes find challenging, that is when I need help (Narri). I have confidence I enjoy mathematics and challenges. I sometimes find challenging, that is when I need help (Narri). Mathematics is one of my favourite subjects and I do it myself most of the times. I seldom ask people for help (Madu). Mathematics is one of my favourite subjects and I do it myself most of the times. I seldom ask people for help (Madu). I understand most of the calculations. Occasionally I ask for help from others Sisi. These are exceptional cases that were found. They were confident and enthusiastic in doing mathematical accounting calculations but were not free from anxiety as they sometimes found it challenging and sought assistance. 2021 39(4): 148-155 148 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Mkhize Pre-service Accounting teachers doing mathematics I find mathematics challenging so if the mathematical accounting calculation is challenging I get lost and confused and not confident in doing it … as long there is someone to assist (Vender). g g g I seek help if lost (Kazi). d K i i di t d th t th ti i t ff t fid d Not confident, mathematical accounting calculations are challenging. In most of the times I seek help if lost (Kazi). er and Kazi indicated that mathematics anxiety affects confidence a ts mathematics anxiety in doing mathematical accounting calculations Vender and Kazi indicated that mathematics anxiety affects confidence and lack of confidence affects mathematics anxiety in doing mathematical accounting calculations. I do need help most of the times because I did mathematics literacy in high scho Buthu is not confident in doing Mathematics as he did Mathematical Literacy in I do need help most of the times because I did mathematics literacy in high school level (Buthu). uthu is not confident in doing Mathematics as he did Mathematical Literacy in ways looks for assistance. Buthu is not confident in doing Mathematics as he did Mathematical Literacy in high school. He always looks for assistance. Interviews on effectance motivation in doing mathematics Most of the participants (15) agreed with the statement, I get the confidence I need to do other problems (Sisi). Yes, I do get motivated and feel determined to work hard in order to improve my understanding (Khoba). Since I like to work with numbers, I always become motivated, happy when I got the right solution because I love accounting. I sometimes assist my friends with calculations. (Sibo) … get lost in the middle of the calculations … get frustrated … mathematical calculations, it is as if you are challenging the teacher … decide to keep quiet even if they do not understand (Phindi). Students sometimes believe that asking a teacher to explain can be misinterpreted by the teacher, as if students are challenging the teacher’s mathematics competence. However, if they want clarification and get a sense that their question is being misconstrued, they resolve to be quiet. Teachers must make a distinction between questions that indicate misunderstanding and questions that are testing the teacher’s competence. 5. DISCUSSION OF RESEARCH RESULTS The mean scores are positive – above average – however, many students had low scores in the factors. The wide spread of scores in Fennema-Sherman Confidence and Motivation in doing mathematics scales indicate that these factors may differ in significance in influencing the learning of accounting by pre-service teachers. This assumption is discussed below, drawing on the results of Fennema-Sherman Motivation and Confidence in doing Mathematics scales, the student interviews and the literature. Interviews on effectance motivation in doing mathematics Most of the participants (15) agreed with the statement, Most of the participants (15) agreed with the statement, “I get motivated towards practising mathematical accounting calculations when I successfully solve the accounting problem”. Only a minority of participants (3) disagreed with this statement. mathematical accounting calculations when I successfully solve the accounting p minority of participants (3) disagreed with this statement. Figure 5: Motivation in doing mathematical accounting calculations Figure 5: Motivation in doing mathematical accounting calculations Number Of Participants Figure 5: Motivation in doing mathematical accounting calculations Figure 5: Motivation in doing mathematical accounting calc The overall results indicated that most of the participants agreed with the statement, while a minority of the participants did not. g g g The overall results indicated that most of the participants agreed with the sta 2021 39(4): 149-155 149 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 18 2021: 39(4) Perspectives in Education The participants gave the following reasons. Makes me feel intelligent, gives me confidence and makes me feel smart (Bima). I feel happy because that is when I pick up information and tips for my understanding of the calculation. I have already attempted the calculation before, when the teacher performs it I am then able to correct my mistakes (Asiya). When the teacher models the process or steps, the students are likely to learn from the rocess and reflect on it. It gives me the motivation to go on solving other problems. I get the confidence I need to do other problems (Sisi). Yes, I do get motivated and feel determined to work hard in order to improve my understanding (Khoba). Since I like to work with numbers, I always become motivated, happy when I got the right solution because I love accounting. I sometimes assist my friends with calculations. (Sibo) … get lost in the middle of the calculations … get frustrated … mathematical calculations, it is as if you are challenging the teacher … decide to keep quiet even if they do not understand (Phindi). I feel happy because that is when I pick up information and tips for my understanding of the calculation. I have already attempted the calculation before, when the teacher performs it I am then able to correct my mistakes (Asiya). When the teacher models the process or steps, the students are likely to learn from the process and reflect on it. It gives me the motivation to go on solving other problems. 5.1 Effectance motivation in doing mathematics domain The students were not discouraged by difficult mathematical problems, which indicate that the challenges of mathematics motivate them. As it was found during the interviews, these students were positively influenced by parents to persevere when faced with challenging mathematics problems. These students were also positively influenced by teachers who gave them tangible rewards (certificates) and intangible rewards (public acknowledgement). These students were intrinsically motivated because they have high autonomous (independent) learning. This finding is supported by Frazier-Kouassi (1999). The majority of the participants agreed that watching and listening to the teacher in an accounting class performing a calculation presents a space for personal reflection. One major reason given by participants was that when the teacher models the process or steps the 2021 39(4): 150-155 150 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 Pre-service Accounting teachers doing mathematics Mkhize students are likely to learn from the process and reflect on it in their own approach. This finding corresponds with Erdogan et al. (2011) that teachers should begin with problems that students understand and solve easily because this may raise the self-efficacy beliefs of students and the teachers can then solve more difficult problems with the students. Only six participants were neutral in regard to the statement that “Watching and listening to the teachers in an accounting class performing a calculation presents a space for personal reflection.” A reason given by participants was that when one asks a teacher to explain the process or steps the question could be misinterpreted by the teacher as challenging the teacher’s mathematics competence; a student wants clarification then gets a sense that the teacher is misinterpreting the question and decides to be quiet. The participants reflected a need for teachers to make use of the classroom environment as a resource for motivation. This result supports the findings by Aruwa (2011). To alleviate mathematics anxiety and increase the motivation level of students, teachers could use top achievers in mathematics as a resource to show easier mathematical accounting calculation methods. These top students benefit by having their abilities affirmed and by learning that they can become a resource for other learners. This finding is consistent with the finding by Vandecandelaere et al. (2012) that the learning environment is linked with the motivation that comes with enjoying the challenges in mathematics. One participant indicated that students in her class were uncomfortable and “not confident to ask questions in front of other learners”. 5.2 Confidence in doing mathematics domain Just two exceptional cases were found where the two participants were confident or enthusiastic in performing mathematical accounting calculations but had a negative disposition towards certain areas of mathematics that affected their learning in accounting. A total of 111 respondents (43.7%) agreed that, for one reason or another, even though they study, mathematics is really hard for them, and 115 agreed that they do fine in most subjects but mess up when it comes to mathematics. It is worrying that the majority of the participants showed lack of confidence in mathematical accounting calculations and rely on assistance from others. They indicated that they become anxious when performing mathematical accounting calculations. This finding is supported by Pajares and Schunk (2001) who found that students with low self-efficacy beliefs tend to be less confident and consequently become more anxious and stressed when attempting tasks. 5.1 Effectance motivation in doing mathematics domain This fear of asking questions lowered students’, self-esteem and personal self-belief. Another participant identified a serious issue facing teaching and learning: she felt that it is essential to understand a theory and set a theoretical foundation before applying it. Consequently, if the students’ theoretical knowledge is solid, there will be less anxiety in the application of theoretical knowledge. Studies by Anthony (2000) and Aruwa (2011) found that self-motivation influenced success in mathematics subjects, that lack of effort was the most likely reason for failure in mathematics subjects and that it appeared to have influenced participants’ achievement and attitudes towards mathematics. 5.3 Relationship between effectance motivation and confidence in doing mathematics and achievement in accounting In first-year accounting, there were low positive and significant connections between accounting 210 and 220 achievement and motivation in doing mathematics as well as confidence in doing mathematics. In fourth-year accounting, strong positive and significant connections 151 http://dx.doi.org/10.18820/2519593X/pie.v39.i4.10 2021 39(4): 151-155 2021: 39(4) Perspectives in Education were found between motivation in doing mathematics, confidence in doing mathematics and achievement in Accounting 420. When students enter their fourth year, there is a relationship between motivation and confidence in doing mathematics and achievement in Accounting 420. Students want to complete their accounting as this is their final year of accounting. were found between motivation in doing mathematics, confidence in doing mathematics and achievement in Accounting 420. When students enter their fourth year, there is a relationship between motivation and confidence in doing mathematics and achievement in Accounting 420. Students want to complete their accounting as this is their final year of accounting. 6. CONCLUSIONS AND IMPLICATIONS FOR HIGHER EDUCATION The quantitative results revealed that more positive attitude towards effectance motivation in doing mathematics came from Indian, English and suburban students (p<0.050). Many of the interview participants agreed that watching and listening to the teacher in an accounting class performing a calculation presents a space for personal reflection. The quantitative results also showed that students have equal positive attitude to confidence in doing mathematics (p>0.050), whereas the qualitative results disclosed that the majority of the participants lack confidence in mathematical accounting calculations and rely on assistance from others. There is a relationship between motivation and confidence in doing mathematics and achievement in fourth year, Accounting 420. Students doing fourth year accounting want to complete their accounting studies as this is their final year of the BEd accounting programme. It is suggested that students in their BEd first year do the foundation courses such as Mathematics for Accounting before registering for an accounting major module in their second year. This mathematics module will ease the transition between high school mathematics and the university mathematics required in accounting. 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https://openalex.org/W2913755939	https://europepmc.org/articles/pmc6384725?pdf=render	English	null	The Role of Saccharomyces cerevisiae Yeast and Lactic Acid Bacteria in the Formation of 2-Propanol from Acetone during Fermentation of Rye Mashes Obtained Using Thermal-Pressure Method of Starch Liberation	Molecules/Molecules online/Molecules annual	2,019	cc-by	10,450	Received: 27 January 2019; Accepted: 7 February 2019; Published: 9 February 2019 Received: 27 January 2019; Accepted: 7 February 2019; Published: 9 February 2019 Received: 27 January 2019; Accepted: 7 February 2019; Published: 9 February 2019 Abstract: This study set out to assess the acetone content in rye sweet mashes prepared using the thermal-pressure method of starch liberation, and to investigate the formation of 2-propanol during the fermentation process. In the ﬁrst set of experiments, we evaluated the correlation between the color and the content of acetone and furfural in industrially produced sweet mashes (n = 37). The L * value was negatively correlated with the content of both acetone and furfural, while chromatic parameters a * and b * and the yellowness index (YI) had strong positive correlations with acetone (r > 0.9) and furfural (r > 0.8 for a * and r > 0.9 for b * and YI). In the second set of experiments, we assessed the concentration of acetone and 2-propanol in distillery rye mashes, fermented by S. cerevisiae yeast and lactic acid bacteria. The inﬂuence of fermentation temperature on the formation of 2-propanol was also evaluated. The presence of 2-propanol in the post-fermentation media was conﬁrmed, while a decrease in acetone content was observed. Fermentation temperature (27 ◦C or 35 ◦C) was found to have a signiﬁcant effect on the concentration of 2-propanol in trials inoculated with lactic bacteria. The content of 2-propanol was more than 11 times higher in trials fermented at the higher temperature. In the case of yeast-fermented mashes, the temperature did not affect 2-propanol content. The acetone in the sweet mash was assumed to be a precursor of 2-propanol, which was found in the fermented mashes. Keywords: 2-propanol; acetone; thermal-pressure method of starch liberation; Maillard reaction products; Saccharomyces cerevisiae yeast; lactic acid bacteria The Role of Saccharomyces cerevisiae Yeast and Lactic Acid Bacteria in the Formation of 2-Propanol from Acetone during Fermentation of Rye Mashes Obtained Using Thermal-Pressure Method of Starch Liberation Katarzyna Pielech-Przybylska 1,* , Maria Balcerek 1 , Urszula Dzieko´nska-Kubczak 1 , Barbara Pacholczyk-Sienicka 2 , Grzegorz Ciepielowski 2, Łukasz Albrecht 2 and Piotr Pat Katarzyna Pielech-Przybylska 1,* , Maria Balcerek 1 , Urszula Dzieko´nska-Kubczak 1 , Barbara Pacholczyk-Sienicka 2 , Grzegorz Ciepielowski 2, Łukasz Albrecht 2 and Piotr Patelski 1 1 Department of Spirit and Yeast Technology, Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland; maria.balcerek@p.lodz.pl (M.B.); urszula.dziekonska-kubczak@p.lodz.pl (U.D.-K.); piotr.patelski@p.lodz.pl (P.P.) 1 Department of Spirit and Yeast Technology, Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland; maria.balcerek@p.lodz.pl (M.B.); urszula.dziekonska-kubczak@p.lodz.pl (U.D.-K.); piotr.patelski@p.lodz.pl (P.P.) p p p p ( ) 2 Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland; barbara.pacholczyk@p.lodz.pl (B.P.-S.); grzegorz.ciepielowski@p.lodz.pl (G.C.); lukasz.albrecht@p.lodz.pl (L.A.) p p p p ( ) 2 Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland; barbara.pacholczyk@p.lodz.pl (B.P.-S.); grzegorz.ciepielowski@p.lodz.pl (G.C.); lukasz.albrecht@p.lodz.pl (L.A.) * C d k i l h b l k @ l d l T l 48 42 631 34 73 p p ( ) * Correspondence: katarzyna.pielech-przybylska@p.lodz.pl; Tel.: +48-42-631-34-73 * Correspondence: katarzyna.pielech-przybylska@p.lodz.pl; Tel.: +48-42-631-34-73 * Correspondence: katarzyna.pielech-przybylska@p.lodz.pl; Tel.: +48-42-631-34-73 molecules molecules www.mdpi.com/journal/molecules 1. Introduction Thermal-pressure processing is a common method in agricultural distilleries which use starchy raw materials. Its technological beneﬁts include sterilization, which eliminates process-dangerous lactic acid bacteria on cereal grains [1], and the liberation of starch from plant cells. Moreover, high temperatures change the physical properties of starch, enabling more effective enzymatic hydrolysis. However, heat treatment of starchy materials can lead to a number of chemical changes, involving Molecules 2019, 24, 610; doi:10.3390/molecules24030610 www.mdpi.com/journal/molecules 2 of 15 Molecules 2019, 24, 610 reducing sugars, amino acids, and peptides, which are abundant in cereal raw materials [2]. During heating, reactions may occur between the carbonyl or hemiacetal groups in reducing sugars and the amino groups in amino acids and peptides. These can then initiate a series of reactions, referred to collectively as Maillard reactions, which produce compounds with strong sensory properties. Maillard reaction products (MRPs) affect the taste, smell, and color of sweet mash obtained using the thermal-pressure method [3–7]. Moreover, Maillard reactions involve reducing sugars, which might otherwise have provided a potential substrate for ethanol production, as well as amino acids with peptides, which could have been a valuable nitrogen source for yeast in the fermentation process. Some MRPs, including furfural and 5-hydroxymethylfurfural (HMF), have been shown to affect the fermentation process [8–11]. Furfural and HMF are sugar degradation products, formed by dehydration of pentoses and hexoses, respectively, at high temperature and pressure [12,13]. Both are known to have a negative effect on Saccharomyces cerevisiae yeast. However, furfural is considered a much stronger inhibitor than HMF in terms of yeast growth and the productivity of ethanol. This is related to its effect on glycolytic activity and the tricarboxylic acid cycle, as well as to its causing oxidative stress and reducing the activity of various dehydrogenases in yeast cells [12,14]. Agricultural distilleries using starchy raw materials therefore need to control the steaming time carefully, to minimize the concentration of MRPs. Steaming of cereal raw materials—such as rye, wheat, or triticale—is carried out at pressures of 0.3–0.4 MPa and temperatures of 144–152 ◦C for about 30–40 min. The steaming of barley grain requires harsher parameters (0.5 MPa, 159 ◦C), due to the presence of an additional ﬁbrous layer around the kernel, which develops as the ﬂower buds grow together with the kernel during the grain’s puberty [15]. 1. Introduction Moreover, lactic acid bacteria are well known for their ability to synthesize volatile compounds such as alcohols, aldehydes, ketones, esters, alkanes, alkenes, 3 of 15 Molecules 2019, 24, 610 terpenes, furans, and sulfur compounds [34–37]. Some of these negatively affect the quality of the agricultural distillates obtained [38,39]. In our previous research [40], we found 2-propanol in distillates obtained from rye- and potato-based mashes prepared using both thermal-pressure (TP) and pressureless (PLS) methods for starch liberation. It was noticed that the concentration of 2-propanol was substantially higher in distillates obtained from mashes prepared using the TP method. The low boiling point of 2-propanol compared to other higher alcohols (C3-C8) and its ability to form azeotropes makes the removal of this compound from the spirit during rectiﬁcation very difﬁcult. Therefore, it is necessary to limit its synthesis during fermentation. According to EU Regulations [41], the higher alcohols level (expressed in 2-methyl-1-propanol) in ethyl alcohol of agricultural origin is limited to a maximum of 0.5 g per hectoliter of 100% vol. alcohol, while to meet non-EU standards [42] the concentration of other alcohols, especially isopropyl alcohol, should be also taken into account, due to the difﬁculty of removing it in the distillation processes. p According to the literature [27,28,36,37], the synthesis of 2-propanol takes place via acetone reduction catalyzed by primary and/or secondary alcohol dehydrogenase. For acetone synthesis, in turn, the main substrate is acetyl-CoA. Two molecules of acetyl-CoA are condensed to one molecule of acetoacetyl-CoA (by acetoacetyl-CoA synthase), from which acetoacetate is formed (by acetoacetyl-CoA transferase) and ﬁnally acetoacetate is decarboxylated to acetone (by acetoacetate decarboxylase) [27]. Acetic acid, which is used by yeast and bacteria to synthesize acetyl-CoA, is already present in sweet mash, and is also formed during the fermentation process [1,43]. According to Martins et al. [5] and Davídek et al. [43], acetic acid is formed during Maillard reaction as the result of the degradation of Amadori products. Based on studies by Rooney et al. [20], Davidek et al. [43], and Cui et al. [18], it can further be assumed that acetone may be present in sweet mash (i.e., before fermentation) prepared by using the TP method, as a result of Maillard reactions. According to Davídek et al. [43], acetone formation occurs as a result of hydrolytic α-dicarbonyl cleavage of 2,4-pentanedione. 1. Introduction A well-steamed mass should have a light-yellow color, and the content of the kernel hulls should ﬂow out freely. Apart from furfural and 5-hydroxymethylfurfural (HMF), MRPs notably include aldehydes, for example phenylacetaldehyde and benzaldehyde, as well as pyrazines [16,17], other furans [18], pyridine, melanoidins [19], and ketones [6]. In research into the formation of carbonyl compounds in Maillard reactions, Rooney et al. [20] conﬁrmed that, in the presence of amino acids—such as alanine, valine, leucine, isoleucine, phenylalanine, and methionine—the corresponding aldehydes are formed—i.e., acetaldehyde, isobutyraldehyde, isovaleraldehyde, 2-methylbutanal, phenylacetaldehyde, and methional. Moreover, small amounts of other carbonyl compounds may be detected, including acetone. Ketones were also identiﬁed among MRPs studied by Cui K. et al. [18]. Mansour et al. [21], in an investigation into the effects of glucose, fructose, and sucrose on the ﬂavor of extruded wheat semolina, likewise detected ketones. In the next stage of the process, ethanol fermentation, the MRPs present in distillery mashes are transformed into other compounds by yeast. Aldehydes will be reduced to corresponding alcohols, which then participate in the synthesis of esters. Acetone is also reduced to the corresponding alcohol, in this case 2-propanol (syn. isopropyl alcohol). 2-propanol belongs to the group of higher alcohols. Higher alcohols are the dominant volatile compounds present in fermented mashes. Some of them, such as 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, and 2-phenylethanol, are synthesized by the yeast from amino acids [22,23] and/or simple sugars [24,25]. However, the ﬁrst pathway is dominant. Synthesis of other higher alcohols, such as 2-butanol, 2-propanol, 1-pentanol, and 1-hexanol, which occur in lower concentrations, only takes place via the latter pathway [26]. Bacteria are also able to synthesize secondary and primary higher alcohols [27,28]. Bacteria, as well as yeast, are present in distillery mashes [1,22]. The most common are lactic acid bacteria [29,30], for which close to optimal growth conditions occur during the fermentation process. Therefore, particular attention should be given to the temperature during fermentation and the initial pH of the sweet mash. High values for each of these parameters affect the growth of lactic acid bacteria [31], which poses a real threat to the proper course of the process, since acetic acid and lactic acid are well known yeast inhibitors [32,33]. 1. Introduction However, the literature does not provide adequate reasons for the synthesis of 2-propanol during the ethanol fermentation of distillery sweet mashes. Differences between the technologies used for sweet mash production, as well as the complexity of the biochemical processes which occur during fermentations involving both yeast and other microorganisms (mainly lactic acid bacteria), combined with the action of multiple factors (including pH, temperature, sugars content, time), make it difﬁcult to clearly explain the presence of 2-propanol. The purpose of the present study was therefore to evaluate whether and in what quantities acetone is present in distillery sweet mashes prepared using the TP method. We then investigated the effects of S. cerevisiae yeast and lactic acid bacteria on acetone metabolism in distillery mashes, and on 2-propanol content in the fermented mashes, depending on the fermentation temperature. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes In our previous study [40], we compared the concentrations of 2-propanol in distillates from mashes prepared using TP and PLS for starch liberation. We demonstrated that the method of processing the starchy raw materials had an effect on the level of isopropyl alcohol. In the present study, we attempted to identify acetone as a possible precursor for the synthesis of isopropanol in mashes prepared using the pressure-thermal method. Cui et al. [18] and Rooney et al. [20] had already reported the presence of acetone among MRPs. We evaluated 37 sweet mash samples prepared in an agricultural distillery. During the preparation of sweet mash samples for gas chromatographic analysis, we observed differences in the colors of some samples (Figure 1). 4 of 15 Molecules 2019, 24, 610 Figure 1. Color of sweet mash samples (A–sample no. 29; B–sample no. 22; C–sample no. 34). Figure 1. Color of sweet mash samples (A–sample no. 29; B–sample no. 22; C–sample no. 34). We therefore undertook to assess the lightness and color of the sweet mashes. To conﬁrm the connection between the observed differences in color and the composition of MRPs, the contents of furfural and HMF were also measured. Hydroxymetylfurfural and furfural are both indicators of Maillard reactions, which occur during the thermal processing of different raw materials. The former indicates reactions involving hexoses, while the latter indicates reactions involving pentoses [44]. In all 37 samples, only furfural was detected in the sweet mashes, and no HMF was observed. The concentrations of furfural in the mash samples were compared to the results of color and brightness measurements. Under the assumption that acetone, which has been identiﬁed as a product of Maillard reactions [18,20], is a potential precursor of 2-propanol and is formed under harsh TP conditions, its concentration and correlation with color was also evaluated. Acetone alone does not affect the color. However, it appears in parallel with colored MRPs as thermal treatment progresses. We therefore attempted to evaluate this correlation. The color parameters and lightness measurements are shown in Table 1, while the results for furfural and acetone content are listed in Table 2. Table 1. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes Color parameters of the sweet mash samples* Sample number L* a* b* YI Mean SD Mean SD Mean SD Mean SD 1 83.15ghijkl 2.58 2.08j 0.06 52.23hij 1.62 89.93hi 2.79 2 85.88ijklm 2.05 -0.53d 0.01 40.20abc 0.96 66.32abc 1.58 3 84.48ghijklm 2.27 1.37hi 0.04 51.68ghi 1.39 87.72gh 2.35 4 82.70efghij 1.91 2.53k 0.06 54.24jkl 1.25 93.89ij 2.17 5 84.47ghijklm 2.01 0.51f 0.01 45.12e 1.08 75.67ef 1.80 6 85.20hijklm 2.28 0.52f 0.01 46.23e 1.23 77.69f 2.08 7 72.01ab 1.66 14.06t 0.32 78.30u 1.81 155.66r 3.59 8 87.59m 2.34 -1.21ab 0.03 42.60cd 1.14 69.64cd 1.86 9 81.50efgh 1.63 3.93m 0.08 57.89n 1.16 101.47k 2.03 10 82.79fghij 2.28 2.15j 0.06 52.05hij 1.43 89.96hi 2.47 11 84.26ghijklm 2.61 0.78g 0.02 49.30fg 1.53 83.75g 2.60 12 83.66ghijklm 1.59 1.14hi 0.02 49.17f 0.94 83.90g 1.60 13 82.69efghij 1.97 1.55i 0.04 49.83fgh 1.19 85.38g 2.03 14 81.34efgh 1.88 3.31l 0.08 55.26klm 1.27 96.92jk 2.23 15 82.39efghi 1.77 2.62k 0.06 53.60ijk 1.15 93.10ij 2.00 16 70.90ab 2.13 14.15t 0.43 74.93t 2.26 150.68p 4.54 Table 1. Color parameters of the sweet mash samples* 5 of 15 Molecules 2019, 24, 610 Table 1. Cont. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes Sample number L* a* b* YI Mean SD Mean SD Mean SD Mean SD 17 78.76def 2.21 6.03n 0.17 61.73o 1.73 112.40l 3.16 18 81.61efgh 2.25 3.74m 0.10 57.48mn 1.58 100.86k 2.77 19 78.71de 2.17 5.95n 0.16 62.70o 1.72 114.06l 3.14 20 76.10cd 2.04 9.10r 0.24 68.88r 1.85 129.79n 3.48 21 81.73efgh 2.13 3.27l 0.09 56.08lmn 1.46 98.33k 2.56 22 77.10cd 1.71 7.83o 0.17 65.81p 1.46 121.96m 2.71 23 81.54efgh 1.88 3.43l 0.08 56.81mn 1.31 99.73k 2.30 24 80.95efg 1.77 3.82m 0.08 57.02mn 1.25 100.60k 2.20 25 86.73jklm 2.37 -0.76de 0.02 41.59bc 1.14 68.81bcd 1.88 26 83.02ghijk 2.22 0.75g 0.02 41.90bc 1.12 72.26de 1.93 27 74.37bc 1.99 11.02s 0.29 71.33s 1.91 137.31o 3.67 28 87.55m 2.10 -1.24ab 0.03 38.32a 0.92 62.43a 1.50 29 87.01klm 2.40 -1.10b 0.03 40.61abc 1.12 66.86abc 1.84 30 87.18lm 2.44 -1.25ab 0.03 39.60ab 1.11 65.00ab 1.82 31 84.09ghijklm 1.87 -0.16e 0.00 44.56de 0.99 75.65ef 1.68 32 86.69jklm 1.99 -0.85c 0.02 39.56ab 0.91 65.26abc 1.50 33 87.58m 2.27 -1.43ab 0.04 38.14a 0.99 62.44a 1.62 34 70.33a 1.39 14.82u 0.29 76.95tu 1.52 156.02r 3.09 35 83.75ghijklm 2.24 1.58i 0.04 50.55fgh 1.35 86.42gh 2.31 36 76.56cd 2.04 8.47p 0.23 66.02p 1.76 123.48m 3.30 37 86.79jklm 2.32 -0.71cd 0.02 40.48abc 1.08 66.78abc 1.78 a–t—Mean values in the column with different lower-case letters are signiﬁcantly different (ANOVA at signiﬁcance level < 0.05); * Average extract content (i.e., soluble solids content) 17.2 ± 0.5 ◦Blg. a–t—Mean values in the column with different lower-case letters are signiﬁcantly different (ANOVA at signiﬁcance level < 0.05); * Average extract content (i.e., soluble solids content) 17.2 ± 0.5 ◦Blg. Table 2. Furfural and acetone content in sweet mash samples. Table 2. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes Furfural and acetone content in sweet mash sample Sample number Furfural (mg/L) Acetone (mg/L) Mean SD Mean SD 1 3.07g 0.10 1.35l 0.04 2 2.01de 0.05 0.78cde 0.02 3 4.09i 0.11 1.06h 0.03 4 4.30ij 0.10 1.07h 0.02 5 2.19ef 0.05 0.81de 0.02 6 0.32a 0.01 1.03gh 0.03 7 22.73t 0.52 1.91p 0.04 8 4.00i 0.11 1.08h 0.03 9 6.47l 0.18 1.24jk 0.03 10 4.58j 0.13 1.18ij 0.03 11 4.03i 0.11 1.24ijk 0.03 12 2.87g 0.08 1.27k 0.04 13 3.67h 0.10 1.18i 0.03 14 2.29ef 0.06 1.22ijk 0.03 15 2.51f 0.07 0.99g 0.03 16 18.80s 0.52 1.91p 0.05 17 7.68m 0.21 1.18ij 0.03 18 6.15k 0.17 1.02gh 0.03 19 7.46m 0.21 1.46m 0.04 20 10.23n 0.28 1.66n 0.05 21 4.30ij 0.12 1.46m 0.04 22 6.67l 0.18 1.84o 0.05 23 3.20g 0.09 1.22ijk 0.03 24 2.50f 0.07 1.02gh 0.03 25 1.85d 0.05 0.74bc 0.02 26 0.45ab 0.01 0.76cd 0.02 27 11.67p 0.32 2.20r 0.06 28 2.19ef 0.06 0.83e 0.02 6 of 15 Molecules 2019, 24, 610 Table 2. Cont. Sample number Furfural (mg/L) Acetone (mg/L) Mean SD Mean SD 29 2.35ef 0.06 0.69b 0.02 30 0.58ab 0.02 0.77cd 0.02 31 1.18c 0.03 0.91f 0.03 32 0.69b 0.02 0.59a 0.02 33 0.47ab 0.01 0.61a 0.02 34 15.49r 0.43 2.40s 0.07 35 3.17g 0.09 1.05h 0.03 36 11.11o 0.31 1.23ijk 0.03 37 3.02g 0.08 0.75bc 0.02 a–t—Mean values with different lower-case letters are signiﬁcantly different (ANOVA at signiﬁcance level < 0.05). Analysis of variance showed statistically signiﬁcant differences in the colors of the sweet mashes. The average values for L , a , and b * parameters ranged from 70.33 to 87.50, from −1.21 to 14.82, and from 78.30 to 42.60, respectively. Of the sweet mash samples, 27 showed positive a * parameter values, signifying a greater proportion of red pigment, as opposed to the remaining 10 samples, which had negative a * parameter values, indicating a higher content of green pigment. The differences in the positive values of this parameter ranged widely, from 0.51 (sample no. 4) to 14.82 (sample no. 34). The highest values for the a * parameter were observed in the cases of samples no. 7, 16, and 27. The values for parameter b * indicated a large share of yellow pigment in all the analyzed samples. However, as in the case of parameter a , samples no. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes 7, 16, 27, and 34 had the highest values for this parameter, in the range of 71.33–78.30. Increases in the levels of red and yellow pigments were reﬂected in the brightness (L ) of the mash samples. The mashes with the highest values for color parameters a * and b * were the darkest. The brightness of samples 7, 16, 27, and 34 ranged from 70.33 to 74.34. The variations in the yellowness index (YI) of the analyzed mash samples were related to the differences in the values of the chromatic index b * and the brightness index L . Statistically signiﬁcant differences were found in the values of the calculated YI, which was also the highest for samples no. 7, 16, 27, and 34. Analysis of variance showed signiﬁcant differences in the concentrations of acetone and furfural (Table 2). The content of these compounds was the highest in the samples with the darkest color parameters, in which the proportions of red and yellow were highest, i.e., samples no. 7, 16, 27, and 34. On this basis, it can be assumed that there is a relationship between the color of the sweet mash and the concentrations of acetone and furfural. Analysis of the correlations between parameters describing the brightness and color components of the sweet mash, the calculated yellowness index, and the acetone and furfural contents (Table 3) revealed a close relationship between these variables. Based on the calculated Pearson correlation coefﬁcients (Table 3), the L value (lightness or darkness) was negatively correlated with both the acetone and the furfural content. This conﬁrms that increases in darkness (a lower L * parameter) are accompanied by higher concentrations of furfural and acetone. The strength of these dependencies was conﬁrmed by a high correlation (r > 0.8). Table 3. Pearson correlation coefﬁcients (r) between color parameters and furfural and acetone content in sweet mashes. Compound L* a* b* YI R p-Value r p-Value r p-Value r p-Value Furfural −0.8955 0.0000 0.9297 0.0000 0.8832 0.0000 0.9105 0.0000 Acetone −0.8963 0.0000 0.9018 0.0000 0.9043 0.0000 0.9117 0.0000 Table 3. Pearson correlation coefﬁcients (r) between color parameters and furfural and acetone content in sweet mashes. Molecules 2019, 24, 610 7 of 15 The chromatic parameters a * and b * had strong positive correlations with acetone (r > 0.9) and furfural (r > 0.8 for a * and r > 0.9 for b ). 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes This suggests that the concentrations of these compounds in the sweet mash rose with increases in the presence of red and yellow pigments. An equally strong positive correlation was observed in the case of the YI (r > 0.9). Arachchi et al. [45], Rooney et al. [20], and Echavarría et al. [46] evaluated the colors of model solutions subjected to heating and their contents of different MRPs. During the heating of glucosamine and cysteine mixtures, Arachchi et al. [45] observed that the Hunter’s b value increased rapidly at higher temperatures, which was associated with the content of MRPs. Rooney et al. [20], in their investigation into the formation of carbonyl compounds in model aqueous systems containing different sugars and amino acids, concluded that color intensity was highest in the presence of glucose with valine, leucine, isoleucine, lysine, phenylalanine, or methionine. In the trials with the highest color intensity, the same authors observed a higher concentration of carbonyl compounds, including acetone. A similar relationship was noted with xylose as a source of sugar. The most intense colors were related to the presence of valine, leucine, or lysine as an amino acid. Echavarría et al. [46] used a and b * values to explain the color development of melanoidins. They noticed that the color development of the melanoidins was inﬂuenced by the type of amino acid and the temperature. Karnagwa et al. [16] reported the presence of ketones (including 2-butanone, 2-pentanone, and 2-heptanone) in model media containing free amino acids and peptides with xylose. Carbonyl compounds are well known products of the decomposition of lipid hydroperoxides, which are themselves products of lipid oxidation [47]. Both low and high molecular weight polymers affect nonzymatic browning [48]. The correlations (Table 3) and the regression model results (Table 4) suggest that higher levels of red and yellow pigments in sweet mash and higher values for YI are accompanied by increases in the concentrations of furfural and acetone. Based on the calculated R2 coefﬁcients, it can be concluded that 80% of the variability in the concentrations of furfural and acetone can be associated with changes in the values of the color parameters for brightness and yellowness index. Regression equations can therefore be used to estimate the concentrations of acetone and furfural. Table 4. Linear regression results Pairs of Relationships r R2 F p-Value Regression Equation Furfural vs. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes L* −0.8955 0.8020 141.7559 0.0000 Furfural = 84.9769 −0.9738 * L* Furfural vs. a* 0.9297 0.8644 223.0314 0.0000 Furfural = 1.7217 + 1.0442 * a* Furfural vs. b* 0.8832 0.7800 124.0878 0.0000 Furfural = -15.8436 + 0.3944 * b* Furfural vs. YI 0.9105 0.8291 169.7743 0.0000 Furfural = −11.3539 + 0.175 * YI Acetone vs. L* −0.8963 0.8034 143.0305 0.0000 Acetone = 7.9397 −0.0824 * L* Acetone vs. a* 0.9018 0.8132 152.3914 0.0000 Acetone = 0.9003 + 0.0857 * a* Acetone vs. b* 0.9043 0.8178 157.0876 0.0000 Acetone = −0.6367 + 0.0342 * b* Acetone vs. YI 0.9117 0.8311 172.2824 0.0000 Acetone = −0.2161 + 0.0148 * YI Table 4. Linear regression results The signiﬁcant differences observed in the colors of the sweet mashes from agricultural distillery are caused by a lack of repeatability during the evaporation process and by the high pressure associated with prolonged steaming. An important stage during the steaming of cereal raw materials is the periodic circulation of the content of the steamer. This is necessary to prevent the grain from overheating, because cereal grain has high bulk density. Circulation is achieved by lowering the pressure in the steamer. By opening the venting valve, the pressure is reduced slightly, which results in the cereal grains being mixed. The pressure and steaming time used in the agricultural distillery from which the sweet mash samples were collected (50 min at 0.42–0.45 MPa) was very long. It is likely that a shorter process time would reduce the concentration of MRPs. In their research, Kłosowski and Błajet-Kosicka [17] increased the pressure gradually during evaporation, to 0.5 MPa with 2–3 min of three-time circulation (at pressures of 0.15, 0.25, and 0.35 MPa). The maximum pressure of 0.5 MPa was maintained for only 5–10 min. A signiﬁcant reduction in MRPs, in this case pyrazines, was observed compared to the control sample (0.5 MPa for 30 min, single circulation at 0.3 MPa). Molecules 2019, 24, 610 8 of 15 2.2. Fermentation Results of Sweet Mashes Using Yeast and Lactic Acid Bacteria at Different Temperatures 2.2. Fermentation Results of Sweet Mashes Using Yeast and Lactic Acid Bacteria at Different Temperatures In the second stage of the study, fermentations of rye distillery sweet mashes were carried out to assess the impact of temperature and the type of microorganisms on the content of acetone and 2-propanol in mashes after fermentation. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes Total sugars content 4.15 ± 0.3 g of glucose/L (yeast) and 47.35 ± 3.1 g of glucose/L (lactic acid bacteria); Fermentation efﬁciency for yeast-provided fermentation reached 88 ± 4 % of the theoretical yield. * Total sugars content 1.30 ± 0.1 g of glucose/L (yeast) and 19.46 ± 2.4 g of glucose/L (lactic acid bacteria); Fermentation efﬁciency for yeast-provided fermentation reached 87 ± 3 % of the theoretical yield. a–c—Mean values for acetone with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p<0.05) a–c—Mean values for 2-propanol with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p < 0.05). A–B—Mean values for ethanol with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p < 0.05). A–B—Mean values for lactic acid with different letters are signiﬁcantly different (results of one-way ANOVA, p < 0.05) Table 5. Acetone, 2-propanol, ethanol, and lactic acid contents in the sweet and fermented mashes Sample Temperature (◦C) Microorganism Added to Sweet Mash (Before Fermentation) Compound Name Concentration Mean (n=3) SD Sweet mash* - - Acetone (mg/L) 1.11c 0.04 2-Propanol (mg/L) nd - Fermented mash 27 Yeast S. cerevisiae (with addition of α-hop acids) Acetone (mg/L) 0.86b 0.04 2-Propanol (mg/L) 0.23b 0.01 Ethanol (g/L) 54.5B 4.2 Lactic acid (g/L) nd - Lactic acid bacteria (with addition of nystatin) Acetone (mg/L) 0.89b 0.04 2-Propanol (mg/L) 0.07a 0.01 Ethanol (g/L) 0.8A 0.0 Lactic acid (g/L) 3.1A 0.4 Fermented mash* 35 Yeast S. cerevisiae (with addition of α-hop acids) Acetone (mg/L) 0.80b 0.04 2-Propanol (mg/L) 0.27b 0.01 Ethanol (g/L) 52.9B 4.4 Lactic acid (g/L) nd - Lactic acid bacteria (with addition of nystatin) Acetone (mg/L) 0.23a 0.02 2-Propanol (mg/L) 0.79c 0.04 Ethanol (g/L) 1.2A 0.1 Lactic acid (g/L) 5.2B 0.6 nd Not detected; SD Standard deviation * Extract of sweet mash (i e soluble solids content) and total sugar Table 5. Acetone, 2-propanol, ethanol, and lactic acid contents in the sweet and fermented mashes Sample Temperature (◦C) Microorganism Added to Sweet Mash (Before Fermentation) Compound Name Concentration Mean (n=3) SD Sweet mash* - - Acetone (mg/L) 1.11c 0.04 2-Propanol (mg/L) nd - Fermented mash 27 Yeast S. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes cerevisiae (with addition of α-hop acids) Acetone (mg/L) 0.86b 0.04 2-Propanol (mg/L) 0.23b 0.01 Ethanol (g/L) 54.5B 4.2 Lactic acid (g/L) nd - Lactic acid bacteria (with addition of nystatin) Acetone (mg/L) 0.89b 0.04 2-Propanol (mg/L) 0.07a 0.01 Ethanol (g/L) 0.8A 0.0 Lactic acid (g/L) 3.1A 0.4 Fermented mash* 35 Yeast S. cerevisiae (with addition of α-hop acids) Acetone (mg/L) 0.80b 0.04 2-Propanol (mg/L) 0.27b 0.01 Ethanol (g/L) 52.9B 4.4 Lactic acid (g/L) nd - Lactic acid bacteria (with addition of nystatin) Acetone (mg/L) 0.23a 0.02 2-Propanol (mg/L) 0.79c 0.04 Ethanol (g/L) 1.2A 0.1 Lactic acid (g/L) 5.2B 0.6 nd—Not detected; SD—Standard deviation. * Extract of sweet mash (i.e., soluble solids content) and total sugars content 14.3 ± 1.1 ◦Blg and 114.0 ± 6.8 g of glucose/L, respectively. Total sugars content 4.15 ± 0.3 g of glucose/L (yeast) and 47.35 ± 3.1 g of glucose/L (lactic acid bacteria); Fermentation efﬁciency for yeast-provided fermentation reached 88 ± 4 % of the theoretical yield. * Total sugars content 1.30 ± 0.1 g of glucose/L (yeast) and 19.46 ± 2.4 g of glucose/L (lactic acid bacteria); Fermentation efﬁciency for yeast-provided fermentation reached 87 ± 3 % of the theoretical yield. a–c—Mean values for acetone with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p<0.05). a–c—Mean values for 2-propanol with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p < 0.05). A–B—Mean values for ethanol with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p < 0.05). A–B—Mean values for lactic acid with different letters are signiﬁcantly different (results of one-way ANOVA, p < 0.05). able 5. Acetone, 2-propanol, ethanol, and lactic acid contents in the sweet and fermented mashes nd—Not detected; SD—Standard deviation. * Extract of sweet mash (i.e., soluble solids content) and total sugars content 14.3 ± 1.1 ◦Blg and 114.0 ± 6.8 g of glucose/L, respectively. Total sugars content 4.15 ± 0.3 g of glucose/L (yeast) and 47.35 ± 3.1 g of glucose/L (lactic acid bacteria); Fermentation efﬁciency for yeast-provided fermentation reached 88 ± 4 % of the theoretical yield. * Total sugars content 1.30 ± 0.1 g of glucose/L (yeast) and 19.46 ± 2.4 g of glucose/L (lactic acid bacteria); Fermentation efﬁciency for yeast-provided fermentation reached 87 ± 3 % of the theoretical yield. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes The concentration of acetone in the laboratory-prepared sweet mash was 1.11 ± 0.04 mg/L (Table 5). The mash samples were inoculated using different microorganisms, S. cerevisiae yeast, or lactic acid bacteria strains, and then fermented in thermostatic rooms with different temperatures (air temperature 27 ◦C or 35 ◦C). The temperatures used in our experiments were selected based on observations (data not published) of the conditions used in agricultural distilleries. It was noticed that, as the temperature in the fermentation tanks increased (reaching up to 38–39 ◦C in the main phase of the fermentation process), the concentration of 2-propanol in the spirit also rose in comparison to samples of spirits obtained from mashes fermented at lower temperatures (maximum temperature in the fermentation tank 33–34 ◦C). Excessive temperatures during fermentation may be caused by setting a high temperature for the mash at the beginning of the process (over 30 ◦C), the lack of a cooling system for the fermentation tanks, or by ambient temperature, which is most often determined by weather conditions. The results of statistical analysis (Table 5) show that both the temperature and the microorg present in the mash affected the concentration of acetone and 2-propanol. Table 5. Acetone, 2-propanol, ethanol, and lactic acid contents in the sweet and fermented mashes Sample Temperature (◦C) Microorganism Added to Sweet Mash (Before Fermentation) Compound Name Concentration Mean (n=3) SD Sweet mash* - - Acetone (mg/L) 1.11c 0.04 2-Propanol (mg/L) nd - Fermented mash 27 Yeast S. cerevisiae (with addition of α-hop acids) Acetone (mg/L) 0.86b 0.04 2-Propanol (mg/L) 0.23b 0.01 Ethanol (g/L) 54.5B 4.2 Lactic acid (g/L) nd - Lactic acid bacteria (with addition of nystatin) Acetone (mg/L) 0.89b 0.04 2-Propanol (mg/L) 0.07a 0.01 Ethanol (g/L) 0.8A 0.0 Lactic acid (g/L) 3.1A 0.4 Fermented mash* 35 Yeast S. cerevisiae (with addition of α-hop acids) Acetone (mg/L) 0.80b 0.04 2-Propanol (mg/L) 0.27b 0.01 Ethanol (g/L) 52.9B 4.4 Lactic acid (g/L) nd - Lactic acid bacteria (with addition of nystatin) Acetone (mg/L) 0.23a 0.02 2-Propanol (mg/L) 0.79c 0.04 Ethanol (g/L) 1.2A 0.1 Lactic acid (g/L) 5.2B 0.6 nd—Not detected; SD—Standard deviation. * Extract of sweet mash (i.e., soluble solids content) and total sugars content 14.3 ± 1.1 ◦Blg and 114.0 ± 6.8 g of glucose/L, respectively. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes a–c—Mean values for acetone with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p<0.05). a–c—Mean values for 2-propanol with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p < 0.05). A–B—Mean values for ethanol with different letters are signiﬁcantly different (results of two-way ANOVA with post-hoc Duncan’s multiple test, p < 0.05). A–B—Mean values for lactic acid with different letters are signiﬁcantly different (results of one-way ANOVA, p < 0.05). 9 of 15 Molecules 2019, 24, 610 As can be seen, in comparison with the sweet mash (before fermentation) the samples fermented using S. cerevisiae yeast showed a decrease in the concentration of acetone. It is worth noting that the reduction was similar in both samples fermented by the yeast. Simultaneously, we detected 2-propanol in these samples, although the temperature did not affect its content. The presence of 2-propanol may conﬁrm the reduction of acetone to isopropyl alcohol. Alcohol dehydrogenases (ADH, EC 1.1.1.1) react with a wide variety of alcohols, aldehydes, and ketones [49]. However, yeast alcohol dehydrogenases are the most active towards acetaldehyde and ethanol, while the oxidation of other alcohols (primary and secondary alcohols) and the reduction of higher aldehydes or methyl ketones catalyze poorly [50]. The lower activity of yeast ADH towards longer chain alcohols, aldehydes, and methyl ketones is due to the smaller size of the substrate binding pocket [51]. Probably for this reason, the acetone was not completely metabolized during the process of ethanol fermentation. Moreover, the temperature in the thermostatic rooms did not affect the ethanol content or fermentation efﬁciency (Table 5). Compared to yeast, bacteria are more often mentioned as producers of secondary alcohols, including isopropanol [52]. Clostridia is the most well researched species [36]. There is less information in the literature concerning lactic acid bacteria, although they are commonly found in distillery mashes [29]. The quantity of bacteria, including lactic acid bacteria, in mash depends on the method of starch liberation. However, there is always some quantity present [1,29]. Therefore, to ascertain whether lactic acid bacteria were responsible for the reduction of acetone to isopropanol, two mash samples were inoculated with two lactic acid bacteria strains, using antifungal protection (nystatin). A decrease in acetone concentration was observed, but it was highly dependent on the temperature. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes During fermentation at 27 ◦C, the concentration of acetone dropped by 0.22 mg/L of mash, while at 35 ◦C it decreased by 0.88 mg/L (by 79%). There were also differences in the concentration of 2-propanol in the mashes after 72 h, depending on the temperature in the thermostatic room. The content of 2-propanol was more than 11 times higher in mash fermented at the higher temperature. Lactic acid bacteria, depending on the genera and species, are categorized as either thermophiles or mesophiles. Lactococcus lactis ssp. lactis are included in the latter group. However, studies have shown [53] that increasing the temperature from 25 ◦C to 38 ◦C increases their speciﬁc growth rate. Lactobacillus casei bacteria belong to the group of thermophiles. The inﬂuence of temperature on the growth and metabolism of lactic acid bacteria was shown by the lactic acid content. The concentration of lactic acid in the mash inoculated with two strains of bacteria was 3.1 g/L when the temperature in the thermostat was set to 27 ◦C, and 5.2 g/L when the temperature in the thermostat was 35 ◦C (Table 5). In our previous research [1], we showed that during the fermentation of mashes not only yeast growth but also bacterial growth may be observed, including the growth of lactic acid bacteria. The source of the bacteria present during ethanol fermentation may be the starchy raw materials, but also the yeast inoculum. In agricultural distilleries, the yeast are propagated in a tank called a ‘yeast tank’, using sweet mash as a medium. To limit bacterial growth during yeast propagation, the initial pH of the sweet mash is set at 3.5–4.0. Propagation starts when the sweet mash is inoculated with dry distillery yeast (after rehydration). The cycle of yeast propagation takes about 10–12 h. Then about 80% of the volume of the inoculum in the yeast tank is used to inoculate the sweet mash in the fermentation tank. Next, a fresh portion of the sweet mash is added to the yeast tank and the next cycle of yeast propagation is carried out. An average of two cycles are carried out per day. How often the yeast is refreshed differs depending on the distillery. It may be every 2–4 weeks, or every 6 months. Lactic acid bacteria are categorized as neutrophils, although some strains show the ability to grow at lower pH. 2.1. Colorimetric and Chromatographic Evaluation of Sweet Mashes The composition of the medium can also have a signiﬁcant effect on their survival at low pH values. LeBlanc et al. [54] observed a slowdown in the growth of Lactobacillus fermentum in medium at pH 4.5 compared to samples with a higher pH. Vrancken et al. [55] showed that the Lactobacillus fermentum IMDO 130201 strain converted sugars present in the medium (glucose, fructose, and sucrose) and form lactic acid in a pH range from 5 to 3.5. In our research, we also detected both lactic and acetic acids in mash samples collected from the yeast tank of an agricultural distillery after the 1st and 10th cycles of yeast propagation. The results (Table 6) show that lactic acid content was two-fold higher in the mash 10 of 15 Molecules 2019, 24, 610 after the 10th cycle of propagation in comparison to the mash after the 1st cycle. A similar relationship was observed for acetic acid content. after the 10th cycle of propagation in comparison to the mash after the 1st cycle. A similar relationship was observed for acetic acid content. Table 6. Lactic acid and acetic acid content in mashes after the ﬁrst and the tenth cycles of yeast inoculation (industrial scale). Cyle of Yeast Inoculation Compound Name Concentration Mean (n = 3) SD 1st Lactic acid (g/L) 1.1a 0.2 Acetic acid (g/L) 0.4A 0.1 10th Lactic acid (g/L) 2.1b 0.2 Acetic acid (g/L) 0.7B 0.1 SD—Standard deviation. a-b—Mean values of lactic acid in column with different lower-case letters are signiﬁcantly different (results of one-way ANOVA, p < 0.05); A-B—Mean values of acetic acid in column with different upper-case letters are signiﬁcantly different (results of one-way ANOVA, p < 0.05). SD—Standard deviation. a-b—Mean values of lactic acid in column with different lower-case letters are signiﬁcantly different (results of one-way ANOVA, p < 0.05); A-B—Mean values of acetic acid in column with different upper-case letters are signiﬁcantly different (results of one-way ANOVA, p < 0.05). An increase in the concentrations of both acids may indicate the growth of lactic acid bacteria during yeast propagation. Inoculation of the sweet mash in the fermentation tank with yeast contaminated with bacteria may cause the growth not of only of yeast, but also of lactic acid bacteria. Moreover, distilleries usually initially set the sweet mash in the fermentation tank to pH 5 or over, which further promotes bacterial growth. 3.1. Industrial Scale Pressure-Thermal Treatment and Mashing Process 3.1. Industrial Scale Pressure-Thermal Treatment and Mashing Process Rye grains were treated on an industrial scale, in a tapered cylindrical steamer (total volume 3.75 m3). The steamer was ﬁlled with water (1.75 m3), which was heated to boiling point by injecting superheated steam. Then, 0.7 t of rye grains was poured in. After venting, the steamer was closed and the pressure inside was increased gradually to 0.42–0.45 MPa. The raw material was steamed at this pressure for 50 min, with periodical circulation of the content. The whole process was carried out with manual control of the steaming parameters (pressure and time) and circulation. Upon completion, the content of the steamer was transferred to a cylindrical steel-mashing vessel. The steamed mass was stirred continuously in a steel mashing vessel and cooled to 90 ◦C, at which point liquefying Termamyl S.C. preparation was added (0.13 L/t of starch, Novozymes, Bagsværd, Denmark). The mixture was kept at this temperature for 15 min, then cooled to 65 ◦C before the addition of saccharifying SAN Extra preparation (0.6 L/t of starch, Novozymes, Denmark). After the addition of SAN Extra, the mash was cooled further, to 32–34 ◦C. This procedure was repeated 37 times. All the mash samples (n = 37) were frozen immediately for transportation from the agricultural distillery to the laboratory. The mash samples were assessed in terms of their color and chemical composition. Mash samples from the yeast tank used for yeast propagation were also frozen and transported to the laboratory (after the 1st and 10th cycles). Prior to inoculation with the yeast, the sweet mash was acidiﬁed to pH 3.5. 3.2. Laboratory Scale Pressure-Thermal Treatment, Mashing, and Fermentation Processes Pressure-thermal processing and mashing (with enzyme preparations) were performed according procedures described previously [1]. Rye grains were used as the raw material. The samples of mash produced on a laboratory scale were used immediately to prepare fermentation trials. The pH of the sweet mash before fermentation was set at 5.0 using sulfuric acid (25% w/w solution). Two variants of the sweet mash were then prepared, with different microorganisms used for inoculation: 11 of 15 Molecules 2019, 24, 610 • Ethanol Red yeast (Saccharomyces cerevisiae, Fermentis Division S.I. Lesaffre, Marcq-en-Barœul, France) (1.3×107 CFU/mL of mash), with the addition of 80 mg/L IsoStab® hop α-acid preparation (BetaTec GmbH, Nürnberg, Germany), to protect the process from bacterial contamination and prevent microbial infections; • a mixture of lactic acid bacteria strains Lactococcus lactis ssp. lactis ŁOCK0877 and Lactobacillus casei ŁOCK0901, obtained from the ŁOCK Pure Culture Collection (ﬁnal quantity 4.0×106 CFU/mL of mash), with the addition of nystatin, to prevent yeast growth. • a mixture of lactic acid bacteria strains Lactococcus lactis ssp. lactis ŁOCK0877 and Lactobacillus casei ŁOCK0901, obtained from the ŁOCK Pure Culture Collection (ﬁnal quantity 4.0×106 CFU/mL of mash), with the addition of nystatin, to prevent yeast growth. Before fermentation, all of the mash samples (1000 mL) were supplemented with diammonium phosphate (0.2 g/L). The glass ﬂasks (2000 mL) were closed with a fermentation tube ﬁlled with glycerin. Fermentations were performed in two thermostatic rooms, with the temperatures set to 27 and 35 ◦C. Fermentation was continued for 72 h. Before and after fermentation, the mash samples were taken for gas chromatographic analysis (HS-GC-MS). All experiments were prepared in triplicate. 3.4. Determination of Total Sugars and Extract Content in the Mashes 3.4. Determination of Total Sugars and Extract Content in the Mashes The concentration of total sugars in the sweet and fermented mashes prepared on the laboratory scale was determined according to the method described previously [1]. The content of extract in the sweet mashes prepared at both the industrial and laboratory scales was measured using a hydrometer, according to the method described previously [57]. 3.5. HPLC Analysis of Fermented Mashes The concentrations of ethanol and lactic acid in the mashes were evaluated using HPLC, according procedures described previously [1]. Fermentation efﬁciency was calculated according to method described previously [57]. Fermentation efﬁciency was calculated according to method described previously [57]. 3.3. Color Measurement Tests were performed using a CHROMA METER CR-5 device (Konica Minolta, Osaka, Japan), using the CIE Lab scale. Three color components were measured: L* (darkness or lightness of color ranges from black (0) to white (100)), a* (+red to −green chromatic components), and b* (+yellow to −blue chromatic components). Based on the L* and b* parameters, the yellowness index (YI) was calculated as Equation (1) [56] YI = 142.86b/L (1) (1) YI = 142.86b/L YI = 142.86b/L Prior to analysis, samples of the mashes were centrifuged at 23,300× g for 10 min at 15 ◦C. The ﬁnal result was the arithmetic mean of three measurements for each sample. 3.6. Gas Chromatographic Analysis (HS-GC-MS) of Sweet and Fermented Mashes Qualitative analysis was performed using a GC apparatus (Agilent 7890A, Agilent Technologies, Santa Clara, CA, USA) coupled to a mass spectrometer (Agilent MSD 5975C, Agilent Technologies, Santa Clara, CA, USA). A capillary column was used to separate the compounds (TGWAX-MS, Thermo, Scientiﬁc Fisher, Pittsburgh, PA, USA; 60 m × 0.32 mm × 0.50 µm). The GC oven temperature was programmed to increase from 40 ◦C (5 min) to 80 ◦C at a rate of 5 ◦C/min, then to 220 ◦C at a rate of 10 ◦C/min, where it was maintained for 5 min. The ﬂow rate of the carrier gas (helium) through the column was 1.1 mL/min. The temperature of the injector (split/splitless) was 250 ◦C. Injections of the tested samples were made in the split mode (10:1) using a headspace analyzer (Agilent 7697A, Agilent Technologies, Santa Clara, CA, USA). The temperatures of the MS ion source, transfer line, and quadrupole were 230, 250, and 150 ◦C, respectively. The ionization energy was 70 eV. Prior to analysis, a 20 mL headspace vial was ﬁlled with 7 mL of mash and closed tightly using an aluminum cap and septa. 12 of 15 Molecules 2019, 24, 610 Headspace conditions: • Temperature settings: oven temperature 50 ◦C, loop temperature 60 ◦C, transfer line temperature 70 ◦C. • Temperature settings: oven temperature 50 ◦C, loop temperature 60 ◦C, transfer line temperature 70 ◦C. • Timing settings: vial equilibration time 20 min, injection duration 0.7 min, GC cycle time 47 m • Timing settings: vial equilibration time 20 min, injection duration 0.7 min, GC cycle time 47 min. • Vial and loop settings: vial shaking 136 shakes/min, ﬁll pressure 15 psi, vial pressurization gas helium. • Vial and loop settings: vial shaking 136 shakes/min, ﬁll pressure 15 psi, vial pressurization gas helium. • Vial and loop settings: vial shaking 136 shakes/min, ﬁll pressure 15 psi, vial pressurization gas helium. Acetone, MRPs (furfural and hydroxymethylfurfural), and 2-propanol were identiﬁed based on a comparison of their mass spectra with those of standard compounds and with the mass spectra in the NIST/EPA/NIH Mass Spectra Library (2012; Version 2.0g). Quantitative analysis of the sweet and fermented mashes was performed to determine the concentrations of the identiﬁed compounds. Headspace and GC conditions were the same those used for qualitative analyses. The acetone, furfural, and 2-propanol were quantiﬁed using calibration curves in the selected ion monitoring mode (SIM). 3.7. Statistical Analysis Statistical calculations were performed using STATISTICA 6.0 software (Tibco Software, Palo Alto, CA, USA). To evaluate the differences between the tested sweet mash samples, analysis of variance (ANOVA) was conducted with a 0.05 signiﬁcance level. When statistical differences were detected (p < 0.05), means were compared using the post hoc Duncan test with a 0.05 signiﬁcance level. Correlation and regression analyses were used to determine the relationship between the test color components and the concentrations of acetone and furfural. Signiﬁcance tests were performed with a 0.05 signiﬁcance level. 3.6. Gas Chromatographic Analysis (HS-GC-MS) of Sweet and Fermented Mashes Quantitative analysis was performed using Agilent MassHunter software (Agilent Technologies, Santa Clara, CA, USA). The results were expressed in mg/L of mash. All analyses were performed in triplicate. Conﬂicts of Interest: The authors declare no conﬂict of interest. Funding: This work was partially supported by the Polish National Center for Research and Development under grant no. PBS2/B8/9/2013. hor Contributions: K.P.-P. conceived and designed the experiments; K.P.-P., M.B., U.D.-K., B.P.-S., G.C., L.A P.P. performed the experiments; K.P.P. analyzed the data and wrote the paper. 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https://openalex.org/W2731504731	https://revistas.upch.edu.pe/index.php/RMH/article/download/3108/3079, https://www.redalyc.org/pdf/3380/338051875003.pdf	es		Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular	Revista médica herediana	2,017	cc-by	4,752	INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH Rev Med Hered. 2017; 28:84-92. Esta obra está bajo una Licencia Creative Commons Atribución 4.0 Internacional. Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular Factors associated with optimal oral anticoagulation in elder patients with atrial fibrillation María T. Gamero1,a, Marilhia Cornejo1,a, Roy Dueñas2,b, Frine Samalvides1,3,c RESUMEN Objetivos: Determinar la calidad del control de la anticoagulación oral y los posibles factores asociados al tiempo de rango terapéutico. Material y métodos: Estudio retrospectivo con grupo control y exploración de posibles variables, realizado en pacientes ≥65 años con fibrilación no valvular e indicación de anticoagulación del Hospital Cayetano Heredia entre el 2011-2013. Se determinó el tiempo de rango terapéutico (TRT) por el método de Rosendaal y se consideró control óptimo (TRT ≥60%) y subóptimo (TRT <60%). Se realizó análisis univariado para los factores. Resultados: La mediana de la edad fue 82,23 años, 74 (51,75%) fueron mujeres. Ciento cuarenta pacientes tuvieron indicación de anticoagulación, 94 (67,14%) iniciaron warfarina, 28 (19,72%) tuvo control óptimo y 66 (46,48%) subóptimo. La edad ≥75 años (OR 25,5; IC: 7,39-155,8), no bajo grado de instrucción (OR 8; IC 3,61-20,61), ≥3 comorbilidades (OR 4,5; IC 2,33-9,39), discontinuidad terapéutica (OR 2,75; IC 1,44-5,52) y polifarmacia (OR 4,2; IC 2,16-8,80) se asociaron con control subóptimo, mientras que independencia funcional (OR 4,3; IC 2,169,47), no deterioro cognitivo (OR 7,8; IC 3,53-20,19) y género femenino (OR 3; IC 1,62-5,81) al control óptimo. Conclusiones: Un gran porcentaje de pacientes con indicación de ACO y con bajo riesgo de sangrado no iniciaron terapia anticoagulante; y el 50% de quienes la iniciaron, tuvo un control subóptimo. Los pacientes de mayor edad, con mayor comorbilidad y el uso de múltiples medicamentos se asociaron con control subóptimo; mientras que el género femenino y la independencia funcional se asociaron con control óptimo. PALABRAS CLAVE: Fibrilación atrial, warfarina, anciano, accidente cerebrovascular. (Fuente: DeCS BIREME). SUMMARY Objective: To determine the quality of oral anticoagulation control and the possible factors associated with time to reach therapeutic range. Methods: Retrospective study with a control group in patients ≥65 years old with nonvalvular atrial fibrillation who had indication for oral anticoagulation at Hospital Cayetano Heredia from 2011 to 2013. Time to therapeutic range (TTR) was determined by the Rosendal´s method, an optimal control was defined as TTR ≥60%. A multivariate analysis was performed. Results: Median age was 82.23 years; 74 (51.75%) were females; 140 patients had indication for anticoagulation; 94 (67.14%) started warfarin; 28 (19.72%) had optimal control and 66 did not (46.48%). Age ≥ 75 years (OR 25.5; CI: 7.39-155.8) and no low degree of instruction (OR 1 2 3 a b c 84 Facultad de Medicina Alberto Hurtado, Universidad Peruana Cayetano Heredia. Lima, Perú. Servicio de Cardiología, Hospital Nacional Cayetano Heredia. Lima, Perú Instituto de Medicina Tropical Alexander von Humboldt, Hospital Nacional Cayetano Heredia. Lima, Perú. Médico Cirujano; Médico Cardiólogo; Magister de Epidemiologia Rev Med Hered. 2017; 28:84-92. Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH Gamero M, y col. 25.5; CI: 7.39-155.8) were associated with sub optimal control, while functional independence (OR 4.3; CI: 2.169.47), no cognitive deterioration (OR 7.8; CI: 3.53-20.19) and female gender (OR 3; CI: 1.62-5.81) were associated with optimal control. Conclusions: A great percentage of patients with indications for oral anticoagulation and at low risk of bleeding did not start it, and 50% of those who started it had suboptimal control. Older patients with comorbidities who used multiple medications had sub optimal control, while females and functional independence were associated with optimal control. KEYWORDS: Atrial fibrillation, warfarin, aged, stroke. (Source: MeSH NLM). INTRODUCCIÓN El envejecimiento demográfico es una realidad global. La población peruana de 60 o más años representa el 8,8% del total de población a nivel nacional (1). El adulto mayor tiene una elevada prevalencia de patologías cardiovasculares, siendo la fibrilación auricular (FA) la arritmia cardiaca más común. Su prevalencia en el adulto mayor de 65 años es 6% y 10% en los de 80 años a más (2). Asimismo, la tasa de incidencia aumenta de manera constante con la edad (2). La tasa de accidente cerebrovascular (ACV), también aumenta con la edad, llegando a 51 por 1000 persona-año entre los 80-89 años (3,4). La FA se produce cuando se presenta una anomalía estructural o electrofisiológica que altera el tejido auricular (5); las anomalías estructurales representan el 70-80% de los casos (6,7). Existen cuatro formas de FA, paroxística si se presenta por menos de 7 días, persistente en caso se mantenga 7 días o más. La FA de larga evolución durará ≥12 meses y busca el control del ritmo; en caso no amerite este último será permanente (5). La FA es responsable del 80% de ACV isquémicos cardioembólicos (6,8). Asimismo, esta arritmia aumenta el riesgo de ACV 5 veces, generando discapacidad severa y mortalidad. De ello radica la importancia del control adecuado de la terapia anticoagulante. La escala CHADS2VASC determina la indicación para iniciar terapia anticoagulante oral (ACO) en pacientes con FA no valvular. Un puntaje ≥2 es indicación para recibir antagonistas de vitamina K (AVK), dabigatran, rivaroxaban o apixaban (5,9). La razón internacional normalizada (INR) entre 2,0 – 3,0 correlaciona con la máxima protección en contra de ACV con sangrado mínimo (10). La eficacia del tratamiento con AVK requiere mantener una intensidad anticoagulante óptima dado que el nivel de Rev Med Hered. 2017; 28:84-92. complicación hemorrágica es alrededor 1,4% (6). La estimación de riesgo de hemorragia puede realizarse mediante la evaluación de la escala HAS-BLED, cuyo puntaje ≥3 indica mayor riesgo de sangrado. En un estudio europeo Nieuwlaat et al, encontraron que sólo el 67% de pacientes con indicación de ACO estaba recibiendo tratamiento (11). Los ensayos clínicos estiman que el promedio de los pacientes puede mantener un rango de INR entre 2-3 durante el 60-65% del tiempo (5). La calidad del estado de la anticoagulación se mide por el método del tiempo en rango terapéutico (TRT). Los pacientes con un TRT < 60 % tienen tasas más altas de mortalidad anual y hemorragia mayor en comparación con los pacientes con buen control del INR, 4,2 % vs 1,69 % y 3,85 % vs 1,58 % respectivamente (12). Se estima que 49,4% se encuentran con un TRT <60% del tiempo de anticoagulación (13). Sin embargo, en nuestro país se desconoce el grado de control de los pacientes en ACO y el nivel de TRT controlado. Es indudable la importancia de identificar los factores asociados al control de la ACO en pacientes con FA (14). Se han descrito factores de riesgo como deterioro cognitivo, vivir en una casa de reposo y no iniciar de ACO durante la hospitalización (15). Asimismo, otros estudios describen como factores asociados al status étnico (no raza blanca), edad, tabaquismo y ≥3 comorbilidades (16,17). El presente estudio tuvo como objetivo determinar la calidad de la anticoagulación y los factores asociados al mismo en pacientes adultos mayores con FA no valvular en un hospital general de nivel III-1. MATERIAL Y MÉTODOS Tipo y diseño general del estudio: Estudio descriptivo retrospectivo con grupo control y exploración de posibles asociaciones. 85 Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH Población: Pacientes con edad ≥65 años, con diagnóstico de FA no valvular atendidos del Hospital Nacional Cayetano Heredia entre los años 2011-2013. Fueron excluidos los pacientes con estenosis mitral significativa (moderada o severa) y portadores de válvula protésica. Muestra: Del registro de atenciones durante los años 2011-2013 se seleccionó a los pacientes ≥65 años, obteniéndose un total de 300 historias clínicas. Mediante la estimación de un tamaño de muestra para frecuencia en una población, se seleccionaron las historias clínicas de 150 pacientes; de las cuales siete se encontraban incompletas, obteniendo un número final de muestra de 143 historias clínicas. Definición de variables: - Grado de instrucción: se consideró bajo grado de instrucción a pacientes analfabetos o con primaria; y no bajo de instrucción a pacientes con secundaria o estudios superiores. - Deterioro cognitivo como perdida de la habilidad mental por > 6 meses. - Discontinuidad terapéutica: paciente que no tiene control de INR en los últimos 6 meses o no ha sido visto por la especialidad en dicho periodo. - Polifarmacia: consumo de ≥3 medicamentos. - Sangrado mayor: asociado a la hemorragia intracraneal, o la disminución de ≥5 g/dl o ≥15% del hematocrito (según TIMI). - Sangrado menor: disminución de 3 gr/dl o 10% del hematocrito (según TIMI). Procedimiento y técnicas: Se seleccionaron a los pacientes con indicación de ACO según score CHADS2VASC, en terapia con AVK. Se calculó el TRT mediante el método de Rosendaal (18,19,20), considerándose como control óptimo TRT ≥60% y control subóptimo TRT <60%. Este es el valor utilizado en los estudios clínicos internacionales (19,20). Los grupos fueron sometidos a análisis para determinar la asociación con los factores edad, género, grado de instrucción, dependencia funcional, deterioro cognitivo, comorbilidades, discontinuidad terapéutica y polifarmacia, así como el porcentaje de ACV y sangrado en ambos grupos. Se contó con la aprobación del Centro de Investigación y Comité de Ética de la Universidad Peruana Cayetano Heredia (CI #60487) así como del Hospital Nacional Cayetano Heredia. Análisis: Se utilizó estadística descriptiva, se determinó la frecuencia absoluta y porcentaje para 86 Gamero M, y col. las variables cualitativas, y la media y desviación estándar para las cuantitativas. La calidad de la anticoagulación se midió mediante el TRT, considerándose como una buena calidad al grupo control (TRT≥60%). Para la exploración se utilizó chi2 para variables cualitativas y t-student para las cuantitativas. Se determinó los factores asociados con un nivel de significancia p<0,05 en ambos grupos mediante el programa EPI INFO versión 7. RESULTADOS Se revisaron un total de 143 historias clínicas de pacientes con edad ³65 años y diagnóstico de fibrilación auricular no valvular, 74 (51,75%) fueron mujeres; la mediana de la edad fue de 82,23 años (RI 76-88 años) (Tabla 1). Las escalas CHADS2VASC y HASBLED dividen a los pacientes en grupos de riesgo cardioembólico y sangrado respectivamente, encontrándose un mayor número de pacientes con un puntaje de 5 (24,48%), y de 2 (46,48%) (Tablas 2 y 3). Asimismo, la media del puntaje de la escala CHADDS2VASC fue 4,58 ± 1,57 y en la escala HASBLED 1,96 ± 0,92. De las 143 historias clínicas revisadas, 140 contaban con indicación de anticoagulación oral. De este grupo, 41 (29,28%) pacientes no iniciaron terapia anticoagulante, 5 (3,57%) iniciaron nuevos anticoagulantes orales, y 94 (67,14%) iniciaron terapia con warfarina. Asimismo, 93 (65,03 %) pacientes no presentaron controles en los últimos 6 meses. La mediana del tiempo para llegar a rango terapéutico fue de 31 días (RI 7,5-93,5). Respecto al TRT general se encontró un valor de 35,40% (RI ±5,35-63%). Se obtuvo 28 (29,78%) pacientes en el grupo de control óptimo, y 66 (70,21%) en el grupo de control subóptimo. La mediana del TRT del grupo con control óptimo fue 79,3% (RI 60-89,4) y en el control subóptimo 15,05% (RI 0-59,3). Dentro de las características sociodemográficas en los grupo con control óptimo y subóptimo se encontró que la edad promedio era de 83,78 años (RI 79-88,5) y 81,3 años (RI 75,5-89) respectivamente. Con respecto al género, la diferencia entre mujeres y varones fue menor en el grupo con control óptimo que en el subóptimo. El nivel de instrucción de bajo grado fue mayor en grupo con control optimo (78,57% vs 72,73). Rev Med Hered. 2017; 28:84-92. Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH Gamero M, y col. Tabla 1. Características sociodemográficas y clínicas en pacientes ≥65 años con fibrilación auricular no valvular en el Hospital Nacional Cayetano Heredia (2011-2013) según género.(%) Femenino Masculino n % n % ≥ 75 años 61 51,70% 57 48,30% 65-74 años 13 52,00% 12 48,00% Bajo Grado 59 55,14% 48 44,85% No Bajo Grado 16 44,44% 20 55,55% Independiente 51 54,84% 42 45,16% Dependiente parcial o total 9 18,00% 41 82,00% 15 41,67% 21 58,33% 45 48,39% 48 51,61% Paroxística 21 43,75% 27 56,25% Persistente 20 48,78% 21 51,22% De larga evolución 4 80,00% 1 20,00% Permanente 29 59,18% 20 40,82% Edad Grado de instrucción Capacidad funcional Deterioro cognitivo Si Discontinuidad terapéutica Si Clasificación de FA Dentro las características clínicas, el índice KATZ encontró 19 (17,86%) pacientes independientes en el grupo con control óptimo, y 39(59,09%) en el grupo de control subóptimo. Respecto al deterioro cognitivo, se encontró 6 (21,43%) pacientes con control óptimo, y 19 (28,79%) con control subóptimo. Con respecto a las comorbilidades, la diferencia entre el grupo con control óptimo y subóptimo fue mínima (64,29% vs 68,18%). Por otra parte, se presentaron 8 casos de ACV isquémicos, siendo la mayoría del control subóptimo (10,6% vs 3,57%); mientras que la diferencia de los casos de sangrado mayor fue menor (10,60% vs 7,14%). La polifarmacia se encontró presente en 18 (64,29%) pacientes del grupo con control óptimo, y 42 (63,64%) del grupo de control subóptimo; además, 16 (57,14%) pacientes del grupo con control óptimo presentaron discontinuidad terapéutica, mientras que en el grupo de control subtóptimo fueron 33 (50%) (Tabla 4). Rev Med Hered. 2017; 28:84-92. Tabla 2. Puntaje CHADS2VASC en pacientes ≥65 años con fibrilación auricular no valvular en el Hospital Nacional Cayetano Heredia (2011-2013). Riesgo de ACV† PUNTAJE N % (%/año) 1 3 2,10% 1,30% 2 11 7,69% 2,20% 3 22 15,38% 3,20% 4 31 21,68% 4,00% 5 35 24,48% 6,70% 6 24 16,78% 9,80% 7 14 9,79% 9,60% 8 3 2,10% 6,70% 9 0 0% 15,20% †Tasa de ACV según puntaje basado en la data de Lip y colegas. 87 Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH Gamero M, y col. Tabla 3. Puntaje HASBLED en pacientes ≥65 años con fibrilación auricular no valvular en el Hospital Nacional Cayetano Heredia (2011-2013). PUNTAJE N % Riesgo de sangrado (%100 pacientes/año)† 1 47 33,10% 1,02 2 66 46,48% 1,88 3 18 12,68% 3,74 4 9 6,34% 8,7 5 2 1,41% Data insuficiente Basado en la cohorte de validación inicial reportada por Pisters, et al, las puntuaciones ≥5 de HAS-BLED dispone tasas reales de accidente cerebrovascular. Estas estimaciones pueden variar en cohortes actuales. † Tabla 4. Factores asociados al control óptimo y subóptimo en pacientes ≥65 años con fibrilación auricular no valvular en el Hospital Nacional Cayetano Heredia (2011-2013). Control óptimo (n=28) Factores Control subóptimo (n=66) N (%) IC (95%) N (%) IC (95%) 26 (92,86%) 76,50-99,12 51 (77,27) 65,30-86,69 F 15(53,57%) 33,87-72,49 39 (59,09) 46.29-71,05 M 13(46,43%) 27,51-66,13 27 (40,91) 28,95-53,71 Edad >75 años Género KATZ Independencia 19(67,86%) 47,65-84,12 39(59,09) 46,29-71,05 Dependencia 9(32,14) 15,88-52,35 27(40,91) 28,95-53,71 Bajo grado 22(78,57%) 59,05-91,70 48 (72,73) 60,36-82,97 No bajo grado 6(21,43%) 8,30-40,95 18(27,27) 17,03-39,64 No 22(78,57%) 59,05-99,91 47 (71,21) 58,75-81,70 Si 6(21,43%) 8,30-40,95 19(28,79) 18,30-41,25 18(64,29%) 44,07-81,36 45(68,18) 55,56-79,11 10(35,71%) 18,64-55,93 21(31,82) 20,89-44,44 Grado de instrucción Deterioro Cognitivo† Comorbilidades ≥3 Si No Discontinuidad terapéutica ‡ Si 16(57,14) 37,18-75,54 33 (50) 37,43-62,57 No 12(42,86) 24,46-62,82 33(50) 37,43-62,57 Polifarmacia‡‡ Si 18(64,29) 44,07-81,36 42(63,64) 50,87-75,13 No 10(35,71) 18,64-55,93 24(36,36) 24,87-49,13 Sangrado Mayor‡‡‡ 2 (7,14%) 7 (10,60%) Menor 1 (3,5%) 5 (7,57%) 1 (3,57%) 7 (10,6%) ACV Si †Deterioro cognitivo como perdida de la habilidad mental por > 6 meses ‡Discontinuidad terapéutica a paciente que no tiene control de INR en los últimos 6 meses o no ha sido visto por la especialidad en dicho periodo ‡‡Consumo de ≥3 medicamentos. ‡‡‡Asociado a hemorragia intracraneal o la disminución de ≥5 g/dl o ≥15 % del hematocrito 88 Rev Med Hered. 2017; 28:84-92. Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH Gamero M, y col. Tabla 5. Factores asociados a control subóptimo y óptimo de anticoagulación en pacientes ≥65 años con fibrilación auricular no valvular en el Hospital Nacional Cayetano Heredia (2011-2013). Factores OR IC Control subóptimo Edad ≥75 años 25,5 (7,39-155,8) No bajo grado de instrucción 8 (3,61-20,61) Comorbilidades 4,5 (2,33-9,39) Discontinuidad terapéutica 2,75 (1,44-5,52) Polifarmacia 4,2 (2,16-8,80) Control óptimo Independencia funcional 4,3 (2,16-9,47) Género femenino 3 (1,62-5,81) No deterioro cognitivo 7,8 (3,53-20,19) En la exploración de posibles factores asociados, se encontró como asociación al control subóptimo: la edad ≥75 años (OR 25,5; IC 7,39-155,8; p< 0,001), no bajo grado de instrucción (OR 8; IC 3,61-20,61; p< 0,001), algún grado de deterioro cognitivo (OR 7,8; IC 3,53-20,19; p< 0,001), la presencia de más de 2 comorbilidades (OR 4,5; IC 2,33-9,39; p< 0,001), la discontinuidad terapéutica (OR 2,75; IC 1,445,52; p< 0,001) y la polifarmacia (OR 4,2; IC 2,168,80; p< 0,001) (Tabla 5). Por otro lado, los factores asociados a un control óptimo fueron la valoración funcional medida según KATZ (OR 3 IC 2,16-9,47; p< 0,001), y el género femenino (OR 3; IC 1,625,81; p< 0,001) (Tabla 5). Las variables donde no se encontró asociación fueron sangrado mayor y menor e interacciones farmacológicas y no farmacológicas. DISCUSIÓN Como es conocido, la fibrilación auricular es la arritmia más común en el adulto mayor, la cual aumenta respecto a la edad (2), lo cual se observó en el presente estudio en el que se encontró un mayor porcentaje de pacientes ≥75 años (82,52%). Ello, en parte al aumento del envejecimiento población con incremento de la esperanza de vida, generando mayores comorbilidades con compromiso cardiaco y por tanto mayor predisposición a este arritmia (5). Asimismo, se observó una ligera predominancia del género femenino 51,75% vs 48,25% a diferencia de otros estudios publicados donde se encontró una mayor prevalencia masculina en <75 años y una similar en ≥75 años (17,21). Dichos hallazgos Rev Med Hered. 2017; 28:84-92. podrían explicarse debido a la mayor esperanza de vida de la mujer así como la mayor asistencia de este grupo a nosocomios en nuestro medio. Como sustento de ello, el reporte del análisis de la situación de salud del año 2013 mostró que el 62,5% de las atenciones registradas en consultorio externo fue por población femenina (22). Por otro lado, al evaluarse el riesgo tanto de ACV como de sangrado, el estudio mostró que la mayoría de pacientes tenía indicación de ACO con un bajo riesgo de sangrado. Por ejemplo, la media del puntaje para la escala CHADS2VASC era superior a 2 y en la escala HASBLED era menor de 3, siendo ideales para la ACO. Sin embargo, de los 140 pacientes con indicación de ACO, 41 (29,2%) no la recibió por razones no registradas, que consideramos alarmante. Igualmente, dos tercios de los pacientes (65,03%) no tuvieron controles posteriores, lo cual podría explicarse a que no iniciaron terapia a pesar de indicación, siguieron controles en otro centro de salud o la descontinuaron. No obstante, existe un desconocimiento de lo que paso con este gran porcentaje de pacientes, que podrían estar en alto riesgo de morbi-mortalidad. En nuestro medio, la medicación más usada para la ACO es la warfarina, principalmente por su precio. En el presente estudio, menos del 5% de los pacientes usaron los nuevos anticoagulantes orales. A pesar del gran uso de esta medicación, es conocido que el tiempo para llegar un INR ideal toma entre 3-5 días, y de no lograrse en los 6 primeros meses de iniciado 89 Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH el tratamiento, se ha demostrado un control subóptimo posterior (23,24). En nuestro estudio se evidenció que el tiempo para llegar al rango terapéutico tuvo una mediana 31 días (RI 7,5-345 días). Ello implica un riesgo en 2 posibles escenarios, riesgo de ACV por no alcanzar el rango terapéutico o riesgo de sangrado por sobrepasarlo. Cabe resaltar también que los controles de INR no se dieron en un intervalo de tiempo constante como es lo ideal y no fueron necesariamente realizados en el laboratorio del hospital con control de calidad adecuado, por lo que podemos considerar un sesgo en la estimación de esta cifra. Además, es importante considerar por cuanto tiempo este valor se encuentra en rangos ideales por medio del TRT. El presente estudio mostró un TRT de 35,40% (RI 5,3-63%), cifra alarmante ya que se encuentra en rango inferior al ideal para la reducción del riesgo de ACV (25). En un estudio realizado a nivel nacional en EEUU que incluyó a 138 319, se reporta que el TRT promedio fue de 53,7%, lo cual demuestra la gran diferencia que existe con nuestra realidad y la situación de la calidad de anticoagulación en la que nos encontramos (13). De igual forma, únicamente el 29,78% de nuestra muestra estuvo en un control óptimo, mientras que estudios a mayor escala mostraron alcanzar dicha meta en el 66,8% de su población (26). Cabe resaltar que mayores episodios de ACV isquémico y sangrados se presentaron en el grupo con control subóptimo de la ACO, los cuales se hubieran podido evitar con un mejor en la ACO. En consecuencia, es de suma importancia determinar los factores que influyen en la calidad de la anticoagulación. Estudios previos describen que la demencia y la estancia en una casa de reposo están asociadas a una anticoagulación subóptima (15), mientras que otros hallaron asociación con edad avanzada, género femenino y presencia de comorbilidades (27). Nosotros encontramos similares asociaciones con algunos de estos factores como edad y presencia de tres o más comorbilidades. Otra variable asociada a este grupo fue el “no bajo grado de instrucción”, lo cual parecería ser antagónico; sin embargo, podría explicarse por una mayor independencia para tomar decisiones, tener menor confianza en el médico o una inapropiada información médica (28). En relación al control óptimo, el género femenino estuvo tres veces más asociado, incongruente a lo hallado en recientes publicaciones, donde el ser mujer implica mayor riesgo para una anticoagulación 90 Gamero M, y col. subóptima (15), mientras que otras sugieren ampliar estudios (27). Por ello, es importante definir si este factor implica riesgo o protección para el control de la anticoagulación dado que es conocida la mayor tendencia de ACV en la mujer. Por otra parte, variables como discontinuidad terapéutica, polifarmacia y dependencia funcional no han sido reportadas en la literatura por lo cual resulta relevante para nuestro estudio que haya mostrado asociación a un control subóptimo. Estudios prospectivos de mayor escala son necesarios. En conclusión, se ha observado que la calidad de la anticoagulación en nuestro medio es deficiente, y todos los factores limitantes no han sido definidos. Asimismo, existe una falencia en nuestros laboratorios, lo que obliga a un gran volumen de pacientes a realizar pruebas en laboratorios externos de dudosa calidad. Por ello, es necesario sistematizar la anticoagulación en centros o unidades con control de calidad adecuada así como controles seriados con el fin de disminuir la morbimortalidad en nuestro medio. Dentro de las limitaciones del estudio, se encuentra el tipo de muestreo por conveniencia usado por la dificultad para la recolección de historias clínicas. Asimismo, al ser un estudio descriptivo muestra tendencias y se requiere de estudio de cohorte para la validación de factores. Declaración de financiamiento y de conflictos de intereses: El estudio fue financiado por los autores. Además, declaran que no tener conflictos de intereses. Contribución de autoría: MTG y MC fueron responsables en la concepción, diseño del estudio y recolección de datos. MTG, MC, RD y FS fueron responsables en el análisis e interpretación de los resultados. MTG y MC fueron responsables redacción del artículo: todos los autores realizaron la revisión crítica y aprobación final del mismo. Correspondencia: María Teresa Gamero Huamán Calle Los ciruelos 153, Lima12, Perú Teléfono: +5101991153271 / +51014357469 E-mail: maria.gamero@upch.pe Rev Med Hered. 2017; 28:84-92. Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH REFERENCIAS BIBLIOGRÁFICAS 1. Ministerio de Salud Plan PAM 2013-2017. Promoviendo el envejecimiento activo y saludable. Lima, Perú: Ministerio de Salud; Octubre 2013. 2. Shen AY, Contreras R, Sobnosky S, et al. Racial/ ethnic differences in the prevalence of atrial fibrillation among older adults: A cross-sectional study. J Natl Med Assoc. 2010; 102(10):906-13. 3. Frost L, Engholm G, Johnsen S, Moller H, Husted S. Incident stroke after discharge from the hospital with a diagnosis of atrial fibrillation. Am J Med. 2000; 108(1):36-40. 4. Chugh S, Blackshear J, Shen WK, Hammill S, Gersh B. Epidemiology and natural history of atrial fibrillation: clinical implications. J Am Coll Cardiol. 2001; 37(2):371-8. 5. January C, Wann S, Alpert JS, et al. 2014 AHA/ ACC/HRS Guideline for the Management of Executive Summary A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the Heart Rhythm Society. J Am Coll Cardiol. 2014; 64(21):2246-2280. 6. Prieto S, Young P, Ceresetto JM, Bullorsky EO. Terapia anticoagulante en fibrilación auricular. Medicina (B. Aires) . 2011(Citado 7 de febrero del 2015); 71(3): 274-282. Disponible en: http://www. scielo.org.ar/scielo.php?script=sci_arttext&pid=S00 2576802011000500017&lng=es 7. Iwasaki Y, Nishida K, Kato T, Nattel S. Atrial fibrillation pathophysiology implications for management. Circulation. 2011; 124(20):2264-74. 8. Stramba M. Atrial fibrillation subtypes, risk of stroke, and antithrombotic therapy. Eur Heart J. 2008; 29:840–42. 9. Werdan K, Braun-Dullaeus R, Presek P. Anticoagulation in atrial fibrillation: NOAC’s the Word Karl. Dtsch Arztebl Int. 2013; 110(31):523−4. 10. Aguilar MI, Hart R. Anticoagulantes orales para la prevención del accidente cerebrovascular en pacientes con fibrilación auricular no-valvular y sin antecedentes de accidente cerebrovascular o accidentes isquémicos transitorios. La Biblioteca Cochrane Plus. 2005; 3:00. doi: https://doi.org/10.1002/14651858.CD001927. pub2 11. Nieuwlaat R, Capucci A, Lip GY, et al. Antithrombotic treatment in real-life atrial fibrillation patients: a report from the Euro Heart Survey on Atrial Fibrillation. Eur Heart J. 2006; 27:3018-26. 12. Molteni M, Cimminiello C. Warfarin and atrial fibrillation: from ideal to real the warfarin affair. Thromb J. 2014; 12(1):5. 13. Dlott JS, George RA, Huang X, et al. National assessment of warfarin anticoagulation therapy for stroke prevention in atrial fibrillation. Circulation. Rev Med Hered. 2017; 28:84-92. Gamero M, y col. 2014; 129(13):1407-14. 14. Lane DA, Lip GY. Barriers to anticoagulation in patients with atrial fibrillation: changing physicianrelated factors. Stroke. 2008; 39 (1):7-9. 15. Gumbinger C, Holstein T, Stock C, Rizos T, Horstmann S, Veltkamp R. Reasons underlying nonadherence to and discontinuation of anticoagulation in secondary stroke prevention among patients with atrial fibrillation. Eur Neurol. 2015; 73(3-4):184-91. 16. Giachini F, Nogueira E, Luz T, Lunelli R, Moraes MA. Esmerio, FG. Uso crônico de anticoagulante oral: implicações para o controle de níveis adequados. Arq Bras Cardiol. 2009; 93(5):549-554. doi: http:// dx.doi.org/10.1590/S0066-782X2009001100017 17. Charles K, Humphries K. Gender-related differences in atrial fibrillation. J Am Coll Cardiol. 2005; 46 (7):1307-1308. 18. Rosendaal FR, Cannegieter SC, van der Meer FJ, Briët E. A method to determine the optimal intensity of oral anticoagulant therapy. Thromb Haemost. 1993; 69(3):236-9. 19. Caldeira D, Cruz I, Morgado G, et al. Is the time in therapeutic range using the ratio of tests equivalent to the Rosendaal method? Blood Coagul Fibrinolysis. 2015; 26(8):972-6. 20. Caldeira D, Cruz I, Morgado G, et al. Evaluation of time in therapeutic range in anticoagulated patients: a single-center, retrospective, observational study. BMC Research Notes. 2014; 7:891. 21. Fang M, Go A, Chang Y, et al. Prevalence of diagnosed atrial fibrillation in adults national implications for rhythm management and stroke prevention: the anticoagulation and Risk Factors In Atrial Fibrillation (ATRIA) Study. J Am Coll Cardiol. 2011; 58(4):395401. 22. Valdez W, Napanga E, Oyola A, et al. Análisis de situación de salud del Perú. Lima: Ministerio de Salud del Perú; Setiembre 2013. p.72-76. 23. Chiang Ch, Naditch-Brûlé L, Murin, J. Distribution and risk profile of paroxysmal, persistent, and permanent atrial fibrillation in routine clinical practice. Circ Arrhythm Electrophysiol. 2012; 5:632639. 24. Phillips KW, Ansell J. Outpatient management of oral vitamin K antagonist therapy: defining and measuring high-quality management. Expert Rev Cardiovasc Ther. 2008; 6(1):57-70. 25. Gallagher A, Setakis E, Plumb J, Clemens A, VanStaa T. Risks of stroke and mortality associated with suboptimal anticoagulation in atrial fibrillation patients. Thromb Haemost. 2011; 106(5):968-77. 26. White HD, Gruber M, Feyzi J, et al. Comparison of outcomes among patients randomized to warfarin therapy according to anticoagulant control: Results from SPORTIF III and V. Arch Intern Med. 2007; 167(3):239-245. 91 Factores asociados a la anticoagulación oral óptima en el adulto mayor con fibrilación auricular INVESTIGACIÓN ORIGINAL / ORIGINAL RESEARCH 27. Melamed O, Horowitz G, Elhayany A, Vinker S. Quality of anticoagulation control among patients with atrial fibrillation. Am J Manag Care. 2011; 17 (3):232-237. Gamero M, y col. 28. Platt A, Russell A, Brensinger C, et al. Risk factors for nonadherence to warfarin: results from the INRANGE study. Pharmacoepidemiol Drug Saf. 2008; 17(9):853–860. Recibido: 13/08/2015 Aceptado: 05/12/2016 92 Rev Med Hered. 2017; 28:84-92.
https://openalex.org/W3179814182	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0009514&type=printable	English	null	A randomised controlled feasibility trial of a BabyWASH household playspace: The CAMPI study	PLoS neglected tropical diseases	2,021	cc-by	11,367	RESEARCH ARTICLE A randomised controlled feasibility trial of a BabyWASH household playspace: The CAMPI study Sophie BudgeID1☯, Paul HutchingsID2☯, Alison Parker1☯, Sean Tyrrel1☯, Sam NortonID3☯, Camila GarbuttID4, Fitsume WoldemedhinID5, Mohammed Yasin Jemal5, Mathewos MogesID6, Siraj Hussen6, Hunachew Beyene6 Sophie BudgeID1☯, Paul HutchingsID2☯, Alison Parker1☯, Sean Tyrrel1☯, Sam NortonID3☯, Camila GarbuttID4, Fitsume WoldemedhinID5, Mohammed Yasin Jemal5, Mathewos MogesID6, Siraj Hussen6, Hunachew Beyene6 1 Cranfield Water Science Institute, Cranfield University, Cranfield, United Kingdom, 2 Faculty of Engineering and Physical Sciences, University of Leeds, Leeds, United Kingdom, 3 Psychology Department, Institute of Psychiatry Psychology & Neuroscience, King’s College London, London, United Kingdom, 4 PIN UK, London, United Kingdom, 5 PIN, Hawassa, Ethiopia, 6 Hawassa University College of Medicine and Health Sciences, Hawassa, Ethiopia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * p.hutchings@leeds.ac.uk Background Citation: Budge S, Hutchings P, Parker A, Tyrrel S, Norton S, Garbutt C, et al. (2021) A randomised controlled feasibility trial of a BabyWASH household playspace: The CAMPI study. PLoS Negl Trop Dis 15(7): e0009514. https://doi.org/ 10.1371/journal.pntd.0009514 Water, sanitation and hygiene (WASH) interventions should support infant growth but trial results are inconsistent. Frequently, interventions do not consider behaviours or transmis- sion pathways specific to age. A household playspace (HPS) is one intervention component which may block faecal-oral transmission. This study was a two-armed, parallel-group, ran- domised, controlled feasibility trial of a HPS in rural Ethiopia. It aimed to recommend pro- ceeding to a definitive trial. Secondary outcomes included effects on infant health, injury prevention and women’s time. Editor: Sitara SR Ajjampur, Christian Medical College and Hospital: Christian Medical College Vellore, INDIA Received: July 28, 2020 Accepted: May 28, 2021 Published: July 14, 2021 Received: July 28, 2020 Accepted: May 28, 2021 Published: July 14, 2021 Methods November 2019−January 2020 106 households were identified and assessed for eligibility. Recruited households (N = 100) were randomised (blinded prior to the trial start) to interven- tion or control (both n = 50). Outcomes included recruitment, attrition, adherence, and acceptability. Data were collected at baseline, two and four weeks. Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pntd.0009514 ☯These authors contributed equally to this work. * p.hutchings@leeds.ac.uk Trial registration RIDIE-ID-5de0b6938afb8. RIDIE-ID-5de0b6938afb8. Author summary This research tested a new way to protect infants and young children from infections that are caused by pathogens in human and animal faeces. It tested the feasibility of using a household playspace to reduce infection by creating a hygienic environment for children to play-in in rural Ethiopia. The results show that the household playspace was well accepted, used regularly and cleaned well by participants in the study. The study also sug- gests a potential positive impact in reducing diarrhoea. Based on these results, we suggest that a larger scale trial be conducted to conclusively assess whether a household playspace can protect young children and infants from infection in rural Ethiopia or similar contexts. Funding People In Need; Czech Development Agency. People In Need; Czech Development Agency. Competing interests: The authors have declared that no competing interests exist. Interpretation Despite failing some a priori criteria, the trial demonstrated mixed adherence and good acceptability among intervention households. A definitive trial to determine efficacy is war- ranted if recommended adjustments are made. Funding: SB is jointly funded as a research student by both Cranfield University and People In Need, who received funding from the Czech Development Agency for the project. No other external funds supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Findings Recruitment met a priori criteria (80%). There was no loss to follow-up, and no non-use, meeting adherence criteria (both 10%). Further, 48.0% (95% CI 33.7−62.6; n = 24) of households appropriately used and 56.0% (41.3−70.0; n = 28) cleaned the HPS over four weeks, partly meeting adherence criteria (50%). For acceptability, 41.0% (31.3−51.3; n = 41) of infants were in the HPS during random visits, failing criteria (50%). Further, the pro- portion of HPS use decreased during some activities, failing criteria (no decrease in use). A modified Barrier Analysis described good acceptability and multiple secondary benefits, including on women’s time burden and infant injury prevention. Copyright: © 2021 Budge et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All underlying data, including study protocol and consent forms, are 1 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES Introduction Final height in adults results from both genetic and environmental factors which support lin- ear growth in childhood [1]. Conversely, adverse influences which begin in utero and continue through puberty can lead to growth failure [1]. This includes the cyclical relationship between infection and nutrition. Symptomatic infection is common during early years in low-income countries, and repeated diarrhoea impairs growth, weight gain and long-term cognitive devel- opment [2]. Moreover, enteric infections which are asymptomatic but which result in subclini- cal enteropathy[3] are also associated with growth shortfalls [4,5]–suggesting infection affects development without overt outcomes like diarrhoea. Population-level nutrition and hygiene status are thus critical for proper growth, but are not sufficient alone: where there are wide- spread infection and inflammation, the effect of nutrition on growth is seriously compromised [1]. Indeed, the modest effects on growth in nutrition interventions suggests that a combina- tion of recurrent infections, chronic inflammation, and gut enteropathy limit the effects of nutrition [6]. Thus randomised controlled trials (RCT) are testing water, sanitation and hygiene (WASH) interventions alongside supplementary nutrition to improve infant health. Despite substantial evidence suggesting safe WASH contributes to good child health in terms of preventing malnutrition and morbidities from infectious diseases[7], RCTs testing improved household WASH (with or without supplementary nutrition) have shown variable, mostly insignificant, effects [8–11]. Whilst it is improbable that interventions at the coverage in these trials will alleviate growth failure, results have prompted discourse on what is neces- sary. The concept ‘Transformative WASH’[12] highlights the necessity of substantially 2 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace improving environmental hygiene amongst the poorest, whom disproportionately experience poor child health. It also recognises the significant burden of contamination from domestic animals–largely unaddressed in WASH trials or programs[13]. In rural, subsistence agriculture settings it is common for domestic animals to share living and sleeping spaces. Acting as natu- ral reservoirs, domestic animals likely contribute substantial contamination to multiple trans- mission routes of zoonotic pathogens such as Cryptosporidium, Campylobacter and E. coli [4,5,13–15] which are associated with growth failure and gut abnormalities [16,17]. Further, a transformative approach will require that interventions (whether technical, structural, or beha- vioural) consider age-related behaviours and transmission pathways to prevent infant infec- tion [18]. One such ‘critical’ WASH intervention component[19] is a household playspace (HPS)–an enclosed, protective play area. Aims The Campylobacter-Associated Malnutrition Playspace Intervention (CAMPI) trial was a ran- domised, controlled feasibility trial to establish the feasibility of a definitive RCT of a HPS in rural Ethiopia. The HPS design (S1 Figure), is described elsewhere [23], underpinned by previ- ous formative research [24–26]. The primary aim of the trial was to establish the feasibility of a future definitive RCT to evaluate the efficacy of a HPS. This involved evaluating the HPS through measures of recruitment, attrition, adherence, and acceptability, and as efficacy meth- ods within a RCT. It also involved evaluating the appropriateness of the study design for rec- ommended adjustments to the intervention and design for future trials. As formal hypothesis testing for effectiveness is not recommended in feasibility studies, the trial did not aim to determine the effect of the HPS on health outcomes, and was not powered for this. However, further evidence was required towards the infection-exposure hypothesis as well as effects on broader outcomes. Thus secondary outcomes aimed to: 1. Confirm the prevalence of Campylobacter infection in the study population 2. Describe effects of the HPS on Campylobacter infection and diarrhoea 3. Describe secondary effects, including on women’s use of time, childcare, or injury prevention. Introduction In rural areas, a HPS may offer some protection from infection during early growth periods by interrupting faecal-oral transmission from ingested soil and faeces[20,21]and contaminated floors during infantplay [22]. Available evidence on the health and non-health benefits of a HPS or playmat has been pre- viously reviewed [23]. This included preventing the ingestion of faeces and contaminated mat- ter (soil, other objects) by the infant and protection from injury. Further, formative data during the participatory design and build of the HPS prototype suggested caregivers liked it and were glad to use it during daily routines [23]. However, there remains a need to assess how long a HPS would be used throughout the day and appropriately maintained and cleaned. Data on infant health outcomes would provide insight into the potential for a HPS to reduce infection from within the home. Moreover, WASH interventions deliver both health and non- health outcomes, all of which contribute to household wellbeing. Thus broader benefits of a HPS, including on women’s’ time and child socioemotional development, also require explora- tion through a definitive RCT. Ethics statement This study is registered with the Registry for International Development Impact Evalua- tions (RIDIE), (RIDIE-STUDY-ID-5de0b6938afb8). The study was approved by Cranfield University Research Ethics Committee (CURES/9357/2019) and Hawassa University Col- lege of Medicine and Health Sciences Institutional Review Board (IRB/010/12). Upon recruitment, PIN staff and HEWs discussed the study with primary caregivers who under- stood data were anonymous. Informed consent and assent on behalf of infant participants was obtained, or thumbprints taken. Surveys were translated to Amharic by PIN staff and administered verbally in Amharic or Sidamo. Various checks throughout the trial assessed HPS safety and monitored for adverse events. This included regular survey checkpoints (data concerns from households and HPS safety and visual inspection of HPS for unsafe use or assembly), and the distribution of feedback response mechanism cards to contact PIN staff. Infants with moderate or severe acute malnutrition measured by MUAC were advised to contact their local health post, which was followed up by a government Health Extension Work (HEW) HEW. Methods This feasibility trial was designed by Cranfield University alongside People in Need (PIN) and Hawassa University and conducted in the Southern Nations, Nationalities and Peoples’ region 3 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace (SNNPR), Ethiopia. It was a two-armed, parallel-group, randomised controlled feasibility trial with equal group allocation. The Consolidated Standards of Reporting Trials (CONSORT) 2010 statement with extension to pilot trials was followed during study design and reporting (S1 PRISMA Checklist). Study intervention The trial was conducted in Sidama zone, SNNPR, Ethiopia between January−March 2020. Two field teams managed intervention and control kebeles. After baseline data collection, caregivers from intervention households were called to the kebele health post for a ‘sensitisa- tion’ day. PIN field team, HEWs and data collectors formally discussed the study rationale, caregiver beliefs around infant faecal-oral transmission and health outcomes, transmission routes and how a HPS might interrupt these to improve infant health. Correct HPS use, main- tenance and cleaning was detailed. Caregivers watched and practiced HPS assembly and dis- cussed potential safety issues. Use was discussed in relation to daily routines and activities and caregivers agreed to use it when possible. Households agreed to clean the HPS at least every other day (and always after defecation or urination) with both soap and water. Playspaces were distributed with safety instructions printed in both Sidamo and Amharic with illustrations. HEWs visited intervention households in the following days to ensure correct HPS assembly. The control group received a HPS upon study completion. Randomisation and masking PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 5 / 20 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Participant data Survey and anthropometry. Households were visited at baseline and at two and four weeks. The primary caregiver present was interviewed, usually the mother. Baseline data included a previously validated survey[25,26] on WASH facilities and use (latrine type; pres- ence and use, presence and availability of soap and water for handwashing; availability of water within the home; water source; person responsible for collecting water; safe storage of water) and animal husbandry practices. Food hygiene, breastfeeding, and diarrhoea incidence were also assessed and again at two and four weeks. Trained data collectors took weight, height and mid-upper arm circumference (MUAC) following standard procedure[27] with a digital mother-child smart scale (Ultratec), a foldable infantometer to 5 mm accuracy (seca 210) and standard MUAC tape to 1 mm accuracy, respectively. Seven-day diarrhoea prevalence was by caregiver report. Laboratory confirmation. Collection and processing of infant faecal samples followed a validated methodology [26]. Briefly, a day prior to household visits data collectors distributed sterile sample collection bags (Whirl-Pak, Sigma-Aldrich, UK) labelled with study time point and study ID and demonstrated sterile sample collection. Faecal samples were collected the morning of deposition and transported on ice within six hours to the laboratory at Hawassa University College of Medicine and Life Sciences. That same day, samples were processed for the isolation of presumptive Campylobacter spp. Microbial culture was performed by trained laboratory staff using CHROMagar selective media and appropriate microaerophilic condi- tions. Samples were processed for all 100 households at each of the three study time points (N = 300). Randomisation and masking As a feasibility trial, a sample size calculation based on power was not performed. A target of 100 households was deemed sufficient to inform researchers about practicalities of running the trial and for sufficient precision to estimate rates of recruitment, retention, and trial out- comes. Specifically, a sample size of 100 was deemed sufficient since to achieve a maximum standard error of ±0.05 for a proportion, which ensures a 95% confidence interval in the esti- mated results with a maximum width of +/-10%. This was deemed achievable with the resources available and is in line with recommendations for feasibility studies where the parameter of interest is a proportion. Eligible households were identified, contacted and enrolled into the trial November 2019−January 2020. Four kebeles (a neighbourhood or small administrative unit; two intervention, two control) were chosen from a woreda (zonal subdivision) representative of rural livelihoods across the region, without geographical overlap. Alongside govern- ment HEW, PIN team members produced a blinded sampling frame from kebeles of all households fulfilling eligibility criteria. Households were sequentially numbered and using statistical software, 25 households were randomly drawn from each frame for a total sample of 100 (50 intervention, 50 control). Inclusion (eligibility) criteria were: 1. Subsis- tence agriculture households raising domestic animals, within PIN intervention scope; 2. With an infant aged 8−16 months (10−18 months at trial commencement); 3. Not partici- pating in other PIN projects. Exclusion criteria: 1. Outside 10−18 month range at trial start; 2. Participating in other PIN projects; 3. Infant was pre-term, low birth weight, or had other birth complications. PIN staff and HEWs approached households with the study information and participants were given time to make an informed decision. Households were then revisited, eligibility was re-verified, and if households were willing, consent was gained. Households were blinded to their status in the trial until after baseline data collec- tion. Fig 1 describes trial enrollment and numbers. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 4 / 20 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Fig 1. Modified version of CONSORT 2010 flow diagram of participants in the CAMPI feasibility trial. https://doi org/10 1371/journal pntd 0009514 g001 Fig 1. Modified version of CONSORT 2010 flow diagram of participants in the CAMPI feasibility trial. Implementation outcomes The adherence outcome included ‘Appropriate use’ and ‘Appropriate cleaning’, created as composite binary outcome variables (described in table footnotes) and described across study time points. Adherence as ‘HPS non-use’ was described as reported non-use after baseline. Acceptability as ‘Infant in playspace upon arrival’ was calculated for both visits. Acceptability as change in HPS use was calculated from reported HPS use during reported daily activities over two and four weeks and the difference in proportions. A Generalised Esti- mating Equation (GEE) was used as a semi-parametric model, using a robust variance estima- tor and an unstructured working correlation matrix. A binary logistic GEE estimated factors associated with ‘Appropriate use’ and ‘Appropriate cleaning’ at two and four weeks. Models were initially run separately: however, the merged composite variable of ‘Appropriate use and cleaning’ showed no difference in parameter estimates between models and is presented. Pre- specified variables included infant sex and age; maternal age; maternal education; number in household; number of children; household owns soap; safe water storage; animal husbandry practices; water availability, and mother collects water. Results are expressed as populated averaged odds ratios (ORs) with estimated 95% CI. As this is a feasibility study, in line with the CONSORT extension for feasibility studies, we do not present p-values. p y p y p p Acceptability as change in HPS use was calculated from reported HPS use during reported daily activities over two and four weeks and the difference in proportions. A Generalised Esti- mating Equation (GEE) was used as a semi-parametric model, using a robust variance estima- tor and an unstructured working correlation matrix. A binary logistic GEE estimated factors associated with ‘Appropriate use’ and ‘Appropriate cleaning’ at two and four weeks. Models were initially run separately: however, the merged composite variable of ‘Appropriate use and cleaning’ showed no difference in parameter estimates between models and is presented. Pre- specified variables included infant sex and age; maternal age; maternal education; number in household; number of children; household owns soap; safe water storage; animal husbandry practices; water availability, and mother collects water. Results are expressed as populated averaged odds ratios (ORs) with estimated 95% CI. As this is a feasibility study, in line with the CONSORT extension for feasibility studies, we do not present p-values. Acceptability was further assessed through a modified Barrier Analysis which explored determinants of use among all participants. Implementation outcomes Evaluation of trial outcomes and proceeding with future definitive trial. Among the intervention group, surveys at two and four weeks assessed feasibility outcomes: Recruitment (number of households contacted who consented); Attrition (the proportion of participants lost to follow-up at the trial end); Adherence (proportion of HPS non-use, as well as Appropri- ate use/maintenance and cleaning), and Acceptability (random observation of HPS use and change in incidence [proportion] of use from two to four weeks). A modified Barrier Analysis at four weeks provided further insight into acceptability. As these outcomes were the main measures to determine whether to proceed to a definitive trial, a priori threshold criteria were established as follows: 1. Recruitment: the proportion of contacted households participating in 6 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace the trial would be 80%; 2. Attrition: the level at the trial end would be 10%; 3. Adherence: the proportion of non-use of HPS would be 10% at both time points and over the trial; 4. Adherence: the proportion of correct HPS use and cleaning would be 50% at both time points and over the trial; 5. Acceptability: the proportion of infants in the HPS at random check would be 50% at both time points and over the trial, and 6. Acceptability: reported incidence of HPS use during daily activities (as a proportion) would not decrease from two to four weeks. Outcomes would also indicate appropriateness of an RCT and provide recommen- dations for adjusting the intervention design. Statistical analysis. Data were managed in Excel and analysed in SPSS (v26.0, IBM). Descriptive statistics summarised survey data and health outcomes. Trial outcomes are dis- played with estimated 95% confidence intervals (CI), calculated using the Wald method for proportions. The adherence outcome included ‘Appropriate use’ and ‘Appropriate cleaning’, created as composite binary outcome variables (described in table footnotes) and described across study time points. Adherence as ‘HPS non-use’ was described as reported non-use after baseline. Acceptability as ‘Infant in playspace upon arrival’ was calculated for both visits. Statistical analysis. Data were managed in Excel and analysed in SPSS (v26.0, IBM). Descriptive statistics summarised survey data and health outcomes. Trial outcomes are dis- played with estimated 95% confidence intervals (CI), calculated using the Wald method for proportions. Implementation outcomes Methods and analysis are described in detail in supplementary information (S1 Text). Derivation of themes was data-driven, where codes resulted from the analysed data as they related to each determinant (See Table D in S1 Data). Coded themes are discussed as either barriers or enablers to the implantation of, and improv- ing outcomes during, a definitive trial. For secondary health outcomes, anthropometric z- scores were calculated (WHO Anthro v3.2.2) and categorised into stunting and wasting using standard cut-off values [28]. Samples positive for presumptive Campylobacter spp., colonies were counted using OpenCFU. Change in diarrhoeal and Campylobacter prevalence between study groups was estimated using a GEE intercept-only model with OR and 95% CI. Baseline characteristics Household demographic characteristics are described in Table 1 for both study groups and as a whole. Characteristics were largely balanced across groups. Average infant age was 10.8 months (median 10.0; range 7−18). Average length-for-age (LAZ) and weight-for-length (WLZ) at baseline did not vary substantially across intervention and control groups at -1.00 and -0.96 (LAZ) and -0.49 and -0.46 (WLZ) respectively. Stunting and wasting affected 33.0% (n = 33) and 13.0% (n = 13) of all infants respectively with some severe acute malnutrition (11.0%, n = 11). Mothers were mostly aged 18−25 (50.0%, n = 25; 62.0%, n = 31, respectively) and educated to second grade (44.0%, n = 22; 52.0%, n = 26, respectively). Whilst most PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 7 / 20 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Table 1. Household demographic characteristics, and water, sanitation and hygiene, animal husbandry and nutri- tion indicators across study groups and as a total at baseline (N = 100). Baseline characteristics Intervention (n = 50) Control (n = 50) Total (N = 100) n % n % n % Demographics Infant sex: Male 28 56.0 24 48.0 52 52.0 Average infant age (months) 10.1 11.6 10.8 Respondent: Mother 45 90.0 48 96.0 93 93.0 Maternal age: <18 1 2.0 0 0.0 1 1.0 18–25 25 50.0 31 62.0 56 56.0 26–35 21 42.0 18 36.0 39 39.0 36–45 3 6.0 0 0.0 3 3.0 >45 0 0.0 1 2.0 1 1.0 Maternal education: Cannot read/write 12 24.0 6 12.0 18 18.0 First grade 13 26.0 15 30.0 28 28.0 Second grade 22 44.0 26 52.0 48 48.0 Secondary and above 3 6.0 3 6.0 6 6.0 Number in household: 1–3 5 10.0 9 18.0 14 14.0 4–6 34 68.0 33 66.0 67 67.0 7+ 11 22.0 8 16.0 19 19.0 Number of children: 1–2 22 44.0 23 46.0 45 45.0 3–4 18 36.0 23 46.0 41 41.0 5–6 9 18.0 4 8.0 13 13.0 7+ 1 2.0 0 0.0 1 1.0 Number of children 5: 1 35 70.0 36 72.0 71 71.0 2 12 24.0 13 26.0 25 25.0 3 3 6.0 1 2.0 4 4.0 Main income: Farming/livestock 48 96.0 49 98.0 97 97.0 Trade 17 34.0 19 38.0 36 36.0 Employee 1 2.0 0 0.0 1 1.0 Household has formal means of saving 9 18.0 8 16.0 17 17.0 House material: Wood and mud 38 76.0 47 94.0 85 85.0 Wood and grass 10 20.0 1 2.0 11 11.0 Concrete 2 4.0 2 4.0 4 4.0 Floor material: Concrete / cement 16 32.0 9 18.0 25 25.0 Mud / soil 34 68.0 41 82.0 75 75.0 Intervention (n = 50) Control (n = 50) Total (N = 100) n % n % n % WASH indicators Latrine type: Defecate in open 8 16.0 11 5.0 19 19.0 Share neighbour’s 6 12.0 8 16.0 14 14.0 Pit latrine without slab 11 22.0 5 10.0 16 16.0 Pit latrine with slab 25 50.0 26 52.0 51 51.0 Water source: Piped water / public tap 50 50.0 50 50.0 100 100.0 Who collects water?: Mother 39 78.0 39 78.0 78 100.0 Father 8 16.0 3 6.0 11 11.0 A grandparent 0 0.0 1 2.0 1 1.0 Female child (15) 11 22.0 10 20.0 21 21.0 (Continued) Table 1. Baseline characteristics Household demographic characteristics, and water, sanitation and hygiene, animal husbandry and nutri- tion indicators across study groups and as a total at baseline (N = 100). 8 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 8 / 20 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Table 1. Calculated as households who were marked ‘Yes’ to all three observation-based questions: Are water containers clean; Do the water containers have a protecting cover; Does the container have a tap or narrow mouth for drawing the water. WASH, water, sanitation and hygiene; LAZ, length-for-age; WLZ, weight-for-length; MUAC, mid-upper arm circumference, where: 135, no risk of undernutrition; 12.5–13.5, at risk of moderate acute undernutrition; 11.5– 12.4, moderate acute undernutrition; 11.5, severe acute undernutrition. WASH, water, sanitation and hygiene; LAZ, length-for-age; WLZ, weight-for-length; MUAC, mid-upper arm circumference, where: 135, no risk of undernutrition; 12.5–13.5, at risk of moderate acute undernutrition; 11.5– circumference, where: 135, no risk of undernutrition; 12.5 13.5, at risk of moderate acute undernutrition; 11.5 12.4, moderate acute undernutrition; 11.5, severe acute undernutrition. https://doi.org/10.1371/journal.pntd.0009514.t001 WASH, water, sanitation and hygiene; LAZ, length-for-age; WLZ, weight-for-length; MUAC, mid-upper arm Baseline characteristics (Continued) Male child (15) 3 6.0 7 14.0 10 10.0 Labourer 1 2.0 11 22.0 12 12.0 Water available inside the home 43 86.0 46 92.0 89 89.0 Household safely stores water 12 24.0 14 28.0 26 26.0 Household owns soap 34 68.0 39 78.0 73 100.0 Animal husbandry Number of cattle: 1–3 31 62.0 25 25.0 56 56.0 4–6 9 18.0 11 22.0 20 20.0 7+ 0 0.0 1 2.0 1 1.0 Number of goats: 1–3 8 16.0 8 16.0 16 16.0 4–6 2 4.0 1 2.0 3 3.0 7+ 1 2.0 1 2.0 2 2.0 Number of donkey: 1–3 1 2.0 6 12.0 7 7.0 Number of sheep: 1–3 0 0.0 10 20.0 10 10.0 Number of chickens: 1–3 11 22.0 16 32.0 27 27.0 4–6 15 30.0 16 32.0 31 31.0 7+ 18 36.0 7 14.0 25 25.0 Animal dwelling during the day Outside, enclosed in a pen 1 2.0 0 0.0 1 1.0 Outside, roaming free 49 98.0 48 96.0 97 97.0 Inside, same room as family 33 66.0 40 80.0 73 73.0 Inside, separate room 1 2.0 2 4.0 3 3.0 Animal dwelling during the night Outside, enclosed in a pen 7 14.0 10 20.0 17 17.0 Outside, roaming free 0 0.0 0 0.0 0 0.0 Inside, same room as family 34 68.0 35 70.0 69 69.0 Inside, separate room 9 18.0 5 10.0 14 14.0 Intervention (n = 50) Control (n = 50) Total (N = 100) n % n % n % Nutrition indicators LAZ z-score (average) -1.00 -0.96 -0.98 LAZ (range) -3.04−0.80 -2.76−0.66 -3.04−0.80 WLZ z-score (average) -0.49 -0.46 -0.47 WLZ (range) -2.30−0.87 -2.41−0.75 -2.41−0.87 MUACa (mm; average) 138.2 138.1 138.2 Stunting (LAZ −2 SD) 17 34.0 16 32.0 33 33.0 Wasting (WLZ −2 SD) 6 12.0 7 14.0 13 13.0 MUACa: 135 36 72.0 39 78.0 75 75.0 125−135 8 16.0 6 12.0 14 13.0 115−124 6 12.0 5 10.0 11 11.0 115 0 0.0 0 0.0 0 0.0 WASH, water, sanitation and hygiene; LAZ, length-for-age; WLZ, weight-for-length; MUAC, mid-upper arm https://doi.org/10.1371/journal.pntd.0009514.t001 9 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 9 / PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace households had a pit latrine with a slab (51.0%, n = 51), open defecation was still common (19.0%, n = 19). Trial outcomes For ease of assessment, study outcomes are described together in Table 2 and individually in sections below. Given that the analysis was exploratory in this feasibility trial and results were preliminary, the 95% CI is expressed without p values. Baseline characteristics Seventy-eight percent of women bore the duty of collecting water which for all households came from a public tap. Only 26.0% of households safely stored their water. Cattle and chickens were the most frequent domestic animal, and husbandry practices indicated ani- mals frequently shared living spaces during the day and night, with infrequent use of pens. Recruitment and attrition Rates for recruitment and attrition are shown in Table 2. One hundred households were recruited from four kebeles. To achieve this, 106 households were assessed for eligibility; four households were then excluded for not meeting infant age criteria at the study start and a fur- ther two did not consent to participate (Fig 1). Thus a recruitment rate of 94.3% (95% CI 88.1 −97.9) met a priori criteria of 80%. All households completed the trial assessments at four weeks and there was no loss to follow-up (0.0%; 95% CI 0.0−3.6), meeting criteria for attrition (10% at trial end). https://doi.org/10.1371/journal.pntd.0009514.t002 Adherence Adherence was first described as the proportion of HPS non-use at both time points and over the study period (Table 2). No households reported not using the HPS at either time point or over the study duration (0.0%, 95% CI 0.0−0.71), meeting a priori criteria 10%. Second, adherence was described through ‘Appropriate use’ and ‘Appropriate cleaning’ and combined, across the study time points and throughout the trial (Table 3). Appropriate use included maintenance, as described in the table footnotes alongside variable components (also in Table A in S1 Data). When considering behaviours and time points separately, Appropriate use and Appropriate cleaning were consistently above the a priori threshold of 50%. However when assessing throughout, findings are mixed. Appropriate use did not meet the threshold (48.0%) whilst cleaning did (56.0%) and only 26.0% of households appropriately used and cleaned the HPS throughout the trial. Variables associated with adherence outcomes across Table 2. Outcomes for the CAMPI trial to determine progression to a future definitive RCT, at two and four weeks and across the trial duration. Quantitative trial outcomes Outcome Definition / Indicator A priori criteria Proportion (N = 50) Baseline Two weeks Four weeks Study duration % 95% CI % 95% CI % 95% CI % 95% CI Recruitment Proportion of contacted houses who consented 80% 94.3 88.1−97.9 - - - - - - Attrition Loss to follow-up 10% - - - - - - 0.0 0.0−3.6 Adherence Non-use of HPS 10% - - 0.0 0.0−0.07 0.0 0.0−0.07 0.0 0.0−0.07 Appropriate use 50% - - 70.0 55.5−82.1 64.0 49.2−77.1 48.0 33.7−62.6 Appropriate cleaning 50% - - 72.0 57.5−83.8 70.0 55.4−82.1 56.0 41.3−70.0 Appropriate use and cleaning 50% - - 52.0 37.4−66.3 48.0 33.7−62.6 26.0 14.6−40.3 Acceptability Infant in HPS upon arrival 50% - - 32.0 19.5−46.7 50.0 35.5−64.5 41.0 31.3−51.3 Proportion of HPS use during daily activities No decrease - - - - - - Decrease during certain activities CAMPI trial to determine progression to a future definitive RCT, at two and four weeks and across the trial duration. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 10 / 2 10 / 20 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Table 3. Adherence: Appropriate playspace use and cleaning across study time points. Adherence Adherence: Appropriate HPS use and cleaning (N = 50) Two weeks Four weeks Both time pointsβ n % 95% CI n % 95% CI n % 95% CI Appropriate use 35 70.0 55.5−82.1 32 64.0 49.2−77.1 24 48.0 33.7−62.6 Appropriate cleaning 36 72.0 57.5−83.8 35 70.0 55.4−82.1 28 56.0 41.3−70.0 Appropriate use and cleaningα 26 52.0 37.4−66.3 24 48.0 33.7−62.6 13 26.0 14.6−40.3 Table 3. Adherence: Appropriate playspace use and cleaning across study time points. HPS, household playspace; CI, confidence interval. HPS, household playspace; CI, confidence interval. Created from the variables: Playspace is assembled correctly (observed), yes; Any changes/modifications to playspace (observed), no, or yes, modifications are safe; Others share playspace (reported), no; Animals in playspace (observed and reported), no; Caregiver leaves infant in playspace when leaving house (reported), no or yes IF; infant is watched by other adult (father, grandparent or child 18). IF; infant is watched by other adult (father, grandparent or child 18). Created from the variables: Frequency of cleaning the playspace (reported), every day, every other day; Cleaning materials used (reported), water and soap; Mattress visibly dirty (observed), no; Urine or faeces on mattress (human or animal; reported), no. αThe sum of households who achieved ‘Yes’ for all criteria for both use and cleaning. βThe sum of households who achieved ‘Yes’ for all criteria across indicators at both two and four weeks. Created from the variables: Frequency of cleaning the playspace (reported), every day, every other day; Cleaning materials used (reported), water and soap; Mattress visibly dirty (observed), no; Urine or faeces on mattress (human or animal; reported), no. Created from the variables: Frequency of cleaning the playspace (reported), every day, every other day; Cleaning materials used (reported), water and soap; Mattress visibly dirty (observed), no; Urine or faeces on mattress (human or animal; reported), no. https://doi.org/10.1371/journal.pntd.0009514.t003 the two time points were assessed using a binary logistic GEE model (Table 4). Results display the 95% CI for the effect size and odds ratio. The only variable to significantly predict Appro- priate use or cleaning was ‘Mother collects water alone’, where an inverse relationship showed a reduced odds of 72.0% (0.28; 95% CI 0.12−0.66). Acceptability Infant in playspace upon arrival, change in playspace use. The first measure noted if the infant was in the HPS during a random visit (Table 2). This increased from 32.0% (95% CI 19.5−46.7, n = 16) at two weeks to 50% (95% CI 35.5−64.5, n = 25) at four weeks, meeting a priori criteria of 50% at this point: however throughout the trial did not reach the threshold Table 4. Adherence: A Generalised Estimation Equation estimating the effect of parameters on the trial outcome of adherence ’Appropriate use and cleaning’ across study time points. Adherence: Appropriate use and cleaning Generalised Estimating Equation (N = 50) Variable 95% Wald Confidence Interval 95% Wald Confidence Interval for Exp(B) B Std. Error Lower Upper Odds Ratio Lower Upper (Intercept) -0.07 1.06 -2.14 2.01 0.94 0.12 7.49 Infant sex = Male 0.42 0.50 -0.57 1.40 1.52 0.57 4.06 Maternal age = 25 -0.65 0.70 -2.02 0.71 0.52 0.13 2.04 Maternal education = Illiterate -0.56 0.61 -1.75 0.63 0.57 0.17 1.88 Number in household = 1−3 -0.0 0.78 -2.52 0.52 0.37 0.08 1.68 Number of children = 1–2 0.45 0.59 -0.71 1.60 1.56 0.49 4.96 Household owns soap = 1 0.63 0.53 -0.41 1.67 1.87 0.66 5.31 Water is safely stored = Yes -0.11 0.54 -1.17 0.95 0.90 0.31 2.59 Animals inside day = Yes -0.22 0.53 -1.26 0.81 0.89 0.28 2.25 Animals inside night = Yes -0.38 0.69 -1.73 0.97 0.68 0.18 2.63 Water available = Yes -0.55 0.75 -2.03 0.93 0.58 0.13 2.54 Mother collects water alone = Yes -1.28 0.44 -2.15 -0.42 0.28 0.12 0.66 Infant age (scale) 0.12 0.10 -0.07 0.31 1.13 0.93 1.37 https://doi org/10 1371/journal pntd 0009514 t004 Table 4. Adherence: A Generalised Estimation Equation estimating the effect of parameters on the trial outcome of adherence ’Appropriate use and cleaning’ across study time points. Equation estimating the effect of parameters on the trial outcome of adherence ’Appropriate use and cleaning’ across Adherence: A Generalised Estimation Equation estimating the effect of parameters on the trial outcome of adherence ’A me points 11 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Table 5. Acceptability: Reported playspace use in the past 24 hours during different daily activities, at two and four weeks, and the change across time points. Acceptability Acceptability: Reported HPS use during specified household activities by 24-hour recall and change in proportion of use across study time points (N = 50) Reported daily activity Total reported activity Reported HPS use Proportion of use Total reported activity Reported HPS use Proportion of use Change in use Change in proportion of useα Two weeks Four weeks Across time points n n % n n % n % Prepared / ate a meal 150 139 92.7 172 139 80.8 0 -11.9 Prepared breakfast 45 40 88.9 47 42 89.4 2 +0.5 Prepared lunch/ snacks 56 54 96.4 51 48 94.1 -6 -2.3 Prepared dinner 49 45 91.8 49 43 87.8 -2 -4.1 Ate a meal 0 0 0.0 25 6 0.0 6 0.0 Prepared coffee 75 69 92.0 95 85 89.5 16 -2.5 Duties within the home 57 46 80.7 73 62 84.9 16 +4.2 Cleaned the house 47 40 85.1 55 47 85.5 7 +0.3 Washed clothes 10 6 60.0 18 15 83.3 9 +23.3 Duties outside of the home 43 35 81.4 49 43 87.8 8 +6.4 Fetched water 41 40 97.6 48 44 91.7 4 -5.9 Prepared enset 30 25 83.3 20 19 95.0 -6 +11.7 Chopped wood 7 6 85.7 8 6 75.0 0 -10.7 Farmed / maintained shop 6 4 66.7 21 18 85.7 14 +19.0 Visits outside home 23 17 73.9 32 15 46.9 -2 -27.0 Went to church / meeting 4 1 25.0 4 0 0.0 -1 -25.0 Went to market 15 14 93.3 16 12 75.0 -2 -18.3 Visited neighbours/ other 4 2 50.0 12 3 25.0 1 -25.0 Breastfed / fed baby 26 15 57.7 30 20 66.7 5 +9.0 Slept / rested 23 5 21.7 18 3 16.7 -2 -5.1 HPS, household playspace. Number represents reported incidence of that activity within the past 24 hours. Households (N = 50) were asked an open-ended question about their daily activities during the past 24 hours. Not every activity was reported by every respondent. Calculated as the proportion of households who reported using the HPS during that daily activity. Calculated as the proportion of households who reported using the HPS during that dail αCalculated as the difference between the proportions of HPS use at two and four weeks (41.0%, 95% CI 31.3−51.3; n = 41). Second, change in incidence (as a proportion) of HPS dur- ing daily activities was assessed. https://doi.org/10.1371/journal.pntd.0009514.t005 Acceptability Primary caregivers were asked open-ended questions to record their activities during the past 24 hours, and if they did or did not use the HPS. Results are shown in Table 5, with activities categorised. Broadly, there was no change in use through- out the trial during food preparation/eating but use increased during other activities inside the home (such as breastfeeding) and outside, such as preparing enset and farming. A full table describing activities and HPS use or non-use is in Table B in S1 Data. Lastly analysing HPS use according to the time of day suggested use was consistently highest in the mornings, although evening use increased at the trial end (Table E in S1 Data). PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 12 / 20 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Modified barrier analysis Acceptability was further assessed through a semi-structured questionnaire as a modified Barrier Analysis. This assessed 12 categories of behavioural determinants, exploring all fac- tors which would act as barriers or enablers during a definitive trial (S1 PRISMA Check- list). Full results are in Table D in S1 Data. The first seven determinants quantitatively assess beliefs and behaviours relating to infant health and HPS use. The further six determi- nants explored attitudes and beliefs through open-ended questions. Many cited advantages, both related and unrelated to infant health, indicated good acceptability of the HPS. Care- givers frequently stated the HPS helped prevent ingestion of dirt and faeces (80.0%, n = 40), 76.0% (n = 38). Further, many suggested the HPS prevented injury from several causes, including from fire, drowning and animals. Over half of caregivers (mothers) asserted that the HPS eased their workload (56.0%, n = 28), reduced time pressures (46.0%, n = 23) and allowed them to carry out their duties without distraction. Mothers reported relief that the HPS alleviated fears and worries over their infant’s safety (52.0%, n = 26), and almost half believed their infant would physically grow better (42.0%, n = 21). Approval within the community was high among neighbours (96.0%, n = 48) husbands (40.0%, n = 20), and both close (66.0%, n = 33) and wider family (36.0%, n = 18). Con- versely, some caregivers mentioned that neighbours (8.0%, n = 4) or friends (12.0%, n = 6) were envious as the common reason for disapproval (‘My friend who does not have one wants one too’), or that money would have been preferable (‘My colleague says better to give the child clothes or money for me’). Barriers to use included the cost of cleaning materials (22.0%, n = 11)–echoed in the Access determinant where caregivers frequently noted the expense of soap (56.0%, n = 28) and clean- ing materials, e.g. brushes (24.0%, n = 12). Importantly, having no older children to watch the infant was a barrier (32.0%, n = 16) and relates to the burden of workload on women. A lack of toys was also a barrier (32.0%, n = 16). Whilst the design appeared largely acceptable, some dif- ficulties included fitting the rope connecting walls (38.0%, n = 19; see S1 Figure). Secondary outcomes: Infant health outcomes Table 6 shows changes in reported seven-day diarrhoeal prevalence and presumptive Campylo- bacter spp. across groups and time periods. Considering change in point prevalence, seven-day diarrhoea declined more markedly within the intervention group from 19 cases (38.0%) at baseline to 5 cases (10.0%) at four weeks, versus 22 cases (44.0%) to 16 (32.0%) amongst con- trols. Considering change in prevalence from baseline, the intervention group showed a reduced odds of reported diarrhoea versus controls (OR 0.57, 95% CI 0.40−0.83). Baseline prevalence of presumptive Campylobacter was high, mirroring a similar prevalence at this site and others [26,29]. However from baseline, point prevalence showed no significant difference between groups or time points. Similarly the intervention group had no reduced odds of a Campylobacter-positive stool versus controls from baseline. Colony counts from positive sam- ples can be viewed in Table D in S1 Data. Harms Secondary health outcomes: Point prevalence across study time points and change in prevalence from baseline f intervention and control. Secondary outcomes: Change in infant health outcomes OR for intervention group 0.49 (95% CI 0.33 0.75) No diarrhoea: No reported diarrhoea at two or four weeks, OR no reported diarrhoea from baseline; Diarrhoea: Reported diarrhoea at two or four weeks, OR reported diarrhoea from baseline. β f Insignificant. αNegative: No suspected Campylobacter at two or four weeks, OR always negative; Positive: Suspected Campylobacter prevalence at two or four weeks, OR always positive Harms No adverse events were observed from HPS use in the intervention group. No household reported any safety concerns associated with use, aside from one household who mentioned the plastic mattress became hot under the sun. HPS use did not increase the risk of any adverse infant health outcome, where the direction of effect does not show an increased risk for the intervention group. 13 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Table 6. Secondary health outcomes: Point prevalence across study time points and change in prevalence from baseline for seven-day diarrhoea and Campylobacter, intervention and control. Secondary outcomes: Change in infant health outcomes Reported seven-day diarrhoea point prevalence across study time points Baseline Two weeks Four weeks Intervention (n = 50) Control (n = 50) Intervention (n = 50) Control (n = 50) Intervention (n = 50) Control (n = 50) n % n % n % n % n % n % No diarrhoea 31 62.0 28 56.0 44 88.0 35 70.0 45 90.0 34 68.0 Diarrhoea 19 38.0 22 44.0 6 12.0 15 30.0 5 10.0 16 32.0 Presumptive Campylobacter point prevalence across study time points No infection 23 46.0 24 48.0 33 66.0 32 64.0 36 72.0 36 72.0 Infection 27 54.0 26 52.0 17 34.0 18 36.0 14 28.0 14 28.0 Change in reported seven-day diarrhoeal prevalence after baseline Intervention (n = 50) Control (n = 50) n % n % No diarrhoea 39 78.0 28 56.0 Diarrhoea 11 22.0 22 44.0 Change in presumptive Campylobacter prevalence after baselineβ No infectionα 30 60.0 28 56.0 Any infection 20 40.0 22 44.0 OR for intervention group 0.49 (95% CI 0.33−0.75) No diarrhoea: No reported diarrhoea at two or four weeks, OR no reported diarrhoea from baseline; Diarrhoea: Reported diarrhoea at two or four weeks, OR reported diarrhoea from baseline. βInsignificant. αNegative: No suspected Campylobacter at two or four weeks, OR always negative; Positive: Suspected Campylobacter prevalence at two or four weeks, OR always i i prevalence across study time points and change in prevalence from baseline for seven-day diarrhoea and Campylobacter, ondary health outcomes: Point prevalence across study time points and change in prevalence from baseline for seven-da and control. Table 6. Discussion The CAMPI trial is the first randomised, controlled feasibility trial of a HPS in rural, subsis- tence agriculture households in Ethiopia. Though trial outcomes did not fully reach a priori criteria, the trial demonstrated mixed adherence and good acceptability. On this basis, a defini- tive RCT for efficacy is feasible if recommended adjustments are made. Results echo two simi- lar studies. In the SHINE trial in Zimbabwe, an imported plastic HPS and locally sourced plastic playmat were included in a WASH intervention to improve growth and anaemia. Whilst fidelity of delivery was high [10], the WASH intervention did not prevent infection [30]. However, the analysis did not estimate a magnitude of effect from the HPS specifically. In Zambia, a community-built HPS was assessed alongside a plastic model for acceptability and feasibility[31]. Reported use was similar between the two types (ranging from 10 minutes to three hours), family and community reactions suggested acceptability was high and caregiver reports suggested the community built space prevented infant ingestion of soil and animal fae- ces. Thus growing evidence supports wide acceptability and feasibility across different contexts and further rigorous assessment of efficacy is merited. Addressing barriers to appropriate use and cleaning of the HPS would improve these out- comes. Data here described a broadly consistent pattern over the four weeks, albeit with a small decline (Tables 2 and 3). The modified Barrier Analysis offered reasons for diminishing use and drops in compliance, including the expense of soap and other cleaning materials. Pro- viding these alongside the HPS would be a key consideration for any future RCT to ensure 14 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace good hygiene. Similarly, contextual WASH factors, such as water quality, availability, and unsafe storage (76.0%, n = 38 in the intervention group; Table 1) must be considered which may result in increased bacterial transmission. Similarly, the team decided not to provide toys during the trial given the potential to become vectors for indirect faecal-oral transmission [18,32].18,32 However, this was a frequently cited barrier for mothers whose infants became bored and cried: thus providing toys or including stimulating features to the HPS is an impor- tant consideration. Alternatively caregivers may be counselled on providing (non-porous), non-hazardous toys and on regular proper cleaning. Discussion Further, during early, critical growth peri- ods there are other important considerations including psychosocial and neurodevelopment. Opportunities for linguistic, socioemotional, and cognitive development are critical and a future RCT should consider if a HPS reduces these opportunities through interruptions to nor- mal play, exploration, and caregiver-infant interaction–all strongly related to contextual norms and traditions. Through random spot checks of HPS use and change in time-use, the trial showed mixed acceptability, partly meeting a priori criteria (Tables 2 and 5). Reported daily use increased for certain activities, suggesting an increasing ease with incorporating the HPS into daily life. However increased use during certain activities (fetching water, farming) may indicate a com- placency with infant safety inside the HPS and present a risk. These increases may account for the reduction in ‘Appropriate use’ at week four which included if the caregiver left the infant alone whilst outside. A key finding from the modified Barrier Analysis were the secondary effects of easing work burden, time restraints, and worries about infant health and safety for many mothers. Thus in the short term, a HPS may hold many benefits including potentially improving women’s empowerment through time availability and choice, reducing anxiety, and even freeing up time to spend with her infant. However any negative long-term impacts will need to balance these. This includes a lack of infant supervision, and the risk of reinforcing women’s roles as sole caregivers alongside a continuing responsibility for other domestic duties. This is reinforced by the GEE model (Table 4) where when the mother bore the duty of collecting water alone, the HPS was less likely to be used or cleaned properly. In many low- income countries, women’s’ ‘triple work burden’ in the productive, reproductive and social domains impedes their well-being and may reduce engagement in childcare[33]–a pattern often inherited by older female siblings. This highlights a trade-off in encouraging more active parenting alongside existing home duties, and any intervention must ensure it does not further encumber women. The CAMPI trial was not powered to detect any differences in health outcomes between groups and results should be interpreted accordingly. Given results were preliminary, results are expressed without claims as to definite direction of effects. However secondary infant health outcomes indicated the potential efficacy of a HPS and appropriateness of these out- comes for a future RCT. Progression to a definitive RCT Progression to a full-scale trial is merited but requires some adjustments. To improve playspace adherence and acceptance, a future definitive RCT should focus on directly addressing the barri- ers whilst promoting the enabling factors as identified in this feasibility trial. Whilst further beha- vioural ‘modules’ and developing caregiver knowledge might have improved outcomes, it is not always practical. During the sensitisation day the HPS was introduced in a ‘scalable’ manner to reduce work burden among households and HEWs who are already overworked. Rather, to achieve behavioural change it is pragmatic to directly address barriers and promote enabling fac- tors. Knowledge alone is unlikely to prevent infant faecal-oral transmission without a material element which breaks contact, and an enabling technology may drive changes in behaviour but still requires addressing factors which support or obstruct change. Factors included in the com- posite variable ‘Appropriate use’ responsible for a decline include another child sharing the HPS. Given the potential to introduce contamination, this might be addressed by a visiting HEW as a risk factor. Similarly, ‘Appropriate cleaning’ declined from every day/every other day to twice a week. The direction of effect and significance in the GEE model (Table 4) is an important con- sideration to improving this: cleaning behaviours will not change without access to soap. To improve time-use, toys (non-porous) might be provided with counselling from HEWs on regu- lar cleaning. Factors not modifiable to counselling are important prognostic factors and might be included as strata in group randomisation in a full RCT. Several contextual factors undoubtedly influenced this trial’s operational success, including ease of recruitment and full retention. The study kebeles, within PIN outreach, may have resulted in higher acquiescence during recruitment and consent. High retention likely results from this plus a high number of data collectors for the sample. However, it is important to note that daily data collection was intense and required serious team dedication. A larger trial would likely expe- rience higher drop out without equivalent input: a 95% CI estimate would be between 96−100% in a power calculation and 95−100% if repeated maintaining the same effort and ratio of study personnel. Over a longer time period, this is likely unsustainable. Future sample size calculations must consider these number requirements for study personnel. Discussion Diarrhoeal prevalence from baseline reduced among the intervention group whilst presumptive Campylobacter did not (Table 6). Beyond the lack of adequate power, substantial methodological limitations may profoundly affect validity. These include the reliability of caregiver-reported diarrhoea and a desirability bias within intervention households; intervention households may have over-reported diarrhoea to claim for improve- ments in infrastructure. No further GEE analysis was performed to explore associated vari- ables. However, aside from a potential lack of effect of the HPS on Campylobacter prevalence, other pathways not interrupted by the HPS likely contributed to pathogen transmission. This includes incorrectly (re-)heated foods[34]; data on this indicated unsafe practices were com- mon (Table F in S1 Data) where across households only 28 safely prepared all meals at both time points (Table G in S1 Data). All infants were given liquids other than breastmilk, includ- ing water, possibly contaminated through unsafe storage or other pathways. Campylobacter PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 15 / 20 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace from domestic free-range poultry appears to present an infection risk to infants[5,26] and here poultry frequently shared living and sleeping areas (Table 1). However, questions remain on what, how and where infants contract Campylobacter, the role of domestic animals in trans- mission and survival time in the environment[34]. The methodology used to isolate Campylo- bacter spp. also holds limitations[26] and a definitive RCT should consider other, more sensitive techniques such as the use of ELISA or quantitative PCR. Progression to a definitive RCT Furthermore, as recipients of pre- vious WASH interventions, the intervention group likely adopted the new intervention modality earlier than might be seen in other contexts, holding implications for external validity. Good uptake may also be seen in other contexts where NGOs have a known presence and have pro- vided multiple interventions for many years, but this does limit the generalisability of findings to other contexts. Generalisability of the efficacy of the intervention would likely be variable across different settings. Lastly it is important to note the extensive HPS design process and the underly- ing formative work. Good contextual understanding is critical for intervention success, which must be culturally acceptable, locally integrated and must consider contextual baseline demo- graphic and WASH characteristics and health status which vary significantly. S1 Fig. The prototype design of the BabyWASH household playspace used in the CAMPI trial. A figure containing four photographs showing the key design features of the BabyWASH household playspace. S1 Data. Supplementary data tables. A file containing supplementary data tables including: Table A. Playspace use behaviours and infant hygiene and playspace cleaning practices included as part of composite variables ‘Appropriate use’ and ‘Appropriate cleaning’. A table containing the full set of behavioural results on appropriate use and appropriate cleaning at two week, four week and both time points. Table B. Reported daily activities and reported use or non-use of the playspace during the past 24 hours, across daily periods and study time points. A table containing the full set of results on daily activities that BabyWASH playspace users engaged with whilst using the playspace at two week and four week time points. Table C. Reported playspace use and non-use during daily activities in the past 24 hours across daily time periods: at two and four weeks. A table containing results for reported use and non-use of the BabyWASH playspace during morning, afternoon and evenings. Table D. Modified Barrier Analysis results among the study intervention group. Two tables containing results from the modified barrier analysis covering key behavioural determinants, including perceived positive consequences, perceived self-efficacy, access and perceived social norms. Table E. Number of samples positive for presumptive Campylobacter spp. under each category of colony count. A table containing results for presumptive Campylobacter spp. colony count within intervention and control arm at baseline, two weeks and four weeks. Table F. Feeding of fresh or reheated foods prepared as recommended, across study groups and time points. A table containing results for reported feeding of fresh and reheated food at different meal times within intervention and control arm at baseline, two weeks and four weeks. Table G. Number of meals safely pre- pared across time points. A table containing results on number of safely prepared meals across intervention and control arm at baseline, two weeks and four weeks. (DOCX) Conclusion The CAMPI trial evaluated feasibility of a BabyWASH HPS and recommendations to progress to a full-scale RCT in a rural, subsistence agriculture setting in Ethiopia. Not all a priori criteria 16 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace were met. However, overall the HPS showed mixed engagement and adherence, good accept- ability and many reported secondary benefits. A larger trial with longer follow-up is feasible to implement and should assess infant health outcomes as primary endpoints. This would help determine a HPS as a viable option to reduce direct faecal-oral transmission and infant infec- tion in this and other similar settings. Addressing identified barriers and promoting enabling factors would be necessary changes and would likely improve adherence and use. S1 PRISMA Checklist. A checklist signposting where key information on the study design is reported in the manuscript. (DOCX) S1 PRISMA Checklist. A checklist signposting where key information on the study design is reported in the manuscript. (DOCX) S1 Text. Modified Barrier Analysis methodology. A description of the modified barrier anal- ysis methodology including questions used to assess behavioural barriers to appropriate use and maintenance of the BabyWASH household playspace. (DOCX) Author Contributions Conceptualization: Sophie Budge, Paul Hutchings, Alison Parker, Camila Garbutt. Conceptualization: Sophie Budge, Paul Hutchings, Alison Parker, Camila Garbutt. Data curation: Sophie Budge, Fitsume Woldemedhin, Mohammed Yasin Jemal, Mathewos Moges, Siraj Hussen, Hunachew Beyene. Formal analysis: Sophie Budge, Sam Norton, Mathewos Moges, Siraj Hussen, Hunachew Beyene. Funding acquisition: Paul Hutchings, Alison Parker, Camila Garbutt. Investigation: Fitsume Woldemedhin, Mohammed Yasin Jemal. Methodology: Sophie Budge, Paul Hutchings, Alison Parker, Sean Tyrrel, Camila Garbutt, Mathewos Moges. Project administration: Fitsume Woldemedhin, Mohammed Yasin Jemal. Supervision: Paul Hutchings, Alison Parker, Sean Tyrrel, Camila Garbutt. Supervision: Paul Hutchings, Alison Parker, Sean Tyrrel, Camila Garbutt. Validation: Sam Norton. Validation: Sam Norton. Writing – original draft: Sophie Budge. Writing – review & editing: Paul Hutchings, Alison Parker, Sean Tyrrel, Sam Norton, Camila Garbutt, Fitsume Woldemedhin, Mohammed Yasin Jemal, Mathewos Moges, Siraj Hussen, Hunachew Beyene. Acknowledgments This study would not have been possible without the tireless efforts of the data collection teams, including Mesfine Melese, Metsinanat Eyoel, Wonsha Bulbula, Dawit Daniso, Deginet 17 / 20 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0009514 July 14, 2021 PLOS NEGLECTED TROPICAL DISEASES A randomised controlled feasibility trial of a household playspace Aklilu and Biruk Solomon. 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https://openalex.org/W2331663238	https://www.nobleresearch.org/Content/PDF/1/2052-4994.2013-15/2052-4994.2013-15.pdf	English	null	Treatment of an atypical metastatic meningioma: a case report	Journal of cancer research & therapy	2,013	cc-by	5,027	Introduction Intracranial meningiomas behave benign (WHO grade I) in about 90% of the cases, with slow growth, and without recurrences after surgical removal. Atypical (WHO grade II) and anaplastic (WHO grade III) meningiomas are more aggressive, and show a high risk of local recurrences, as well as, more rarely, of extra cranial dissemination [1, 2, 3]. Metastatic meningiomas are usually resistant to chemotherapy and target therapy. The purpose of this case report is to increase the knowledge about the diagnosis and treatment of metastatic meningiomas. Treatment of an atypical metastatic meningioma: a case report Conca R1,, Roviello G1, Cerase A2, Mazzei MA4, Miracco C3, Mastrogiulio MG3, Marsili S1 and Francini G1, Conca R1,, Roviello G1, Cerase A2, Mazzei MA4, Miracco C3, Mastrogiulio MG3, Marsili S1 and Francini G1, 1Medical Oncology, University of Siena, Policlinico Le Scotte, Viale Bracci 11, 53100 Siena, Italy 2Unit NINT Neuroimaging and Neurointervention, Department of Neurological and Sensorial Sciences, University of Siena, Italy 3Department of Human Pathology and Oncology, Section of Pathological Anatomy, University of Siena, Italy 4Department of Human Pathology and Oncology, Section of Radiological Sciences, University of Siena, Italy Abstract Meningiomas are common intracranial tumors which usually pursue a benign course. Extracranial metastases from meningiomas are very rare and the lung is the most common site. We report a 27 year old girl with an intracranial atypical meningioma with pulmonary metastasis which had been misdiagnosed as lung sequestration upon chest CT examination. She underwent subtotal surgical resection of the meningioma in 2006 and surgical removal of the lung metastasis in 2009. Then, the patient developed pleural, lung, periesophageal and diaphragmatic nodal metastases, despite only subtle increase of the intracranial residual meningioma. Therefore, she was treated by Doxil (pegylated liposomal doxorubicin) and bevacizumab with a progression of disease after three cycles and then with hydroxyurea for two months with a dimensional increase of metastatic lesions. After adding sorafenib to hydroxyurea the patient showed a further progression disease and finally died for respiratory insufficiency. Metastatic meningiomas have been rarely reported. Hydroxyurea is one of the most used drug in recurrent and metastatic meningiomas, despite modest results are obtained. Further chemotherapy strategies or biological agent must be investigated in clinical trials. Keywords: metastatic meningioma; chemotherapeutic agents; doxorubicin; bevacizumab; hydroxy basal opacity. Upon chest CT, this lesion was 65 mm in maximum diameter, showed a wide pleural contact, smooth and sharp borders, and sporadic calcifications on the pleural surface (Figure 2). A diagnosis of pulmonary intralobar sequestration was made because of the identification of the venous drainage into the left inferior pulmonary vein, but underestimating the lack of the identification of an arterial supply. The patient underwent Copyright: © 2013 Conca R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Corresponding authors: Conca R and Francini G, Medical Oncology, University of Siena, Policlinico Le Scotte, Viale Bracci 11, 53100 Siena, Italy, Fax: +39 0577 586429; E-mail: raffaeleconca@hotmail.it; giandomenicoroviello@hotmail.it Corresponding authors: Conca R and Francini G, Medical Oncology, University of Siena, Policlinico Le Scotte, Viale Bracci 11, 53100 Siena, Italy, Fax: +39 0577 586429; E-mail: raffaeleconca@hotmail.it; giandomenicoroviello@hotmail.it Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 http://dx.doi.org/10.14312/2052-4994.2013-15 Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 http://dx.doi.org/10.14312/2052-4994.2013-15 Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 http://dx.doi.org/10.14312/2052-4994.2013-15 Case Report Received 17 November 2012 Revised 5 March 2013 Accepted 12 March 2013 Published 19 March 2013 Journal of Cancer Research & Therapy NobleResearch www.nobleresearch.org Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 http://dx.doi.org/10.14312/2052-4994.2013-15 Case report Received 17 November 2012 Revised 5 March 2013 Accepted 12 March 2013 Published 19 March 2013 In November 2006, an otherwise healthy 27-year-old girl presented with headache, vague balance difficulties, right hemisensory and hemiparetic deficits. Head computed tomography (CT), magnetic resonance imaging (MRI), and MR angiography (MRA) (Figure 1) showed a large left frontal and parietal extra-axial mass, infiltrating the superior sagittal sinus, consistent with meningioma. Preoperative chest radiographs revealed a left posterior Citation: Conca R, Roviello G, Cerase A, Mazzei MA, Miracco C, Mastrogiulio MG, Marsili S, Francini G (2013) Treatment of an atypical metastatic meningioma: a case report. J Cancer Res Ther 1: 99-104. doi:10.14312/2052-4994.2013-15 Copyright: © 2013 Conca R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 100 a subtotal resection of the intracranial mass through a left frontoparietal approach. Pathologic evaluation showed a meningioma with high to moderate cellularity, pattern less areas, atypical nuclear features, with prominence Figure 1 Magnetic resonance (MR) imaging and angiography of the head. (a) Unenhanced T2-weighted; (b) diffusion-weighted; (c) apparent diffusion coefficient; (d) gadolinium-enhanced T1-weighted, axial MR images; (e) 2D phase contrast sagittal MR angiogram; (f) unenhanced T2-weighted and T1- weighted; (h) gadolinium-enhanced T1-weighted, coronal MR images clearly shows the infiltration of the middle and posterior portion of the superior sagittal sinus. The lesion shows some degree of inhomogeneous signal intensity, and homogeneous gadolinium-enhancement a meningioma with high to moderate cellularity, pattern less areas, atypical nuclear features, with prominence a subtotal resection of the intracranial mass through a left frontoparietal approach. Pathologic evaluation showed Figure 1 Magnetic resonance (MR) imaging and angiography of the head. (a) Unenhanced T2-weighted; (b) diffusion-weighted; (c) apparent diffusion coefficient; (d) gadolinium-enhanced T1-weighted, axial MR images; (e) 2D phase contrast sagittal MR angiogram; (f) unenhanced T2-weighted and T1- weighted; (h) gadolinium-enhanced T1-weighted, coronal MR images clearly shows the infiltration of the middle and posterior portion of the superior sagittal sinus. Case report The lesion shows some degree of inhomogeneous signal intensity, and homogeneous gadolinium-enhancement Figure 2 (a) axial CT image after iv contrast material administration showed an ipervascular mass with wide pleural contact; (b) presenting focal calcifications on the pleural surface on the unenhanced CT scan (arrow); (c) Sagittal-oblique reformatted, demonstrated the venous drainage of the mass into the left inferior pulmonary vein (white arrow), suggesting the possible diagnosis of intralobar pulmonary sequestration; (d) A sagittal reformat of the CT examination performed three years later, showed a significant increase in size of the lesion with calcifications (white arrow) and the appearance of a new lesion, located beneath the first, displacing the diaphragm (arrow head) (a) (b) (a) (b) of nucleoli, and rare mitotic figures (less than 1/1mm2). Tumor cell proliferation index, such as evaluated by anti-ki-67 MIB1 antibody (rabbit monoclonal antibody, diluted 1: 100; NeoMarkers, Inc, Fremont, CA, USA), was about 7%; tumor cells positive to progestinic receptors, and EGFR were 50%, and 70%, respectively, p53 was negative. A diagnosis of grade II meningioma, according to WHO 2007 grading system, was rendered (Figure 3). Considering the very young age of patient, the absence of symptoms and the unfavourable risk/benefit ratio, the external beam radiotherapy (EBRT) of the residual lesion wasn’t performed. The intracranial mass remained stable for three years. In December 2009, brain MRI and MRA showed a subtle degree of growth of the residual disease. In the same period, the patient referred abdominal pain associated to short breathing. A chest CT showed a dramatic increase in volume of the left lung lower lobe lesion (maximum diameter 72 mm), showing calcifications and necrotic areas, associated with ipsilateral pleural effusion with total atelectasis of the ipsilateral lower lobe lung. Discussion Therefore, considering the over mm2), a higher proliferation index (Ki-67 labeling index: about 20% vs 7%). Additionally an immunohistochemical staining demonstrated a lack of expression of c-Erb-B2, CD117 (c-kit) and estrogen receptors, while EGFR was expressed. Chest CT images obtained at diagnosis were revised, and the possibility of lung metastases already at the diagnosis was considered. The issue of the radiological and CT differential diagnosis of the thoracic mass is beyond the scope of this paper, and will be addressed elsewhere. The patient began a close follow-up until September 2010 when a chest and abdomen CT revealed the presence of multiple pleural lesions in left hemithorax (maximum diameter, 38 mm) and a large lesion near diaphragmatic hiatus and left diaphragmatic pilar with some pathological diaphragmatic thickenings in left costal-phrenic margin (maximum diameter, 36 mm). A millimetric parenchymal lung lesion of the left lower lobe was evident too. Excluding any local treatment for the systemic dissemination of the tumour, in August 2010, she began treatment with Doxil (pegylated liposomal doxorubicin) 50 mg/m2 and bevacizumab 75 mg/m2 every 21 days [4, 5]. After three cycles of this regimen, a total body CT showed a dimensional increase of pleural and periesophageal lesion (maximum diameter of the last one, 75 mm), with some border line para-aortic lymph nodes. From October 2010 treatment with hydroxyurea 20 mg/m2/die was undertaken. In December 2010, after two months of this treatment a total body CT showed a volumetric growth of pleural lesions with a major central necrotic component. Therefore, considering the over Meningioma preferentially disseminate through the cerebrospinal or lymphatic route [15, 16]. Metastatic spread to the lung and pleura (the most frequent sites) [17, 18], and to other extracranial organs is thought to occur through the blood route, following invasion of dural venous sinuses, as probably occurred in our case, and cranial veins [3, 12, 19]. Some authors have also considered surgery as a risk factor for extracranial spreading [20]. Radical surgery is the treatment choice, after taking into account its usually slow growth, lack of aggressiveness, and the benefit-risk ratio for the patient [21]. A radical resection is not always possible, in particular for tumours in tight contact with vital structures or with high infiltration of venous sinus and cranial base such as in the patient reported herein. Case report When compared to the primary lesion, metastatic deposits showed huge areas with a higher degree of malignancy, showing severe nuclear atypia, a higher number of mitoses (12/1 mm2 vs 1/1 Figure 3 (a) Necrosis; (b) as well as patternless areas with nuclear atypia, scattered, anaplastic nuclei; (c) white arrows and mitoses (black arrow) were observable in primary meningioma; (d) A metastatic meningioma deposit to the lung (the asterisk indicates lung alveoli) Case report (a) (b) ( ) ( ) Figure 2 (a) axial CT image after iv contrast material administration showed an ipervascular mass with wide pleural contact; (b) presenting focal calcifications on the pleural surface on the unenhanced CT scan (arrow); (c) Sagittal-oblique reformatted, demonstrated the venous drainage of the mass into the left inferior pulmonary vein (white arrow), suggesting the possible diagnosis of intralobar pulmonary sequestration; (d) A sagittal reformat of the CT examination performed three years later, showed a significant increase in size of the lesion with calcifications (white arrow) and the appearance of a new lesion, located beneath the first, displacing the diaphragm (arrow head) Figure 2 (a) axial CT image after iv contrast material administration showed an ipervascular mass with wide pleural contact; (b) presenting focal calcifications on the pleural surface on the unenhanced CT scan (arrow); (c) Sagittal-oblique reformatted, demonstrated the venous drainage of the mass into the left inferior pulmonary vein (white arrow), suggesting the possible diagnosis of intralobar pulmonary sequestration; (d) A sagittal reformat of the CT examination performed three years later, showed a significant increase in size of the lesion with calcifications (white arrow) and the appearance of a new lesion, located beneath the first, displacing the diaphragm (arrow head) Another lesion (maximum diameter 125 mm) with similar features was evident caudally, between the lesion and the dorsal vertebral column, in the left costophrenic sinus, apparently arising in the parietal Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 101 expression of EGFR in meningioma and its involvement in the proliferation and/or differentiation of meningothelial cells [6, 7], a treatment with sorafenib (EGFR inhibitor) 400 mg/die and hydroxyurea 20 mg/m2/die was undertaken [8, 9]. The patient performed this treatment for 4 months with no severe toxicity. Then, a total body CT after 4 months of the tyrosine kinase inhibitor and hydroxyurea showed a new further dimensional increase of pleural and meningeal lesions, a modest increase of para-aortic lymph nodes with appearance of ascites. Considering the severe clinical conditions of the patient, we opted for best supportive care. After 2 months the patient died for respiratory insufficiency. pleura, shifting the diaphragm forward and down (Figure 2). A transthoracic biopsy was performed, and histology demonstrated meningioma metastasis. In January 2010, both the pleuropulmonary masses were surgically removed (Figure 2). Discussion Meningiomas represent approximately 13-26% of all intracranial tumours [10]. The most common locations are along the sagittal sinus, and over the cerebral convexity such as in the case reported herein [11]. The WHO grade I meningiomas have usually a benign behaviour. The atypical (WHO grade II) and anaplastic (WHO grade III) meningiomas are more aggressive, with a greater proliferative capacity [12]. The local recurrence rates of meningiomas, even after complete resection varies from 9 to 32% [13]. Metastatic spread outside the skull is rare, and correlates with the histological grade. Exceptionally reported in grade I, it raises to 5% in grade II, and to 30% in grade III [14]. Figure 3 (a) Necrosis; (b) as well as patternless areas with nuclear atypia, scattered, anaplastic nuclei; (c) white arrows and mitoses (black arrow) were observable in primary meningioma; (d) A metastatic meningioma deposit to the lung (the asterisk indicates lung alveoli) mm2), a higher proliferation index (Ki-67 labeling index: about 20% vs 7%). Additionally an immunohistochemical staining demonstrated a lack of expression of c-Erb-B2, CD117 (c-kit) and estrogen receptors, while EGFR was expressed. Chest CT images obtained at diagnosis were revised, and the possibility of lung metastases already at the diagnosis was considered. The issue of the radiological and CT differential diagnosis of the thoracic mass is beyond the scope of this paper, and will be addressed elsewhere. The patient began a close follow-up until September 2010 when a chest and abdomen CT revealed the presence of multiple pleural lesions in left hemithorax (maximum diameter, 38 mm) and a large lesion near diaphragmatic hiatus and left diaphragmatic pilar with some pathological diaphragmatic thickenings in left costal-phrenic margin (maximum diameter, 36 mm). A millimetric parenchymal lung lesion of the left lower lobe was evident too. Excluding any local treatment for the systemic dissemination of the tumour, in August 2010, she began treatment with Doxil (pegylated liposomal doxorubicin) 50 mg/m2 and bevacizumab 75 mg/m2 every 21 days [4, 5]. After three cycles of this regimen, a total body CT showed a dimensional increase of pleural and periesophageal lesion (maximum diameter of the last one, 75 mm), with some border line para-aortic lymph nodes. From October 2010 treatment with hydroxyurea 20 mg/m2/die was undertaken. In December 2010, after two months of this treatment a total body CT showed a volumetric growth of pleural lesions with a major central necrotic component. Discussion The treatment was well tolerated, but the two molecules did not show significant benefit. Although other EGFR inhibitors exist, very few trials have examined their therapeutic potential for treating meningiomas. Therefore, we opted for a multi-target and EGFR inhibitor as sorafenib that may decrease the growth-stimulatory mechanism in this kind of tumor. However, in the patient reported herein neither bevacizumab nor sorafenib showed significant activity. In 2010, Wernicke et al. reported an inverse correlation between EGFR expression and tumor grade in human meningioma [7]. EGFR activation increase resistance to apoptosis, promotes angiogenesis and impairs immune surveillance. A recent small phase II trial has examined the efficacy of gefitinib and erlotinib, EGFR inhibitors, in 25 patients with recurrent meningiomas [9]. The treatment was well tolerated, but the two molecules did not show significant benefit. Although other EGFR inhibitors exist, very few trials have examined their therapeutic potential for treating meningiomas. Therefore, we opted for a multi-target and EGFR inhibitor as sorafenib that may decrease the growth-stimulatory mechanism in this kind of tumor. However, in the patient reported herein neither bevacizumab nor sorafenib showed significant activity. Doxil is a formulation of pegylated liposomal doxorubicin with a prolonged circulation time and selective accumulation in ipervascularized tissue as solid tumors. It appears to have citostatic and radiosensitizing effect in brain tumor [38, 39]. Travitzky et al. reported a single case of a 60 year old woman with massive pleuropulmonary metastases from recurrent cranial meningioma treated with Doxil (50-37.5 mg/m2) for 18 months with regression of pleuropulmonary metastases and disappearance of pleural fluid [4]. In the patient reported herein, treatment with those chemotherapeutic agents has provided no benefit. Somatostatin is a neuropeptide produced in hypothalamus that regulates the endocrine end exocrine secretions as well as the gastrointestinal mobility. Somatostatin and its synthetic analogs, binding their receptors, can have e antitumor effect by stimulation of apoptosis, inhibition of angiogenesis and suppression of tumor-stimulating growth factors [40]. Somatostatin receptors, in particular sst2A subtype, are overexpressed in most menigiomas and their inhibition can suppress the meningioma cell growth [41]. A prospective pilot trial of sustained-release somatostatin (Sandostatin LAR) in 16 patients with recurrent meningiomas was conducted obtaining a partial response in thirty one percent of patients with acceptable toxicity [42, 43]. A recent phase II trial testing a new somatostatin analogue with a wide receptor spectrum has recently opened [44]. Discussion An attractive alternative therapeutic approach is the inhibition of platelet derived growth factor receptor (PDGF) pathway. Because of the high expression of PDGF ligand AA and BB and the PDGF beta receptor in human meningioma cells [49–54]. Imatinib mesylate (inhibitor of PDGF receptor alfa e beta, c-Kit tyrosine kinases and Bcl-Abl) has been investigated as a possible therapeutic option for recurrent meningiomas in some phase II trials with modest results [55–57] Discussion Radiotherapy is usually not performed in grade I meningiomas, being recommended only after partial resection, to prevent local recurrences, or in unresectable forms [22, 23, 24] also considering the variety of adverse effects, including radiation necrosis, which may occur after SNC irradiation [25, 26]. Postoperative radiotherapy was found to improve the progression free survival in grade II [27] (poorly defined) and III meningiomas [28, 29, 30]. Stereotactic radiation, with a high-dose radiation to a target volume, is an alternative way to reduce adverse effects of radiotherapy. Radiotherapy was not performed in our case, considering the very young age of patient, with Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 102 an unfavourable risk/benefit ratio [31]. Furthermore, indication for radiation therapy in meningiomas involving large vessels is still debated, since there are not long term follow up [32]. with recurrent glioblastoma. Additionally, it can induce radiation sensitivity in solid cancers inhibiting the angiogenesis [48]. In the literature there are few case report on the treatment of meningiomas with Bevacizumab but the treatment of recurrent or metastatic meningioma with VEGF inhibitor with other chemotherapeutic agents may be very effective. EGFR (epidermal growth factor receptor) is a member of the receptor tyrosine kinase HER family that is crucial for the regulation of cell division and tumor growth. It’s over expression is associated with a poor prognosis in various epithelial tumor. In 1987 Weisman et al, demonstrated that EGFR is involved in the proliferation and differentiation of meningothelial cells [6]. Chemotherapy is usually applied to inoperable lesions, especially in the setting of tumor progression or recurrence, with modest results. Among the traditional cytotoxic drugs, hydroxyurea is the most experienced. It is an oral antimetabolite commonly used in the treatment of haematologic malignancies whose mechanism of action is to inhibit ribonucleotide reductase and causes apoptosis in phase S of the cell cycle [33]. Preliminary results of clinical studies suggest that hydroxyurea has modest activity against recurrent and inoperable meningiomas, and can induce long term stabilization in some patients, with an acceptable safety [34, 35, 36, 37]. In 2010, Wernicke et al. reported an inverse correlation between EGFR expression and tumor grade in human meningioma [7]. 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Neuropathology 27:448–452. with liposomal doxorubicin. Cancer 92:1936–1942. [40] Kvols LK, Woltering EA (2006) Role of somatostatin analogs in the clinical management of non-neuroendocrine solid tumors. Anticancer Drugs 17:601–608. Conclusions Histological features of atypia or malignancy, as well as large vessel invasion may be associated with an unfavourable course in meningioma, and the occurrence of extra-cranial spread, although infrequent, must always be considered. Currently, the traditional chemotherapeutic agents have provided minimal benefit in meningiomas recurring after surgery. In spite of the negative results of the alternative treatments reported in this case, we believe that new strategies are required in metastatic meningioma. Recent publications report an intense search for new molecular markers that may serve as potential therapeutic target. VEGF (vascular endothelial growth factor) is an important regulator of angiogenesis and has been implicated in pathologic angiogenesis associated with tumor growth [45]. Meningiomas are extremely vascular tumor and VEGF may have a critical role in this characteristic [46, 47]. Bevacizumab is humanized monoclonal antibody that inhibits VEGF and it has been the first antiangiogenic therapy to be approved in solid tumor. Many trials have showed its efficacy and good safety as single agent or with chemotherapy for patients There are very few open trials testing target therapies in this pathology probably because the molecular pathways driving meningioma progression are still mostly unknown. Only a better elucidation of these pathways can help us to modify treatment paradigms in this uncommon tumor. Conca R et al., J Cancer Res Ther 2013, 1(2): 99-104 103 The authors wish to express that they have no conflict of interest. The authors wish to express that they have no conflict of interest. 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https://openalex.org/W1991660531	https://europepmc.org/articles/pmc3094350?pdf=render	English	null	Downregulation of FIP200 Induces Apoptosis of Glioblastoma Cells and Microvascular Endothelial Cells by Enhancing Pyk2 Activity	PloS one	2,011	cc-by	11,840	Abstract The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors. Received November 12, 2010; Accepted April 11, 2011; Published May 13, 2011 Received November 12, 2010; Accepted April 11, 2011; Published May 13, 2011 ght: 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Licen cted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants DK46199 to PWS, HL58655 to MAO, CA109748 and CA127620 to CLG, and the Melvin Burkhardt chair in Neurosurgical Oncology and the Karen Colina Wilson research endowment within the Brain Tumor and Neuro-Oncology Center at the Cleveland Clinic for support of RJW. Abstract The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. C i I Th h h d l d h i i i Competing Interests: The authors have declared that no competing interests exist. * E-mail: gladsoc@ccf.org Downregulation of FIP200 Induces Apoptosis of Glioblastoma Cells and Microvascular Endothelial Cells by Enhancing Pyk2 Activity Dongyan Wang1, Mitchell A. Olman2, Jerry Stewart Jr.1, Russell Tipps5, Ping Huang5, Paul W. Sanders3, Eric Toline1, Richard A. Prayson7, Jeongwu Lee6, Robert J.Weil8, Cheryl A. Palmer1, G. Yancey Gillespie4, Wei Michael Liu1,5, Russell O. Pieper9, Jun-Lin Guan10, Candece L. Gladson1,5,8* 1 Division of Neuropathology, Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 2 Division of Pulmonary/ Critical Care, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 3 Department of Nephrology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 4 Neurosurgery Division, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 5 Department of Cancer Biology, Cleveland Clinic, Cleveland, Ohio, United States of America, 6 Department of Stem Cell and Regenerative Medicine, Cleveland Clinic, Cleveland, Ohio, United States of America, 7 Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio, United States of America, 8 Brain Tumor and Neuro-Oncology Center, Cleveland Clinic, Cleveland, Ohio, United States of America, 9 Department of Neurosurgery, University of California San Francisco, San Francisco, California, United States of America, 10 Division of Molecular Medicine and Genetics, Cell and Developmental Biology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States of America Citation: Wang D, Olman MA, Stewart J Jr, Tipps R, Huang P, et al. (2011) Downregulation of FIP200 Induces Apoptosis of Glioblastoma Cells and Microvascular Endothelial Cells by Enhancing Pyk2 Activity. PLoS ONE 6(5): e19629. doi:10.1371/journal.pone.0019629 Editor: Robert E. Means, Yale Medical School, United States of America Introduction Specifi- cally, Pyk2 can effectively compete with FAK for binding to Src and/or Fyn and thereby inhibit ERK activation [13]. The analysis of the potential role of FIP200 in tumor development has focused, for the most part, on breast cancer [7,22,23], and there has been one report of the effects of transient downregulation of FIP200 in breast tumor cells [7]. We investigated whether endogenous FIP200 protein regulates the proliferation of cells found in glioblastoma tumors and whether it does so by regulating Pyk2 and/or FAK activity. We and others have shown previously that Pyk2 and FAK can regulate cell proliferation and survival in glioblastoma cells, with overexpression of Pyk2 decreasing and overexpression of FAK enhancing cell proliferation [17,24,25]. In the current studies, we found that FIP200 is expressed in tumor cells and occasional glial cells in glioblastoma tumor biopsies. It also was present in the endothelial cells in the tumor-associated vessels, although it is not detectable in the endothelial cells in the vessels of normal brain. siRNA downregulation of endogenous FIP200 protein inhibited cell proliferation and induced apoptosis in U-87MG human glioblastoma cells, immortalized human astrocytes, and human brain microvessel endothelial cells (MvECs) propagated in complete media, which are reactive and commonly used as a surrogate model of tumor-associated MvECs. FIP200 downregula- tion increased Pyk2 activity, without affecting FAK activity. The pro- apoptotic effect was blocked by a fusion protein containing the Pyk2 autophosphorylation site in the glioblastoma cells and the MvECs. This preferential effect of the FIP200 downregulation on Pyk2 activity may be explained by the greater association of FIP200 with Pyk2, rather than FAK. Our data suggest that FIP200 may be necessary for the survival of multiple cell types found in glioblastoma tumors, and that FIP200 inhibition of Pyk2 activity may play a prominent role in this promotion of survival. p ( g ) On examining the gliobastoma biopsies, we observed apparent staining of FIP200 in the endothelial cells of the tumor-associated blood vessels although such staining had not been noted on examination of the normal brain samples. We therefore assessed FIP200 staining in the vessels using a semi-quantitative immuno- histochemical approach. The pathological identification of the vessels was confirmed by staining of the von Willebrand factor (vWf) in the endothelial cells in the adjacent sections and only small and medium sized vessels were counted in case the FIP200 staining reflected differences in the size of the vessels. Introduction In the tumor cells in the glioblastoma biopsies, the FIP200 staining usually was located in the cytoplasm but occasionally was seen in the nucleus, which is consistent with the staining patterns described for FIP200 expression in breast cancer biopsies [26]. In the glial cells it was localized in the cytoplasm. Similar results were obtained on immunohistochemical analysis of 15 additional frozen biopsies (Fig. 1G). FIP200 staining of tumor cells was observed in 14 of these biopsies, and in an occasional glial cell in all of them. The reactivity of the rabbit antibody with FIP200 in the brain biopsies was confirmed by immunoblotting detergent lysates of 17 glioblastoma and five anaplastic astrocytoma, as well as nine normal brain cortex or white matter biopsies (Figure S1 and data not shown). Immunoblotting of U-87MG and SNB19 human glioblastoma cells and 827 and 905 human glioblastoma stem cells propagated as neurospheres with the rabbit antibody showed that these cell lines express FIP200 (Figure S2A). functions of these proteins [1,2]. Both Pyk2 and FAK regulate cell proliferation, apoptosis, and the cell cycle; in general, Pyk2 acts to downregulate cell survival whereas FAK acts to promote it [11–17]. The forced overexpression of Pyk2 induces apoptosis, with the N-terminal and kinase domains of Pyk2 being necessary for this effect [11,18]. Some apoptosis signals [12,19] activate Pyk2 including TNFa [19]. Clues to the associated signaling pathways are provided by the findings that methylmethane sulfonate induction of apoptosis requires Pyk2 activation of JNK [20] and expression of the transcription factor Smad4 can increase Pyk2 expression with a subsequent promotion of apoptosis in MDA- MB468 cells [21]. Evidence that Pyk2 can modulate FAK activity has been generated through analysis of FAK-mediated promotion of cell cycle progression. FAK can promote cell cycle progression through activation of ERK and induction of the transcription factor Kruppel-like factor 8 (KLF8), which activates transcription of cyclin D1, as well as through inhibition of p27(Kip1) expression in glioblastoma cells [13–15,17]. The C-terminal domain of Pyk2 is necessary for its inhibition of the cell cycle [13] and expression of Pyk2 in cells with a low level of endogenous Pyk2 inhibits cell cycle progression through the differential activation of JNK and ERK [13]. Notably, Pyk2 also can inhibit cell cycle progression by competing with endogenous FAK for binding partners. Introduction and is expressed in a wide variety of tissues and cell lines [2,6]; however, its effects appear to be highly dependent on the cell type and the experimental conditions [3]. The focal adhesion kinase family interacting protein of 200-kDa (FIP200), which is also known as retinoblastoma coiled coil protein 1 (Rb1CC1), can interact with and regulate the activity of several different proteins that are involved in the signaling of proliferation, apoptosis, autophagy, and cell cycle progression [1–4] and is therefore particularly well-positioned to regulate cell survival. The FIP200-nullizygous state is embryonic lethal in mice and E14.5– E15.5 day embryos show histologic evidence of heart failure and liver degeneration that is associated with high levels of apoptosis in the heart and liver tissue [5]. The FIP200 gene is highly conserved Several different regulatory mechanisms have been identified that may affect FIP200 activity, including its level of expression [7], localization (cytoplasmic vs. nuclear) [1,3,8–10], and its interaction with other proteins [3,4,10]. FIP200 can interact directly with the kinase domains of both focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2, also known as related adhesion focal tyrosine kinase, calcium-dependent tyrosine kinase, and focal adhesion kinase-2). Overexpression of FIP200 inhibits the kinase activity and the kinase-associated cellular PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 1 May 2011 \| Volume 6 \| Issue 5 \| e19629 FIP200 in Glioblastoma Tumors expression in neurons in the brain [10]. Representative photomi- crographs are shown in Figure 1. In all of the 20 normal brain biopsies, FIP200 was detected in the cytoplasm of a population of neurons (Figure 1B) and in the cytoplasm of occasional glial cells (data not shown); with the FIP200 being largely localized to the cortex. The detection of FIP200 in a population of neurons in the normal brain cortex is consistent with its reported role in maintaining normal homeostasis of neurons [10]. In the tumor biopsies from 14 of 21 patients with glioblastoma, FIP200 was detectable in the tumor cells. As can be seen in the representative photomicrographs shown in Figure 1, panel F, the levels of expression varied considerably among the tumor cells in any one glioblastoma biopsy as well as among the biopsies from different patients. FIP200 also was detectable in an occasional glial cell in 19 of the 21 glioblastoma biopsy samples (data not shown). Introduction The tumor- associated vessels exhibited the histologic abnormalities described previously [27], i.e., dilated, tortuous, and with reduced pericyte coverage. FIP200 was not detectable in the endothelial cells in the small and medium-sized vessels in 19 of the 20 normal brain biopsies (Figures 1B–D). (In the one biopsy in which staining of the endothelial cells in the vessels was observed, approximately 5% of the blood vessels contained endothelial cells that stained weakly for FIP200.) In marked contrast, FIP200 was detectable in the endothelial cells of the tumor-associated vessels in 16 of the 21 formalin-fixed, paraffin-embedded glioblastoma biopsies and in 14 of the 15 additional frozen biopsies (Fig. 1F–H). (In the five formalin-fixed, paraffin-embedded biopsies in which the endothe- lial cells in the tumor-associated vessels did not stain for FIP200, the tumor cells also were negative for FIP200 and in the one frozen biopsy that was negative for staining of the endothelial cells, only 10% of the tumor cells were positive for staining of FIP200.) This unexpected difference in expression of FIP200 in the endothelial cells in the glioblastoma tumors and normal brain was significant (p value,0.0001, Fisher exact test). Immunoblot analysis of cultured primary normal human brain endothelial cells propagated in complete media, which are commonly used as a surrogate model for the endothelial cells in brain tumor-associated microvessels, indicated that the rabbit antibody was reacting with FIP200 Protein is Expressed in Tumor Cells, Glial Cells and the Endothelial Cells of Tumor-Associated Vessels in Glioblastoma Biopsies Immunohistochemical analysis of the expression of FIP200 in formalin-fixed, paraffin-embedded biopsies was carried out using a rabbit anti-FIP200 antibody used previously to identify FIP200 PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 2 FIP200 in Glioblastoma Tumors FIP200 in Glioblastoma Tumors Figure 1. FIP200 is expressed in tumor-associated endothelial cells as well as tumor cells in glio fixed and paraffin-embedded sections of surgical biopsies of normal brain (n = 20) and glioblastomas glioblastoma (n = 15) were subjected to immunohistochemical analysis using diaminobenzidene as th hematoxylin. The sections were reacted with a commercial rabbit anti-FIP200 (0.2 microgram/ml) or rabbit Ig D, representative photomicrographs of sections of normal brain reacted with control rabbit IgG (A) or rabb neurons; open arrows, unstained endothelial cells. Magnification = 4006. E–H, representative photomicro reacted with 0.2 microgram/ml control rabbit IgG (E) or rabbit anti-FIP200. F–G, representative micrograph PLoS ONE \| www.plosone.org 3 May Figure 1. FIP200 is expressed in tumor-associated endothelial cells as well as tumor cells in glioblastoma tumor biopsies. Formalin- fixed and paraffin-embedded sections of surgical biopsies of normal brain (n = 20) and glioblastomas (n = 21), as well as frozen sections of glioblastoma (n = 15) were subjected to immunohistochemical analysis using diaminobenzidene as the substrate and counterstaining with hematoxylin. The sections were reacted with a commercial rabbit anti-FIP200 (0.2 microgram/ml) or rabbit IgG (0.2 microgram/ml) for 24 h (4uC). A– D, representative photomicrographs of sections of normal brain reacted with control rabbit IgG (A) or rabbit anti-FIP200 (B–D). Open arrowheads, neurons; open arrows, unstained endothelial cells. Magnification = 4006. E–H, representative photomicrographs of glioblastoma tumor sections reacted with 0.2 microgram/ml control rabbit IgG (E) or rabbit anti-FIP200. F–G, representative micrographs of sections of glioblastoma tumors in Figure 1. FIP200 is expressed in tumor-associated endothelial cells as well as tumor cells in glioblastoma tumor biopsies. Formalin- fixed and paraffin-embedded sections of surgical biopsies of normal brain (n = 20) and glioblastomas (n = 21), as well as frozen sections of glioblastoma (n = 15) were subjected to immunohistochemical analysis using diaminobenzidene as the substrate and counterstaining with hematoxylin. The sections were reacted with a commercial rabbit anti-FIP200 (0.2 microgram/ml) or rabbit IgG (0.2 microgram/ml) for 24 h (4uC). A– D, representative photomicrographs of sections of normal brain reacted with control rabbit IgG (A) or rabbit anti-FIP200 (B–D). Open arrowheads, neurons; open arrows, unstained endothelial cells. FIP200 Downregulation Increases Pyk2 Activity, but not FAK activity, in Glioblastoma Cells, Immortalized Astrocytes, and Primary Human Brain MvEC Somewhat surprisingly, we found that FIP200 downregulation in U-87MG cells, E6/E7 immortalized astrocytes, and primary human brain MvEC propagated in complete media did not affect either the levels of FAK activity (as determined by the level of autophosphorylation at Y397) or the levels of FAK protein (Figures 4A & 4B). In contrast, downregulation of FIP200 enhanced the activity of Pyk2 in all three cell types although it did not enhance the levels of Pyk2 protein (Figures 4C and 5). The levels of autophosphorylation on Y402 relative to normalized Pyk2 protein were approximately 2-fold higher in the U-87MG cells and the primary human brain MvEC, and 4-fold higher in the E6/E7 cells Greater Amounts of FIP200 Protein are Co- Immunoprecipitated with Pyk2 than with FAK Immunoprecipitation of Pyk2 from lysates of U-87MG cells, resulted in co-immunoprecipitation of FIP200 (Figure 7A, lane 1) and downregulation of FIP200 by treatment of the cells with siFIP200 reduced the 200-kDa band detected in the Pyk2 immunoprecipitate by an estimated 70% (based on densitometry) (Figure 7A, lane 2). Immunoprecipitation of FAK from lysates of U-87MG cells did not result in detectable coprecipitation of FIP200 (Figure 7B) even after prolonged exposure of the autoradiographs (data not shown). Thus, there is an apparent preferential association of FIP200 with Pyk2 in these cells. Transfection of all three cell types with either siFIP200 #1 or #2 for 96 h induced caspase-7 cleavage (Figure 3A) with higher levels of caspase-7 cleavage occurring at day 7 (data not shown). Increased caspase-7 cleavage was observed in the primary and the immortalized human astrocytes on siFIP200 #1 or #2 treatment for 96 h. Similarly, transfection of all three cell types with siFIP200#1 or #2 for 96 h resulted in significantly higher numbers of TUNEL-positive cells than transfection with the mutant siFIP200 #1 or #2 RNAs (Figures 3B & 3C, and data not shown). In contrast, FIP200 co-immunoprecipitated with both Pyk2 and FAK from lysates of the primary human brain MvEC (Figure 7C, lane 1, and Figure 7D, lane 1, respectively), although lesser amounts of FIP200 were associated with FAK than Pyk2. Treatment of the cells with siFIP200 reduced the 200-kDa band associated with Pyk2 (Figure 7C, lane 2) and with FAK (Figure 7D, lane 2) by an estimated 70% and 60% respectively (based on densitometric analysis). The FIP200 downregulation increased Pyk2 activity (pPyk2(Y402) blot) (Figure 7C, lane 2), but not FAK activity (pFAK(Y397) blot) (Figure 7D, lane 2) in the MvEC used in this assay. Thus, there also is an apparent preferential association of FIP200 with Pyk2 in the MvEC. The levels of total Pyk2 protein and total FAK protein in the U-87MG and MvEC cell lysates are shown in Figure 7E. FIP200 Downregulation Inhibits Proliferation and Induces Apoptosis of Glioblastoma Cells, Immortalized Astrocytes, and Cultured Primary Human Brain MvEC Two duplex oligonucleotides (FIP200 siRNA #1 and #2) were used to determine the effects of downregulation of endogenous FIP200 protein on proliferation and apoptosis in the three cell types found in glioblastomas. The U-87MG cell line was selected for these studies as it expressed intermediate levels of FIP200 as compared to the other cell lines tested (Figure S2A), immortalized human astrocytes (E6/E7 cells) were used as a model for the tumor- associated glial cells [28], and cultured human brain microvessel endothelial cells were used as a model for the endothelial cells in the tumor-associated vessels [29,30]. All experiments were carried out using cells propagated in complete media. Transfection of all three cell types with either of the two duplex oligonucleotides used in the experiments effectively downregulated FIP200 protein as assessed by densitometric analysis of immunoblots (Table S1). Controls included a random scrambled siRNA or siFIP200 #1 and #2 duplex oligonucleotides with one or two base pair mutations (mutant siFIP200). FIP200 also has been shown to promote the activity of S6 kinase by associating with and inhibiting the function of the TSC1&2 complex of proteins [32]. To determine the effects of FIP200 downregulation on S6 kinase activity, the cell lysates were blotted for phospho-p70 S6 kinase (Thr389). We found a small reduction (<30% based on densitometry when normalized to the loading control cortactin) in S6 kinase activity on FIP200 downregulation in the U-87MG cells at 96 h (data not shown), suggesting that a reduction in S6 kinase activity may contribute in part to the decreased proliferation we observed on FIP200 downregulation. Treatment of the U-87MG cells with either siFIP200 #1 or siFIP200 #2 resulted in significant inhibition of proliferation as compared to treatment with the random scrambled siRNA (Figure 2A) or the mutant siFIP200 #1 and #2 RNAs (Figure 2B). There also was significant inhibition of proliferation when the cells were plated at a higher confluency prior to transfection with siFIP200 #1 or #2 for 7 days (data not shown). Downregulation of FIP200 by treatment of the immortalized human astrocytes (E6/E7 cells) (Figure 2C) and the cultured primary normal human brain endothelial cells (Figure 2D) also resulted in a significant reduction in proliferation. FIP200 Protein is Expressed in Tumor Cells, Glial Cells and the Endothelial Cells of Tumor-Associated Vessels in Glioblastoma Biopsies Magnification = 4006. E–H, representative photomicrographs of glioblastoma tumor sections reacted with 0.2 microgram/ml control rabbit IgG (E) or rabbit anti-FIP200. F–G, representative micrographs of sections of glioblastoma tumors in PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 3 FIP200 in Glioblastoma Tumors which tumor cells expressed FIP200 (28 of 36). Filled arrow-head, tumor cell expressing FIP200; filled arrows, endothelial cells expressing FIP200. Sections were viewed and photographed using a Leica DM4000 microscope. Magnification, 4006. doi:10 1371/journal pone 0019629 g001 which tumor cells expressed FIP200 (28 of 36). Filled arrow-head, tumor cell expressing FIP200; filled arrows, endothelial cells expressing FIP200. Sections were viewed and photographed using a Leica DM4000 microscope. Magnification, 4006. doi:10.1371/journal.pone.0019629.g001 which tumor cells expressed FIP200 (28 of 36). Filled arrow-head, tumor cell expressing FIP200; filled arrows, endothelial cells expressing FIP200. Sections were viewed and photographed using a Leica DM4000 microscope. Magnification, 4006. doi:10.1371/journal.pone.0019629.g001 FIP200 in these cells (Figure S2B). In the endothelial cells in the tumor-associated vessels in the glioblastoma tumor biopsies, the FIP200 staining was cytoplasmic. at 48h after knockdown of FIP200. At the same time-point, the levels of autophosphorylation were approximately 4-fold higher in U-87MG cells propagated in suspension (data not shown). at 48h after knockdown of FIP200. At the same time-point, the levels of autophosphorylation were approximately 4-fold higher in U-87MG cells propagated in suspension (data not shown). As the Pyk2 autophosphorylation site has been shown to be necessary for the pro-apoptotic effect of Pyk2 [11,18], we investigated whether a TAT-Pyk2 fusion protein containing the autophosphorylation site (abbreviated TAT-Pyk2-AP) [31] could block the pro-apoptotic effect of downregulation of FIP200. A TAT-Pyk2 fusion protein containing the Grb2 binding site (Y881) was used as a control [31]. The TAT-Pyk2-AP fusion protein (150 nM) significantly reduced the pro-apoptotic effect of downregulation of FIP200 in both the U-87MG glioblastoma cells (Figure 6A) and the primary human brain MvEC (Figure 6B). These data suggest that the apoptosis associated with the downregulation of FIP200 protein in the U-87MG cells and primary brain MvECs propagated in complete media most likely requires activation of Pyk2. PLoS ONE \| www.plosone.org Figure 2. Cell proliferation is inhibited in glioblastoma cells, immortalized astrocytes, and primary human brain MvECs on downregulation of FIP200. U-87MG human glioblastoma cells (A & B) and E6/E7 immortalized human astrocytes (C) were plated in complete media (16104 cells/well) in replicas of three or four (20 h) then treated with siFIP200 #1 or siFIP200 #2, or with non-silencing siRNA (denoted as liposome) or with mutant siFIP200 #1 or mutant siFIP200 #2 for the indicated time periods with the cell media and siRNA being replaced every three to four days. The cells were harvested and adherent cells counted. (D) Primary human brain MvEC, which are a surrogate model of tumor-associated MvECs and express FIP200, were plated in complete media (10% FBS, 10 ng/ml VEGF, 5 ng/ml bFGF) and treated with siFIP200 #1 or #2 or with mutant siFIP200 #1 or #2 and the adherent cells counted. The means 6 S.E.M were calculated and the data are plotted as the percentage of proliferation (cell number) relative to control cells. The experiment was repeated 26 and representative results are shown. (* probability = 0.03; ** probability #0.01 as compared to the control at that day; unpaired t test.) doi:10.1371/journal.pone.0019629.g002 on FIP200 downregulation is that pPyk2 is competing with FAK for Src family binding partners, but we have not ruled out other possible explanations. cells [11,13]. It has been demonstrated that Pyk2 activation is associated with a reduction in the amounts of cellular Src or Fyn associated with FAK in other cell types [11,13]. As we have shown previously that Fyn, Lyn and c-Src are the predominant Src family members that are expressed in glioblastoma tumors and in the U- 87MG cell line [33,34], we examined the effects of FIP200 downregulation on the association of these molecules with FAK. We found that FIP200 downregulation reduced the amount of Fyn associated with FAK in the U-87MG cells (60% decrease by normalized densitometry) (Figure 7F), but did not reduce the association of Lyn or c-Src with FAK (data not shown). Similarly, in the human brain MvEC, we found a reduction in the amount of Fyn (25% decrease) associated with FAK on FIP200 downregu- lation, and no decrease in Lyn or c-Src association with FAK (data not shown). Thus, one possible explanation for the observed effects A Reduction in the Association of Fyn with FAK Occurs on FIP200 Downregulation Activated Pyk2 may compete with FAK for binding partners and thereby decrease the FAK-promoted pro-survival signal in PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 4 FIP200 in Glioblastoma Tumors Figure 2. Cell proliferation is inhibited in glioblastoma cells, immortalized astrocytes, and primary human brain MvECs on downregulation of FIP200. U-87MG human glioblastoma cells (A & B) and E6/E7 immortalized human astrocytes (C) were plated in complete media (16104 cells/well) in replicas of three or four (20 h) then treated with siFIP200 #1 or siFIP200 #2, or with non-silencing siRNA (denoted as liposome) or with mutant siFIP200 #1 or mutant siFIP200 #2 for the indicated time periods with the cell media and siRNA being replaced every three to four days. The cells were harvested and adherent cells counted. (D) Primary human brain MvEC, which are a surrogate model of tumor-associated MvECs and express FIP200, were plated in complete media (10% FBS, 10 ng/ml VEGF, 5 ng/ml bFGF) and treated with siFIP200 #1 or #2 or with mutant siFIP200 #1 or #2 and the adherent cells counted. The means 6 S.E.M were calculated and the data are plotted as the percentage of proliferation (cell number) relative to control cells. The experiment was repeated 26 and representative results are shown. (* probability = 0.03; ** probability #0.01 as compared to the control at that day; unpaired t test.) doi:10.1371/journal.pone.0019629.g002 FIP200 and Pyk2 Localization is Largely Cytoplasmic and Diffuse On analysis of the cellular distribution of FIP200 using double- label immunofluorescence, we found that the expression was largely cytoplasmic and diffuse in the U-87MG cells and primary human brain MvEC (Figures S3A & S3C, arrows) as reported previously in other cell types [13]. We did not observe localization of FIP200 to focal adhesions. Similar to FIP200, we found that total Pyk2 localization was cytoplasmic and diffuse (Figures S3A– D), independent of FIP200 downregulation and consistent with that reported previously in fibroblasts and PC12 cells [13,35]. PLoS ONE \| www.plosone.org P PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 5 FIP200 in Glioblastoma Tumors FIP200 in Glioblastoma Tumors Figure 3. Apoptotic cell death is induced in glioblastoma cells, astrocytes, and primary h FIP200. A, Human astrocytes, U-87MG glioblastoma cells and human brain MvEC (HBMvEC) were p with FIP200 siRNA, a non-silencing control siRNA (control #1), or mutant siFIP200 (control #2) for 96 reduced 12% SDS-PAGE and blotted with the indicated antibodies as described in the Materials and the three different cell types and a representative experiment is shown. B & C, U-87MG and E6/E replicas of two, treated with siFIP200 or mutant siFIP200 as described for Panel A for 96 h and the cells were counted in 10 fields per well in a blinded manner and the data analyzed and presented a seen with HBMvEC plated and treated as above with siFIP200 and then subjected to TUNEL assay a representative result is shown. doi:10.1371/journal.pone.0019629.g003 Figure 3. Apoptotic cell death is induced in glioblastoma cells, astrocytes, and primary human brain MvECs on downregulation of FIP200. A, Human astrocytes, U-87MG glioblastoma cells and human brain MvEC (HBMvEC) were plated in complete media (20 h) and then treated with FIP200 siRNA, a non-silencing control siRNA (control #1), or mutant siFIP200 (control #2) for 96 h. Whole cell lysates were resolved on disulfide- reduced 12% SDS-PAGE and blotted with the indicated antibodies as described in the Materials and Methods. The experiment was repeated 26with the three different cell types and a representative experiment is shown. B & C, U-87MG and E6/E7 E6/E7 immortalized astrocytes were plated in replicas of two, treated with siFIP200 or mutant siFIP200 as described for Panel A for 96 h and then subjected to the TUNEL assay. FIP200 and Pyk2 Localization is Largely Cytoplasmic and Diffuse TUNEL-positive cells were counted in 10 fields per well in a blinded manner and the data analyzed and presented as the mean 6 S.E.M. Highly similar results were seen with HBMvEC plated and treated as above with siFIP200 and then subjected to TUNEL assay at 96 h. The experiment was repeated 26and a representative result is shown. doi:10.1371/journal.pone.0019629.g003 Figure 3. Apoptotic cell death is induced in glioblastoma cells, astrocytes, and primary human brain MvECs on downregulation of FIP200. A, Human astrocytes, U-87MG glioblastoma cells and human brain MvEC (HBMvEC) were plated in complete media (20 h) and then treated with FIP200 siRNA, a non-silencing control siRNA (control #1), or mutant siFIP200 (control #2) for 96 h. Whole cell lysates were resolved on disulfide- reduced 12% SDS-PAGE and blotted with the indicated antibodies as described in the Materials and Methods. The experiment was repeated 26with the three different cell types and a representative experiment is shown. B & C, U-87MG and E6/E7 E6/E7 immortalized astrocytes were plated in replicas of two, treated with siFIP200 or mutant siFIP200 as described for Panel A for 96 h and then subjected to the TUNEL assay. TUNEL-positive cells were counted in 10 fields per well in a blinded manner and the data analyzed and presented as the mean 6 S.E.M. Highly similar results were seen with HBMvEC plated and treated as above with siFIP200 and then subjected to TUNEL assay at 96 h. The experiment was repeated 26and a representative result is shown. doi:10.1371/journal.pone.0019629.g003 Analysis of the cellular localization of these proteins in four frozen glioblastoma biopsy samples by triple-label immunofluo- rescence indicated cytoplasmic co-expression of FIP200 and Pyk2 in endothelial cells in tumor-associated blood vessels identified by FIP200 downregulation did not appear to affect the localization of Pyk2. As expected, FAK protein largely co-localized with vinculin to focal adhesions in both cell types, independent of FIP200 downregulation (data not shown). PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 PLoS ONE \| www.plosone.org 6 FIP200 in Glioblastoma Tumors Figure 4. Downregulation of FIP200 protein with siRNA enhances Pyk2 activity without affecting FAK activity and E6/E7 immortalized astrocytes. FIP200 and Pyk2 Localization is Largely Cytoplasmic and Diffuse A–C, Primary human brain MvEC were plated in complete media (10% FSB) and treated with siFIP200 #1, #2, or #4, or with control liposomes or mutant siFIP200 #1 for 96 h, detergent lysed, the lysate resolved on disulfide-reduced 7.5% SDS- PAGE and blotted with the indicated antibodies. In Panel C, the densitometric ratio of pPyk2/normalized to Pyk2 was set at 1.0 for the cells treated with mutant siFIP200, and the ratio for the cells treated with specific siFIP200 expressed relative to the ratio for the cells treated with mutant siFIP200. The experiment was repeated and representative blots are shown. d i 10 1371/j l 0019629 005 survival. Collectively, our results indicate that FIP200 regulation of Pyk2 can play an important role in the survival of glioblastoma tumor cells and suggests the intriguing possibility that this mechanism may contribute to sustained tumor growth by promoting not only the survival of tumor cells but also the intra- tumor glial cells and the tumor-associated endothelial cells. doi:10.1371/journal.pone.0019629.g005 These results underscore the importance of FIP200 in the regulation of cell survival in that they demonstrate that downregulation of FIP200 can induce apoptosis. The potential role of pPyk2 in promoting the apoptosis induced by FIP200 downregulation was indicated by the ability of a TAT-Pyk2 fusion protein containing the autophosphorylation site to inhibit the pro- apoptotic effect, whereas a TAT-Pyk2 fusion protein containing the potential Grb2 binding site (Y881) did not. This is consistent with the prior report that the N-terminus and the kinase domains of Pyk2, but not the C-terminus, are necessary for the pro- apoptotic effect of Pyk2 [11]. Several other mechanisms have been implicated in FIP200 regulation of apoptosis in other cell types. In the FIP200-null mouse embryos, no changes in Pyk2 activity were detected in the hepatocytes or cardiomyocytes and the enhanced apoptosis was attributed to a decrease in S6 kinase activity [5]. The TSC1&2 complex is thought to negatively regulate cell proliferation through S6 kinase, mTOR, and 4E-BP1 [5,32]. On analysis of the p70 S6 kinase activity in the glioblastoma cells we did find that there was a slight reduction (an estimated 30%) in this vWf expression (Figure 8A1&2), suggesting that FIP200 and Pyk2 are localized in a manner that is consistent with their potential association. FIP200 and Pyk2 Localization is Largely Cytoplasmic and Diffuse A–C, Primary human brain MvEC were plated in complete media (10% FSB) and treated with siFIP200 #1, #2, or #4, or with control liposomes or mutant siFIP200 #1 for 96 h, detergent lysed, the lysate resolved on disulfide-reduced 7.5% SDS- PAGE and blotted with the indicated antibodies. In Panel C, the densitometric ratio of pPyk2/normalized to Pyk2 was set at 1.0 for the cells treated with mutant siFIP200, and the ratio for the cells treated with specific siFIP200 expressed relative to the ratio for the cells treated with mutant siFIP200. The experiment was repeated and representative blots are shown. doi:10.1371/journal.pone.0019629.g005 Figure 5. Downregulation of FIP200 protein with siRNA enhances Pyk2 activity without affecting FAK activity in primary human brain MvEC. A–C, Primary human brain MvEC were plated in complete media (10% FSB) and treated with siFIP200 #1, #2, or #4, or with control liposomes or mutant siFIP200 #1 for 96 h, detergent lysed, the lysate resolved on disulfide-reduced 7.5% SDS- PAGE and blotted with the indicated antibodies. In Panel C, the densitometric ratio of pPyk2/normalized to Pyk2 was set at 1.0 for the cells treated with mutant siFIP200, and the ratio for the cells treated with specific siFIP200 expressed relative to the ratio for the cells treated with mutant siFIP200. The experiment was repeated and representative blots are shown. doi:10.1371/journal.pone.0019629.g005 Figure 6. A TAT-Pyk2 fusion protein containing the autopho- sphorylation site inhibits the pro-apoptotic effect observed with the downregulation of FIP200-protein. (A) U-87MG glioblas- toma cells plated in complete media (10% FBS) (20 h) were treated with the indicated siRNA for 48 h, the media removed and the cell monolayer treated with 150 nM TAT-Pyk2 fusion protein containing the autophosphorylation site (AP) or the potential Grb2 binding site (Grb2) for 1 h, followed by fresh media and siRNA for 38 h, detergent lysis, and immunoblotting with the indicated antibodies. B, Human brain MvEC (HBMvEC) were plated in complete media (20 h), treated with the indicated siRNA (50 h), the media removed and the cell monolayer treated with 75 nM TAT-Pyk2-fusion protein (1 h), followed by fresh media and siRNA for 36 h, detergent lysis and immunoblotting with the indicated antibodies. The experiment was repeated and representative blots are shown. doi:10.1371/journal.pone.0019629.g006 Figure 5. Downregulation of FIP200 protein with siRNA enhances Pyk2 activity without affecting FAK activity in primary human brain MvEC. FIP200 and Pyk2 Localization is Largely Cytoplasmic and Diffuse In the tumor cell compartment of all samples, we found cytoplasmic co-expression of FIP200 and Pyk2 in some tumor cells (Figure 8B), also suggesting that FIP200 and Pyk2 are localized in a manner that is consistent with potential association. Nuclear localization of FIP200 also was observed in some tumor cells (data not shown). The cytoplasmic localization of Pyk2 in the tumor-associated endothelial cells and in the tumor cells in frozen glioblastoma biopsies is consistent with the report of another group that examined Pyk2 expression in glioblastoma biopsies [36]. FIP200 and Pyk2 Localization is Largely Cytoplasmic and Diffuse A, B, U-87MG glioblastoma cells and immortalized human astrocytes were plated in FBS), treated with 20 nM FIP200 siRNA (#1, or #2) or control non-silencing siRNA for the indicated times, detergent lysed lysate resolved on disulfide-reduced 6% SDS-PAGE, and then blotted with the indicated antibodies. C, U-87MG glioblastoma human astrocytes were plated in complete media (10% FBS), and treated with different concentrations of FIP200 siRNA (#1 silencing siRNA for 53 h, detergent lysed, equivalent amount of lysate resolved on disulfide-reduced 6% SDS-PAGE, and indicated antibodies. In Panel C all lanes are from the same gel and the densitometric ratio of pPyk2/normalized Pyk2 was treated with control non-silencing siRNA, and the ratio for the cells treated with siFIP200 expressed relative to the ratio fo control non-silencing siRNA. The experiments were repeated 26 and representative blots are shown. doi:10.1371/journal.pone.0019629.g004 Figure 4. Downregulation of FIP200 protein with siRNA enhances Pyk2 activity without affecting FAK activity in gliobastoma cells and E6/E7 immortalized astrocytes. A, B, U-87MG glioblastoma cells and immortalized human astrocytes were plated in complete media (10% FBS), treated with 20 nM FIP200 siRNA (#1, or #2) or control non-silencing siRNA for the indicated times, detergent lysed, equivalent amount of lysate resolved on disulfide-reduced 6% SDS-PAGE, and then blotted with the indicated antibodies. C, U-87MG glioblastoma cells and immortalized human astrocytes were plated in complete media (10% FBS), and treated with different concentrations of FIP200 siRNA (#1, or #2) or control non- silencing siRNA for 53 h, detergent lysed, equivalent amount of lysate resolved on disulfide-reduced 6% SDS-PAGE, and then blotted with the indicated antibodies. In Panel C all lanes are from the same gel and the densitometric ratio of pPyk2/normalized Pyk2 was set at 1.0 for the cells treated with control non-silencing siRNA, and the ratio for the cells treated with siFIP200 expressed relative to the ratio for the cells treated with control non-silencing siRNA. The experiments were repeated 26 and representative blots are shown. doi:10.1371/journal.pone.0019629.g004 May 2011 \| Volume 6 \| Issue 5 \| e19629 PLoS ONE \| www.plosone.org 7 FIP200 in Glioblastoma Tumors Figure 5. Downregulation of FIP200 protein with siRNA enhances Pyk2 activity without affecting FAK activity in primary human brain MvEC. Discussion Our demonstration that specific downregulation of endogenous FIP200 protein can increase Pyk2 activity and induce apoptosis adds to the body of evidence that FIP200 can regulate Pyk2 and underscores the importance of FIP200 in the regulation of cell PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 8 FIP200 in Glioblastoma Tumors Figure 7. FIP200 protein predominantly associates with Pyk2 in the glioblastoma cells and primary human brain MvEC. A–D, U-87MG glioblastoma cells (A&B) and primary human brain MvEC (C&D) were plated in complete media for 20 h, treated with siFIP200 or mutant siFIP200 for 96 h, detergent lysed, and immunoprecipitated with the indicated antibody. The immunoprecipitates were subjected to 7.5% disulfide-reduced SDS- PAGE and immunoblotted with the indicated antibodies, as described in the Materials and Methods. Panels A & B are from the same gel. (E) U-87MG cells and human brain MvEC plated in complete media were detergent lysed, subjected to 8% SDS PAGE, and immunoblotted with the indicated antibodies. (F) U-87MG cells and human brain MvEC plated as above and treated with the indicated siRNA for 72 h were detergent lysed, immunoprecipitated with anti-FAK antibody, the immunoprecipitate subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. The experiment was repeated 26 and representative blots are shown. doi:10.1371/journal.pone.0019629.g007 FIP200 in Glioblastoma Tumors Figure 7. FIP200 protein predominantly associates with Pyk2 in the glioblastoma cells and primary human brain MvEC. A–D, U-87MG glioblastoma cells (A&B) and primary human brain MvEC (C&D) were plated in complete media for 20 h, treated with siFIP200 or mutant siFIP200 for 96 h, detergent lysed, and immunoprecipitated with the indicated antibody. The immunoprecipitates were subjected to 7.5% disulfide-reduced SDS- PAGE and immunoblotted with the indicated antibodies, as described in the Materials and Methods. Panels A & B are from the same gel. (E) U-87MG cells and human brain MvEC plated in complete media were detergent lysed, subjected to 8% SDS PAGE, and immunoblotted with the indicated antibodies. (F) U-87MG cells and human brain MvEC plated as above and treated with the indicated siRNA for 72 h were detergent lysed, immunoprecipitated with anti-FAK antibody, the immunoprecipitate subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. The experiment was repeated 26 and representative blots are shown. doi:10.1371/journal.pone.0019629.g007 activity on downregulation of FIP200. Discussion This small decrease in S6 kinase activity could contribute to the inhibition of proliferation we observed on FIP200 downregulation but the current results suggest that the mechanism by which FIP200 regulates apoptosis in glioblastoma tumor cells and brain MvECs differs from that in the embryonic murine cardiocytes and hepatocytes in that it requires Pyk2 activity. It also has been reported that FIP200 upregulates the expression of the retinoblastoma (Rb) gene [26,37], thus the downregulation of endogenous FIP200 protein in our cells could potentially inhibit cell proliferation by decreasing the Rb gene expression. Although the association of FAK with the death domain kinase receptor interacting protein (RIP) is thought to prevent the pro-apoptotic signal of RIP [38], we could not detect an association of FAK with RIP in the U-87MG cells by co- immunoprecipitation (D. Wang and C.L. Gladson unpublished observation). Although both Pyk2 and FAK were expressed in the glio- blastoma cells and human brain MvEC, we found that FIP200 May 2011 \| Volume 6 \| Issue 5 \| e19629 May 2011 \| Volume 6 \| Issue 5 \| e19629 PLoS ONE \| www.plosone.org 9 FIP200 in Glioblastoma Tumors Figure 8. Co-Expression of FIP200 and Pyk2 in the cytoplasm of tumor cells and of tumor-associated endothelial cells. Frozen sections of four glioblastoma tumors were reacted with antibodies directed toward vWf (pink), FIP200 (green) and Pyk2 (red), followed by reaction with Alexa 633-, Alexa 563- and Alexa 488-conjugated secondary antibodies, and DAPI staining, as described in the Materials and Methods. Images were viewed and photographed on a Leica DMB microscope at 4006 magnification. A1 & A2, triple-labeled tumor-associated endothelial cells; B, double-labeled tumor cells; and C, secondary antibodies alone followed by DAPI staining. Merged images are shown at the reader’s far right. doi:10.1371/journal.pone.0019629.g008 Figure 8. Co-Expression of FIP200 and Pyk2 in the cytoplasm of tumor cells and of tumor-associated endothelial cells. Frozen sections of four glioblastoma tumors were reacted with antibodies directed toward vWf (pink), FIP200 (green) and Pyk2 (red), followed by reaction with Alexa 633-, Alexa 563- and Alexa 488-conjugated secondary antibodies, and DAPI staining, as described in the Materials and Methods. Images were viewed and photographed on a Leica DMB microscope at 4006 magnification. A1 & A2, triple-labeled tumor-associated endothelial cells; B, double-labeled tumor cells; and C, secondary antibodies alone followed by DAPI staining. Merged images are shown at the reader’s far right. doi:10.1371/journal.pone.0019629.g008 Figure 8. Discussion Co-Expression of FIP200 and Pyk2 in the cytoplasm of tumor cells and of tumor-associated endothelial cells. Frozen sections of four glioblastoma tumors were reacted with antibodies directed toward vWf (pink), FIP200 (green) and Pyk2 (red), followed by reaction with Alexa 633-, Alexa 563- and Alexa 488-conjugated secondary antibodies, and DAPI staining, as described in the Materials and Methods. Images were viewed and photographed on a Leica DMB microscope at 4006 magnification. A1 & A2, triple-labeled tumor-associated endothelial cells; B, double-labeled tumor cells; and C, secondary antibodies alone followed by DAPI staining. Merged images are shown at the reader’s far right. doi:10.1371/journal.pone.0019629.g008 consistent with the preferential co-precipitation of FIP200 with Pyk2 we observed, whereas FAK was largely localized to the focal adhesions and FIP200 was not detectable at these sites. The regulation of localization of FAK and Pyk2 to focal adhesions can be independently influenced by the presence or absence of specific binding partners, for example, gelsolin has been reported to bind Pyk2 but not FAK [40] as well as differences in the affinity of FAK and Pyk2 for shared binding partners within the focal adhesions, such as paxillin [41–43]. FIP200 localization to the nucleus has been reported in other cell types [26], emphasizing a potential role for FIP200 in regulation of transcription through, for example, its potential interaction with Rb, which is a regulator of transcription. We did not observe a clear-cut association of FIP200 with the nuclei of the U-87MG or primary human MvECs, but we did not utilize biochemical assays to explore this possibility and cannot rule out a partial nuclear localization. However, the pattern of expression of FIP200 in the tumor cells in the glioblastoma biopsies was heterogeneous. Although the pattern was predomi- nantly cytoplasmic, nuclear staining was observed and partial FIP200 localization to the nucleus was observed in tumor cells in 10 of 36 biopsy samples. In contrast, in the tumor-associated preferentially co-immunoprecipitated with Pyk2 in the absence of FIP200 downregulation in the unmanipulated U-87MG glioblas- toma and brain MvEC cells propagated in complete media. The association of the endogenous FIP200 with Pyk2 in the lysates of the unmanipulated glioblastoma cells and human brain MvEC propagated in complete media suggests that the FIP200 regulation of Pyk2 activity in these cells may be a direct effect. PLoS ONE \| www.plosone.org FIP200 in Glioblastoma Tumors endothelial cells of glioblastoma biopsies, the distribution of FIP200 was largely cytoplasmic with only rare nuclear staining. (Upstate Biotechnology), mAb anti-glyceraldehyde-3-phosphate- dehydrogenase (GAPDH) (Fitzgerald Laboratories), mouse anti-a- tubulin (GeneTex, DM1A) and rabbit antibodies toward Fyn, Lyn and c-Src (Santa Cruz Biotechnology) also were purchased. Control normal rabbit IgG and normal IgG were purchased from Santa Cruz Biotechnology. We observed a reduced association of Fyn with FAK on FIP200 downregulation in both U-87MG cells and the brain MvEC although the levels of activity of FAK and its expression were unaffected by the downregulation. This supports the concept that activated Pyk2 competes with FAK for binding partners that affect downstream signaling pathways, as shown in other cell types by Zhao and colleagues [13]. Activated Pyk2 can signal the activation of p38MAP kinase [20], a MAP kinase that can promote apoptosis [44], and Pyk2 induction of apoptosis in cardiomyocytes requires Src activation of p38MAP kinase [18]. FAK can promote cell proliferation and cell survival through the activation of other MAP kinase(s) in a cell-type and experimental context-dependent manner reviewed in [3,45]. Immunohistochemistry Paraffin and frozen sections (7 microns thick) were stained as described previously [34] with the Avidin/Biotin Kit (Vector Laboratories) as recommended. Rabbit anti-FIP200 was utilized at 0.2 mg/ml for paraffin sections and 1.13 mg/ml for frozen sections as described previously [10], and the following antibodies mAb anti-Pyk2, rabbit anti-vWf, goat anti-vWf, and the negative control IgG were utilized at a 1:100 dilution of the stock. Sections were viewed and photographed using a Leica DM4000B microscope. Cell culture U-87MG human glioblastoma cells were obtained from the ATCC and propagated as described [33]. Human astrocytes immortalized with the telomerase gene and the E6 and E7 viral genes have been characterized previously and were propagated as described [28], and primary human astrocytes were purchased from Cell Systems (Kirkland, WA) and propagated as recom- mended. Highly similar results were found on FIP200 downreg- ulation in both the immortalized and the primary human astrocytes. SNB19 human glioblastoma cells were cultured as described previously [46], and were a kind gift from Dr. Jasti Rao (University of Illinois, Peoria, IL). The 827 and 905 human glioblastoma stem cells were cultured as described previously [47]. Primary human brain MvEC were obtained from Cell Systems and propagated as directed [29,30]. It should be emphasized that there was heterogeneity in the levels of expression of FIP200 in the tumor cells and that FIP200 was not detectable in all of the tumor cells within a single biopsy specimen. In addition, we did not detect FIP200 protein in tumor cells in 8 of 36 of the glioblastoma biopsies, although FIP200 was detected in scattered glial cells and the endothelial cells in the tumor-associated vessels in 3 of these 8 tumors. Currently, it is not known whether the failure to detect FIP200 protein in tumor cells in eight of the glioblastoma biopsy samples is due to a gene deletion in these cells or an environmentally triggered reduction in the levels of FIP200 mRNA or protein. Human tissues Frozen glioblastoma and anaplastic astrocytoma tumor tissue, paraffin sections of glioblastoma tumor, and normal brain tissue (tissue adjacent to seizure foci resected to gain access to the seizure foci), were obtained from surgical samples with the approval of the Institutional Review Board of the University of Alabama at Birmingham (IRB number: X050415007) or the Cleveland Clinic (IRB number: 2559). All tissue banked at the University of Alabama at Birmingham and the Cleveland Clinic was with the written consent from the patient. De-identified tissue was provided by the tissue bank at the University of Alabama at Birmingham or the Cleveland Clinic. Glioblastoma tumor (Grade IV of IV) and anaplastic astrocytoma tumor (Grade III of IV) were diagnosed according to the most recent World Health Organization Classification of Tumors of the Central Nervous System [48]. Frozen tumor and normal brain tissues were lysed in RIPA lysis buffer with protease inhibitors as described [34]. p The location of the FIP200 gene on chromosome 8q11 [6,8,9] has led other investigators to formulate the hypothesis that FIP200 could be an ‘‘anti-tumor’’ or ‘‘tumor suppressor’’ gene. Recently, however, Wei and colleagues [23] created a conditional knockout of FIP200 in the MMTV-Cre model of breast cancer and showed no promotion of tumorigenesis, suggesting that FIP200 inactiva- tion alone does not promote tumor formation. In addition, a prior report demonstrated that overexpression of FIP200 in breast cancer cells resulted in an inhibition of both cell cycle progression and clonogenic cell survival due to FIP200 promotion of p21 expression [7]. It is possible that the level of FIP200 protein in cells, and the cell type, influences its function. For example, the kinetics of FIP200 association with some of its binding partners, such as Pyk2, FAK or the p21 or Rb promoter, could be altered when FIP200 is overexpressed and this could be affected by the experimental context and the specific cell-type. Taken together with the expression of FIP200 in the tumor-associated endothelial cells in the glioblastoma tumors but not the normal brain, these data underscore the importance of elucidation of the factors that regulate the expression of FIP200. Reagents g The following antibodies were purchased, rabbit anti-FAK IgG (Upstate Biotechnology, Cat# 06-543); mouse mAb anti-FAK (Santa Cruz Biotechnology; clone # H-1); rabbit anti-phospho- FAK (pY397) (Chemicon, Inc.); mouse mAb anti-Pyk2 (BD Biosciences-Transduction Laboratories; clone 11); rabbit anti Pyk2 (Santa Cruz, #SC9019), and rabbit anti-phospho-Pyk2 (pY402) (BioSource International and Upstate Biotechnology). Two rabbit anti-FIP200 antibodies were used. One was purchased from (ProteinTech, Chicago, IL) and the other, a rabbit anti- FIP200 antibody directed toward the C-terminus, has been described previously [2]. Mouse mAb anti-death domain kinase receptor interacting protein (RIP) (BD Transduction Laborato- ries); rabbit anti-cleaved caspases 7 (Calbiochem); mAb anti-actin (Sigma), rabbit monoclonal anti-phospho-p70 S6 kinase (Thr389) Discussion The interaction of FIP200 with Pyk2 has been shown to inhibit Pyk2 activity in other cell types [2] and Pyk2 activation has been shown to promote its dissociation from FIP200. Thus, it is possible that downregulation of the endogenous FIP200 may lead to exposure of the autophosphorylation site permitting autophosphorylation of Pyk2 [2,13,39]. The activities of Pyk2 and FAK and their contribution to specific cell functions, including apoptosis, are regulated in large part by their shuttling between different compartments of the cell. The largely cytoplasmic and diffuse pattern of FIP200 that we observed on immunofluorescence analysis of both the U-87MG cells and the primary human brain MvECs is consistent with the pattern reported for fibroblasts [2,13]. The distribution of Pyk2 also was predominantly cytoplasmic and diffuse, which is May 2011 \| Volume 6 \| Issue 5 \| e19629 10 FIP200 in Glioblastoma Tumors (TIF) Table S1 FIP200 protein expression in cells treated with siFIP200. The cells were propagated in complete media and treated with siFIP200 #1 or #2 at 20 nM, empty liposomes or mutant siFIP200 as described in the Materials and Methods section for the indicated time periods. The cells were then harvested and FIP200 protein expression evaluated by western blotting. Densitometric readings of the FIP200 band on the blots were normalized to the loading control protein (actin, GAPDH or Immunoprecipitation U-87MG cells or human brain MvEC were plated in complete media at 80% confluence for 20 h, followed by siRNA treatment for the indicated number of days. The cells were then lysed using 1% NP40 Lysis Buffer with protease inhibitors as described [33]. For immunoprecipitation analysis equivalent amount of protein lysate (50 to 200 mg) from each sample was incubated with 5– 10 mg of the appropriate antibody followed by pull-down using TrueBlot anti-rabbit IP beads (eBiosciences) overnight at 4uC. The immunoprecipitates were washed 3 times with PTO buffer (PBS with 0.1% ovalbumin and 0.5% Tween 20) and 3 times with PT buffer (PBS with 0.5% Tween 20) and subjected to 7.5% disulfide- reduced SDS-PAGE, transferred to PVDF membrane and immunoblotted as described [33]. Immunoblot analysis Immunoblotting was carried out as described previously [17]. The cells were lysed in the Cell Lytic-M reagent lysis buffer (Sigma) or with 1% NP40 buffer with protease inhibitors [17]. Immunoblotting after lysis of the cells in RIPA lysis buffer yielded similar results except that the blots were not as clean. The rabbit anti-FIP200 antibody directed toward the C-terminus [2] was used at a 1:1000 dilution and the commercial anti-FIP200 at 0.2 microgram/ml. Band intensities were measured by densitom- etry and normalized to the intensity of the loading control, i.e., antibody directed to actin, a-tubulin or cortactin. FAK activity was detected by blotting using an antibody specific for pFAK(Y397), and then stripping and reprobing for total FAK and a loading control. Pyk2 activity was detected by blotting using an antibody specific for pPyk2(Y402), then stripping and reprobing for total Pyk2 and a loading control. The activity of FAK and Pyk2 was determined by normalizing the intensity of the pFAK(Y397) and pPyk2(Y402) bands to total FAK or Pyk2 bands, respectively. Supporting Information Figure S1 FIP200 expression in glioblastoma tumor and normal brain biopsies. Tissues were detergent lysed and 200 micrograms of each sample electrophoresed on an 6% disulfide-reduced SDS-PAGE, transferred to Immobilon mem- brane, and then immunoblotted with the indicated antibodies, as described in the Materials and Methods. The densitometirc reading for the FIP200 band in each sample was normalized to the actin band that served as a loading control. (TIF) Figure S2 FIP200 expression in glioblastoma cells and primary brain microvessel endothelial cells (MvEC). U- 87MG and SNB19 human glioblastoma cells plated as a monolayer in complete media (A), 905 and 827 human glioblastoma stem cells propagated as neurospheres in serum-free media (A), as well as primary human brain MvEC plated as a monolayer in complete media (B), were detergent lysed, equivalent amount of lysate subjected to 7.5% SDS-PAGE, and the gels immunoblotted with the indicated antibodies as described in the Materials and Methods. The experiment was repeated and representative blots are shown. (TIF) Figure S2 FIP200 expression in glioblastoma cells and primary brain microvessel endothelial cells (MvEC). U- 87MG and SNB19 human glioblastoma cells plated as a monolayer in complete media (A), 905 and 827 human glioblastoma stem cells propagated as neurospheres in serum-free media (A), as well as primary human brain MvEC plated as a monolayer in complete media (B), were detergent lysed, equivalent amount of lysate subjected to 7.5% SDS-PAGE, and the gels immunoblotted with the indicated antibodies as described in the Materials and Methods. The experiment was repeated and representative blots are shown. (TIF) Cell proliferation and apoptosis assays Immortalized astrocytes, U-87MG cells, and human brain MvEC were plated on 12- or 24-well plates in complete media (10% FBS) and then treated with siRNA for the indicated times; the medium was changed and fresh siRNA added every three or four days. To assess proliferation, adherent cells were harvested with trypsin/IEDTA, stained with trypan blue and viable cells counted using a hematocytometer. The TUNEL apoptosis assay was performed as described previously [29,30]. Figure S3 FIP200 and Pyk2 are localized in a cytoplas- mic, diffuse manner in glioblastoma cells and human brain MvEC. U-87MG glioblastoma cells or primary human brain MvEC were plated onto chamberslides in complete media (10% FBS) or in complete media with 10 ng/ml VEGF, and 5 ng/ ml bFGF, respectively, for 20 h, treated with siFIP200 or mutant siFIP200 (labeled control siRNA) for 48 h, fixed, and subjected to double-label immunofluorescence, as described in the Materials and Methods. MAb anti-vinculin was detected with Alexa 488- conjugated anti-mouse antibody (green fluorescence), and rabbit anti-FIP200, and anti-total Pyk2 antibodies were detected with anti-rabbit Alexa 594-conjugated antibody (red fluorescence). Merged images are shown. Arrows denote a diffuse cytoplasmic localization (FIP200 and Pyk2), and arrowheads denote a localization at focal adhesions (vinculin). Cells were viewed and photographed using a Leica DMR microscope. All panels, magnification 4006. The experiment was repeated and represen- tative photomicrographs are shown. (TIF) Downregulation of FIP200 with siRNA Downregulation of FIP200 with siRNA Three validated duplex siRNAs for FIP200 were purchased from Qiagen (Valencia, CA): siFIP200 #1 (Cat. # SI02664571), target sequence CTGGGACGCATACAAATCCAA; siFIP200#2 (Cat #SI02664578), target sequence ACGCAAATCAGTTGAT- GATTA; and siFIP200 #4 (Cat #SI00108122). To further substantiate specific downregulation of FIP200, siFIP200 #1 and #2 duplex oligonucleotides with one or two base pair mutations (mutant siFIP200) were purchased from Qiagen and used as controls. The target of the mutated siFIP200#1 is CTGGGACG- GATACAAATCCAA; and the target of mutated siFIP200 #2, ACGCAAGTCCGTTGATGATTA. Control non-silencing PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 11 FIP200 in Glioblastoma Tumors siRNA also was purchased from Qiagen. Transient transfection of siRNA was carried out using the HiPerfect transfection reagent (Qiagen), according to the manufacturer’s instructions, with empty liposomes being used as a negative control. The efficiency of the downregulation of FIP200 in the different cell types was confirmed and monitored by western blot analysis. No effects on the morphology of the cells were observed. 365–518 and containing the autophosphorylation site (abbreviated AP), nt 1741–2099, coding for amino acids 581–700 and containing the putative phosphatidyl inositol 39 phosphate (PI3) kinase binding domain (abbreviated PBM); and nt 2617–2986, coding for amino acids 873–995 and containing the Grb2 binding site (abbreviated Grb2BS) [31]. The constructs were grown in BL21 DE3 E. coli, and the recombinant proteins purified as described [31]. TAT fusion proteins were used for these studies as they have been shown to transduce nearly 100% of mammalian cells regardless of cell type [50,51]. Both the TAT-Pyk2 fusion proteins are well characterized and do not exhibit toxicity in various in vitro and in vivo models [31,51]. Immunofluorescence analysis Cells were plated in complete media (10% FBS) and treated with siRNA for the indicated times. Frozen tissue was sectioned at 7 microns. Immunofluorescence was performed as described previously [49]. A Leica DMF microscope was used for viewing and photomicrographs were obtained using a 406 oil objective. References (2002) Identification of RB1CC1, a novel human gene that can induce RB1 in various human cells. Oncogene 21: 1295–8. 30. Rege TA, Stewart J, Jr., Dranka B, Benveniste EN, Silverstein RL, et al. (2009) Thrombospondin-1-induced apoptosis of brain microvascular endothelial cells can be mediated by TNF-R1. J Cell Physiol 218: 94–103. 9. Chano T, Kontani K, Teramoto K, Okabe H, Ikegawa S (2002) Truncating mutations of RB1CC1 in human breast cancer. Nat Genet 31: 285–8. 31. Han H, Fuortes M, Nathan C (2003) Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation. J Exp Med 197: 63–75. 10. Liang CC, Wang C, Peng X, Gan B, Guan JL (2010) Neural-specific deletion of FIP200 leads to cerebellar degeneration caused by increased neuronal death and axon degeneration. J Biol Chem 285: 3499–509. 32. Gan B, Melkoumian ZK, Wu X, Guan KL, Guan JL (2005) Identification of FIP200 interaction with the TSC1-TSC2 complex and its role in regulation of cell size control. J Cell Biol 170: 379–89. 11. Xiong W-C, Parsons JT (1997) Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase. J Cell Biol 139: 529–39. 33. Ding Q, Stewart JE, Jr., Olman MA, Klobe MR, Gladson CL (2003) The pattern of enhancement of Src kinase activity on platelet-derived growth factor stimulation of glioblastoma cells is affected by the integrin engaged. J Biol Chem 278: 39882–91. 12. Chauhan D, Hideshima T, Pandey P, Treon S, Teoh G, et al. (1999) RAFTK/ PYK2-dependent and -independent apoptosis in multiple myeloma cells. Oncogene 18: 6733–40. 34. Stettner MR, Wang W, Nabors LB, Bharara S, Flynn DC, et al. (2005) Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells. Cancer Res 65: 5535–43. g 13. Zhao J, Zheng C, Guan J (2000) Pyk2 and FAK differentially regulate progression of the cell cycle. J Cell Sci 113: 3063–72. 14. Zhao J, Pestell R, Guan JL (2001) Transcriptional activation of cyclin D1 promoter by FAK contributes to cell cycle progression. Mol Biol Cell 12: 4066–77. 35. Park SY, Avraham H, Avraham S (2000) Characterization of the tyrosine kinases RAFTK/Pyk2 and FAK in nerve growth factor-induced neuronal differentiation. J Biol Chem 275: 19768–77. 15. Zhao J, Bian ZC, Yee K, Chen BP, Chien S, et al. References determining glioblastoma migration and proliferation. Mol Cancer Res 1: 323–32. determining glioblastoma migration and proliferation. Mol Cancer Res 1: 323–32. 1. Abbi S, Ueda H, Zheng C, Cooper LA, Zhao J, et al. (2002) Regulation of focal adhesion kinase by a novel protein inhibitor FIP200. Mol Biol Cell 13: 3178–91. 2. Ueda H, Abbi S, Zheng C, Guan JL (2000) Suppression of Pyk2 kinase and cellular activities by FIP200. J Cell Biol 149: 423–30. 25. Wang D, Grammer JR, Cobbs CS, Stewart JE, Jr., Liu Z, et al. (2000) p125 focal adhesion kinase promotes malignant astrocytoma cell proliferation in vivo. J Cell Sci 113 Pt 23: 4221–30. 3. Gan B, Guan JL (2008) FIP200, a key signaling node to coordinately regulate various cellular processes. Cell Signal 8; 20: 787–94. 26. Ikebuchi K, Chano T, Ochi Y, Tameno H, Shimada T, et al. (2009) RB1CC1 activates the promoter and expression of RB1 in human cancer. Int J Cancer 125: 861–7. 4. Hara T, Takamura A, Kishi C, Lemura S, Natsume T, et al. (2008) FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J Cell Biol 181: 497–510. 27. Nagy JA, Chang S-H, Shih S-C, Dvorak AM, Dvorak HF (2010) Heterogeneity of the tumor vasculature. Seminars in Thrombosis and Hemostasis 36: 321–331. 5. Gan B, Peng X, Nagy T, Alcaraz A, Gu H, et al. (2006) Role of FIP200 in cardiac and liver development and its regulation of TNFalpha and TSC-mTOR signaling pathways. J Cell Biol 175: 121–33. 28. Sonoda Y, Ozawa T, Hirose Y, Aldape KD, McMahon M, et al. (2001) Formation of intracranial tumors by genetically modified human astrocytes defines four pathways critical in the development of human anaplastic astrocytoma. Cancer Res 61: 4956–60. 6. Chano T, Ikegawa S, Saito-Ohara F, Inazawa J, Mabuchi A, et al. (2002) Isolation, characterization and mapping of the mouse and human RB1CC1 genes. Gene 291: 29–34. 29. McFarland BC, Stewart J, Jr., Hamza A, Nordal R, Davidson DJ, et al. (2009) Plasminogen kringle 5 induces apoptosis of brain microvessel endothelial cells: sensitization by radiation and requirement for GRP78 and LRP1. Cancer Res 69: 5537–45. g 7. Melkoumian ZK, Peng X, Gan B, Wu X, Guan JL (2005) Mechanism of cell cycle regulation by FIP200 in human breast cancer cells. Cancer Res 65: 6676–84. 8. Chano T, Ikegawa S, Kontani K, Okabe H, Baldini N, et al. Acknowledgments We thank Dr. Amy Nowacki for assistance with the statistical analysis and Dr. Fiona Hunter for critical reading of this manuscript. We thank Dr. Amy Nowacki for assistance with the statistical analysis and Dr. Fiona Hunter for critical reading of this manuscript. References (2003) Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Mol Cell 11: 1503–15. J 36. Gutenberg A, Bruck W, Buchfelder M, Ludwig HC (2004) Expression of tyrosine kinases FAK and Pyk2 in 331 human astrocytomas. Acta Neuropathol 108: 224–30. 16. Kuwabara K, Nakaoka T, Sato K, Nishishita T, Sasaki T, et al. (2004) Differential regulation of cell migration and proliferation through proline-rich tyrosine kinase 2 in endothelial cells. Endocrin 145: 3324–30. 37. Chano T, Ikebuchi K, Ochi Y, Tameno H, Tomita Y, et al. (2010) RB1CC1 activates RB1 pathway and inhibits proliferation and cologenic survival in human cancer. PLoS ONE 5(6): e11404. doi:10.1371/journal.pone.0011404. 17. Ding Q, Grammer JR, Nelson MA, Guan JL, Stewart JE, Jr., et al. (2005) p27Kip1 and cyclin D1 are necessary for focal adhesion kinase regulation of cell cycle progression in glioblastoma cells propagated in vitro and in vivo in the scid mouse brain. J Biol Chem 280: 6802–15. 38. Kurenova E, Xu LH, Yang X, Baldwin AS, Jr., Craven RJ, et al. (2004) Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein. Mol Cell Biol 24: 4361–71. p g p 39. Park SY, Avraham HK, Avraham S (2004) RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem 279: 33315–22. 18. Melendez J, Turner C, Avraham H, Steinberg SF, Schaefer E, et al. (2004) Cardiomyocyte apoptosis triggered by RAFTK/pyk2 via Src kinase is antagonized by paxillin. J Biol Chem 279: 53516–23. 40. Wang Q, Xie Y, Du QS, Wu XJ, Feng X, et al. (2003) Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin. J Cell Biol 160: 565–75. 19. Avdi NJ, Nick JA, Whitlock BB, Billstrom MA, Henson PM, et al. (2001) Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis. J Biol Chem 276: 2189–99. g J 41. Brown MC, Turner CE (2004) Paxillin: Adapting to change. Physiol Rev 84: 1315–39. 20. Pandey P, Avraham S, Kumar S, Nakazawa A, Place A, et al. (1999) Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism. J Biol Chem 274: 10140–4. 42. Avraham H, Park SY, Schinkmann K, Avraham S (2000) RAFTK/Pyk2- mediated cellular signalling. Author Contributions Conceived and designed the experiments: DW CLG. Performed the experiments: DW MAO JSJ RT PH ET. Analyzed the data: DW CLG MAO WML JSJ RT PH C ib d / i l / l i l Conceived and designed the experiments: DW CLG. Performed the experiments: DW MAO JSJ RT PH ET. Analyzed the data: DW CLG MAO WML JSJ RT PH. Contributed reagents/materials/analysis tools: JLG PWS RAP JL RJW CAP GYG ROP. Wrote the paper: DW CLG. TAT-Pyk2 Fusion Proteins The following constructs in the pTatHA bacterial expression vector were provided by Dr. Carl Nathan (Weil Medical College of Cornell University, NY): nt 1102–1557, coding for amino acids PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 12 FIP200 in Glioblastoma Tumors cortactin). The normalized FIP200 protein expression in the siFIP200-treated cells is shown as a percentage of the normalized FIP200 expression in the control cells treated with liposome or +mutant siFIP200. Abbreviations: HBMvEC, primary human brain microvessel endothelial cells. (TIF) cortactin). The normalized FIP200 protein expression in the siFIP200-treated cells is shown as a percentage of the normalized FIP200 expression in the control cells treated with liposome or +mutant siFIP200. Abbreviations: HBMvEC, primary human brain microvessel endothelial cells. (TIF) 47. Son MJ, Woolard K, Nam DH, Lee J, Fine HA (2009) SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell Stem Cell 4: 440–52. 48. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, eds (2007) WHO Classification of Tumours of the Central Nervous System. 4th ed. IARC: Lyon. urokinase-type plasminogen activator (uPA) receptor and uPA retards angiogenesis and inhibits intracranial tumor growth in nude mice. Clin Cancer Res 13: 4051–60. 51. Ying WZ, Aaron K, Sanders PW (2008) Mechanism of dietary salt-mediated increase in intravascular production of TGF-ss1. Am J Physiol Renal Physiol 295: F406–14. References Cell Signal 12: 123–33. 43. Lulo J, Yuzawa S, Schlessinger J (2009) Crystal structures of free and ligand- bound focal adhesion targeting domain of Pyk2. Biochem Biophys Res Com 383: 347–52. 21. Ramachandra M, Atencio I, Rahman A, Vaillancourt M, Zou A, et al. (2002) Restoration of transforming growth factor Beta signaling by functional expression of smad4 induces anoikis. Cancer Res 62: 6045–51. 44. Katsoulidis E, Li Y, Mears H, Platanias LC (2005) The p38 mitogen-activated protein kinase pathway in interferon signal transduction. J Interferon Cytokine Res 25: 749–56. 22. Dahiya R, Perinchery G, Deng G, Lee C (1998) Multiple sites of loss of heterozygosity on chromosome 8 in human breast cancer has differential correlation with clinical parameters. Int J Oncol 12: 811–6. 45. Cox BD, Natarajan M, Stettner MR, Gladson CL (2006) New concepts regarding focal adhesion kinase promotion of cell migration and proliferation. J Cell Biochem 99: 35–52. 23. Wei H, Gan B, Wu X, Guan JL (2009) Inactivation of FIP200 leads to inflammatory skin disorder, but not tumorigenesis, in conditional knock-out mouse models. J Biol Chem 284: 6004–13. J 46. Gondi CS, Lakka SS, Dinh DH, Olivero WC, Gujrati M, et al. (2007) Intraperitoneal injection of a hairpin RNA-expressing plasmid targeting 24. Lipinski CA, Tran NL, Bay C, Kloss J, McDonough WS, et al. (2003) Differential role of proline-rich tyrosine kinase 2 and focal adhesion kinase in PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 May 2011 \| Volume 6 \| Issue 5 \| e19629 13 49. Natarajan M, Stewart JE, Golemis EA, Pugacheva EN, Alexandropoulos K, et al. (2006) HEF1 is a necessary and specific downstream effector of FAK that promotes the migration of glioblastoma cells. Oncogene 25: 1721–32. 50. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, et al. (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT- p27Kip1 induces cell migration. Nat Med 4: 1449–52. urokinase-type plasminogen activator (uPA) receptor and uPA retards angiogenesis and inhibits intracranial tumor growth in nude mice. Clin Cancer Res 13: 4051–60. FIP200 in Glioblastoma Tumors 49. Natarajan M, Stewart JE, Golemis EA, Pugacheva EN, Alexandropoulos K, et al. (2006) HEF1 is a necessary and specific downstream effector of FAK that promotes the migration of glioblastoma cells. Oncogene 25: 1721–32. p g g g 50. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, et al. (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT- p27Kip1 induces cell migration. Nat Med 4: 1449–52. PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e19629 14
https://openalex.org/W4323342075	https://pubs.rsc.org/en/content/articlepdf/2023/na/d2na00956k	English	null	Epitaxial growth of crystal phase quantum dots in III–V semiconductor nanowires	Nanoscale advances	2,023	cc-by	19,402	Nanophotonics Technology Center, Universitat Polit`ecnica de Val`encia, Camino de Vera, s/n Building 8F, 2a Floor, 46022 Valencia, Spain. E-mail: vjgomher@ntc.upv.es Nanoscale Advances View Article Online View Journal \| View Issue Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6 This article is licensed under a Creative Commons Attribution 3.0 U Received 30th December 2022 Accepted 6th March 2023 DOI: 10.1039/d2na00956k rsc.li/nanoscale-advances rst be introduced. Then, the growth mechanism and control over the nucleation, layer growth, and crystal phase selection will be reviewed together with the process of selective core/shell growth. Following this, the review will cover the optical and electronic properties including specic examples and case studies. 1. Introduction 1. This review is focused on crystal phase QDs formed in III–V semiconductor NWs by stacking, in the axial direction, WZ or ZB crystal phases of the same material by the bottom-up VLS growth method. General concepts of WZ and ZB polytypes will Nanoscale structures, such as NWs, enable heterostructures to overcome the limitations related to lattice mismatch and strain relief, whereas the growth of their planar counterparts is Nanophotonics Technology Center, Universitat Polit`ecnica de Val`encia, Camino de Vera, s/n Building 8F, 2a Floor, 46022 Valencia, Spain. E-mail: vjgomher@ntc.upv.es V´ıctor Jes´us G´omez Hern´andez joined the Nanophotonics Tech- nology Center, Universitat Polit`ecnica de Val`encia, as a Distinguished Researcher where he leads the III–V semi- conductors and graphene group. He received his PhD at Uni- versidad Polit´ecnica de Madrid in 2017 and did 2 postdocs at Cardiﬀand Lund University for a total of 3 years. Dr V´ıctor J. G´omez main scientic goal is to develop new material architectures through high-level scientic research. V´ıctor has already participated in interdisciplinary and international projects, for a total of 13 diﬀerent projects, 5 of them as Principal Investigator (∼1 MV) and leads two infrastructure projects (>3 MV). V´ıctor Jes´us G´omez Hern´andez joined the Nanophotonics Tech- nology Center, Universitat Polit`ecnica de Val`encia, as a Distinguished Researcher where he leads the III–V semi- conductors and graphene group. He received his PhD at Uni- versidad Polit´ecnica de Madrid in 2017 and did 2 postdocs at Cardiﬀand Lund University for a total of 3 years. Dr V´ıctor J. G´omez main scientic goal is to develop new material architectures through high-level scientic research. V´ıctor has already participated in interdisciplinary and international projects, for a total of 13 diﬀerent projects, 5 of them as Principal Investigator (∼1 MV) and leads two infrastructure projects (>3 MV). Miguel Sinusia Lozano got his Bachelor and Master’s degree in Materials Science and Engi- neering in 2014 and 2016 respecively from the Universidad Polit´ecnica de Madrid. In 2020 he graduated from the Uni- versidad Polit´ecnica de Madrid where he pursued his PhD focusing on the piezoelectric ScAlN compound and its usage in surface acoustic wave (SAW) devices. He is currently working at the Nanophotonics Technology Center (NTC), Universitat Polit`ecnica de Val`encia. His work is devoted to the study of III–V semiconductors for sensing and quantum-related technologies. Epitaxial growth of crystal phase quantum dots in III–V semiconductor nanowires Cite this: Nanoscale Adv., 2023, 5, 1890 Miguel Sinusia Lozano and V´ıctor J. G´omez * Miguel Sinusia Lozano and V´ıctor J. G´omez * Crystal phase quantum dots (QDs) are formed during the axial growth of III–V semiconductor nanowires (NWs) by stacking diﬀerent crystal phases of the same material. In III–V semiconductor NWs, both zinc blende (ZB) and wurtzite (WZ) crystal phases can coexist. The band structure diﬀerence between both crystal phases can lead to quantum conﬁnement. Thanks to the precise control in III–V semiconductor NW growth conditions and the deep knowledge on the epitaxial growth mechanisms, it is nowadays possible to control, down to the atomic level, the switching between crystal phases in NWs forming the so-called crystal phase NW-based QDs (NWQDs). The shape and size of the NW bridge the gap between QDs and the macroscopic world. This review is focused on crystal phase NWQDs based on III–V NWs obtained by the bottom-up vapor–liquid–solid (VLS) method and their optical and electronic properties. Crystal phase switching can be achieved in the axial direction. In contrast, in the core/shell growth, the diﬀerence in surface energies between diﬀerent polytypes can enable selective shell growth. One reason for the very intense research in this ﬁeld is motivated by their excellent optical and electronic properties both appealing for applications in nanophotonics and quantum technologies. 1.2 Bottom-up fabrication of NWQDs However, in the bottom-up approach chemical or physical forces operating at the nanoscale during the growth process are employed to assemble the NWs where the QDs are included. This can be achieved by changing the growth conditions, precursor species or precursor ows. The bottom-up approach oﬀers a way to grow NWQDs with smooth sidewalls with axial and/or radial heterostructures or doping proles. Due to the nanowire geometry, lattice mismatch and the resulting stress can be accommodated via strain relaxation in the radial direc- tion without the introduction of defects, i.e., dislocations. This allows us to obtain NWQDs in both the axial and radial directions.22–24 An additional advantage of NWQDs to be considered is their relatively high surface-to-volume ratio compared to their planar counterparts that allows the existence of other crystal phases not stable in the bulk (polytypism).25–27 In III–V NWs, ZB and WZ alternating segments can be successfully controlled to conne charges and obtain type-II NWQDs within the same material.28–31 Two diﬀerent approaches can be distinguished for the fabrication of NWQDs: top-down and bottom-up. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. y p The combination of NWs and QDs is crucial for photonics due to the enhanced spontaneous emission rate by coupling generated light with cavity modes (Purcell eﬀect), waveguiding, and suppression of the total internal reection so that the eﬃciency of light extraction can theoretically approach 100%.2–6 Additionally, the directionality of emissions can be controlled by engineering the shape of the tip of the NW,.7 thus resulting in the realization of novel nanophotonic devices based on nano- wires. In agreement with this, NWQD-based single-photon subwavelength detectors8,9 and emitters10,11 have been demon- strated. NWQDs can also easily be addressed via electrical contacts. Therefore, QDs can be electrically pumped when embedded in an NW axial p–n junction. This allows the injec- tion of electron–hole pairs and their subsequent radiative recombination into photons emitted by the QD.12 In addition, virtual QDs can be induced by localized electrical gating, allowing a versatile manipulation of the density of states in the QD.13,14 In this case, the QD-like connement is achieved by local tuning of the Fermi level by applying the right voltage by means of precisely positioned gate electrodes instead of engi- neering the QD by a local diﬀerence either in material, composition or crystal phase. 1. Introduction Miguel Sinusia Lozano got his Bachelor and Master’s degree in Materials Science and Engi- neering in 2014 and 2016 respecively from the Universidad Polit´ecnica de Madrid. In 2020 he graduated from the Uni- versidad Polit´ecnica de Madrid where he pursued his PhD focusing on the piezoelectric ScAlN compound and its usage in surface acoustic wave (SAW) devices. He is currently working at the Nanophotonics Technology Center (NTC), Universitat Polit`ecnica de Val`encia. His work is devoted to the study of III–V semiconductors for sensing and quantum-related technologies. © 2023 The Author(s). Published by the Royal Society of Chemistry 1890 \| Nanoscale Adv., 2023, 5, 1890–1909 Nanoscale Advances View Article Online Nanoscale Advances View Article Online Review strongly constrained to a small lattice parameter window, therefore, oﬀering additional degrees of freedom, for example in terms of materials combination, for the formation of low dimensional heterostructures, including quantum wells (QW), quantum wires (QWRs), and QDs embedded in NWs, also known as nanowire quantum dots (NWQDs). The three- dimensional carrier connement and localized/discrete carrier states in QDs are predicted1 to introduce new physical phenomena to diode lasers and other photonic devices. Tradi- tionally NWQDs can be produced via heterostructured inter- faces between materials with diﬀerent band gaps (i.e. switching materials and/or alloy composition) or by changing the crystal structure without changing the material. Epitaxially grown NWs with embedded QDs oﬀer a way for fully deterministic posi- tioning of the QDs. Moreover, the shape and aspect ratio of the NWs guarantee the link of the QD with the macroscopic world, either by electrical contacts or photon emission. strongly constrained to a small lattice parameter window, therefore, oﬀering additional degrees of freedom, for example in terms of materials combination, for the formation of low dimensional heterostructures, including quantum wells (QW), quantum wires (QWRs), and QDs embedded in NWs, also known as nanowire quantum dots (NWQDs). The three- dimensional carrier connement and localized/discrete carrier states in QDs are predicted1 to introduce new physical phenomena to diode lasers and other photonic devices. Tradi- tionally NWQDs can be produced via heterostructured inter- faces between materials with diﬀerent band gaps (i.e. switching materials and/or alloy composition) or by changing the crystal structure without changing the material. Epitaxially grown NWs with embedded QDs oﬀer a way for fully deterministic posi- tioning of the QDs. © 2023 The Author(s). Published by the Royal Society of Chemistry 1. Introduction Moreover, the shape and aspect ratio of the NWs guarantee the link of the QD with the macroscopic world, either by electrical contacts or photon emission. In general, self-assembled semiconductor QDs are grown in the Stranski–Krastanov (SK) growth mode. The SK growth mode is driven by the lattice mismatch between the substrate and the growing layer. Aer growing a few monolayers (wetting layer), the accumulated strain is relieved via the random nucleation and growth of island-like formation (QDs).18 The resulting QDs can then be embedded in the material aer the epitaxial growth of a new epi-layer called the capping layer. The optical proper- ties of the QDs depend on the composition (in the case of ternary or quaternary alloys), size, and strain. It is worth high- lighting that the composition of the alloy denes the lattice parameter, which is related to the strain and, therefore, to the size of the QDs. Moreover, the emission energy of the QDs is directly related to the surrounding material (wetting and capping layers), determining the charge carrier connement in the QD. One of the main drawbacks of self-assembled QDs is the lack of uniformity in heights and lateral size, ranging typically between 1–10 and 10–70 nm respectively.19 The density and size distribution can be controlled up to some extent by varying the growth conditions.20 Another disadvantage is the material inter- mixing between the QD and capping layer that can take place during the growth, which further increases the variation in the optical properties of the QDs.21 Therefore, the top-down approach must deal with QDs ensembles with limited control in location, size, density, and material uniformity. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 1.1 Top-down fabrication of NWQDs The top-down approach involves the breaking down of a substrate including QDs into nanostructures such us NWs. By using this fabrication method precisely positioned one- dimensional structures can be obtained. Depending on the density of QDs in the substrate and the NW diameter (dened via lithography), the resulting NWQDs can embed one or several QDs.15–17 One of the main drawbacks of this approach is the impact of etching on exposed surfaces, also referred to as etch damage, hindering the material quality and the subsequent device performance. Moreover, as this approach needs a substrate containing QDs, this technique is limited to mate- rials and material combinations which already exist in bulk, which is not generally the case for diﬀerent crystal phases. The bottom-up NW growth approach relies either on the VLS (Fig. 1A) or on the selective-area epitaxy (SAE, Fig. 1B). It is worth mentioning that the main diﬀerence between the two growth modes is in the axial direction, being the radial growth domi- nated by the vapor–solid growth mode which is similar in both cases. Both growth modes, VLS or SAE, allow forming NWQDs. Compared to the top-down fabrication, there are some major advantages when using the bottom-up growth of NWQDs: a wider material compatibility, deterministic fabrication down to a single QD per NW, and natural alignment of the QD along the NW axis. Nanoscale Adv., 2023, 5, 1890–1909 \| 1891 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online Nanoscale Advances Fig. 1 Schematic representation of the NW growth modes. (A) VLS growth mode where the axial growth is catalysed by a metallic droplet (typically Au). (B) SAE growth mode where the growth takes place at an opening in a mask layer. Fig. 1 Schematic representation of the NW growth modes. (A) VLS growth mode where the axial growth is catalysed by a metallic droplet (typically Au). (B) SAE growth mode where the growth takes place at an opening in a mask layer. Fig. 1 Schematic representation of the NW growth modes. (A) VLS growth mode where the axial grow (typically Au). (B) SAE growth mode where the growth takes place at an opening in a mask layer. 1.1 Top-down fabrication of NWQDs Additionally, the NW growth sites can be lithographically dened showing the upscaling possibilities of this technology.52–55 The VLS NW growth mode,32 originally introduced by Wag- ner and Ellis in 1964,33 is mediated by a metallic droplet in the liquid phase, which collects the growth species introduced in the reactor chamber in the vapor phase, leading to the nucle- ation of the crystal in the solid phase, below the droplet. Then, the growing NW lis the droplet that remains at the tip of the NW. In 1991, Hiruma and co-workers34 grew, for the rst time, III–V compound nanowires. One year later, in 1992, the same group reported the co-existence of both ZB and WZ crystal structures in indium arsenide NWs.35 In 1994, Murayama and co-workers reported the calculations of the band oﬀsets at WZ– ZB interfaces.36 However, it was not until 2008 that the rst successful attempts to control the crystal structure during NW growth37,38 and the rst optical studies39–42 were reported. This progress practically enables engineering of the electronic structure of a single material. For instance, a WZ/ZB/WZ structure can dene a quantum well in a single material. Growing such a structure in a NW will also conne charges in two other dimensions, forming a crystal phase NWQD. During the past few decades, a huge eﬀort has been made in order to understand the VLS growth process, thus leading to NWs with high crystal quality,43 controlled crystal phase,27,44 controlled axial doping,45 and to obtain axial and radial hetero- structures.22,46,47 Despite the signicant progress made, the detailed growth mechanism is not fully understood yet and research in this area is going on. Since metal contamination from the metallic droplet in the NWQD could be detrimental to its optical and electrical prop- erties, self-catalysed or catalyst-free growth methods have been developed.56–58 Also in this case, arrays of NWs can be grown on a lithographically patterned substrate.58 The SAE is a catalyst- free direct vapor–solid growth and is based on growth mask layers with openings that dene the NW growth. The growth is driven by diﬀerences in the nucleation and crystal growth on diﬀerent crystal facets mainly due to diﬀerences in surface energies.59 The openings in the growth mask expose the underlying substrate and dene the locations where the epitaxy of the NWs starts. © 2023 The Author(s). Published by the Royal Society of Chemistry 1.1 Top-down fabrication of NWQDs For the axial growth along the h111i direction, the conditions are chosen such that the {110} facets have signicantly lower growth rates than the {111} facets. Then, the NW grows with {110} lateral facets and the NW shows a hexag- onal cross-section. Under the appropriate conditions, the radial growth can be fully suppressed, and the NW diameter remains equal to the mask opening. Fig. 1B shows the schematic representation of the SAE growth. As with VLS growth, it is possible to obtain high crystal quality,60 control the crystal phase,61 switch between axial and radial growth,62 dope the NW during growth,62 and obtain axial and radial heterostructures.62,63 In principle, SAE growth is less complex than VLS growth, as only two phases, vapor and solid, are involved. Nevertheless, the growth dynamics are highly complex, for example the dependence on the diameter of the mask openings and distance between openings, on the NW geometry, and on the relative supply of group III (or V) constituents in the growth of ternaries.64–66 Therefore, the main The metallic droplet material can either be one of the group- III growth species (leading to a self-catalysed or self-assisted growth) or a foreign material, being Au the most widespread and studied.48 The metallic droplets, catalysts, can be randomly deposited on the substrate by means of spark discharge,49 aerosol generation,50 or annealing of Au thin lms.51 1892 \| Nanoscale Adv., 2023, 5, 1890–1909 © 2023 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances View Article Online Review challenges in the SAE growth method are to obtain a high degree of control in NW growth and in understanding its subtleties. changed instead of the material composition28,29,84 and the crystal structure can be controlled using basic growth parame- ters43,44 creating crystal phase QDs, which, in some aspects, supersede material QDs. As the interfaces are dened between diﬀerent crystal phases in the same material, no atom inter- mixing can occur, creating atomically sharp WZ–ZB inter- faces.28,85 Moreover, there is minimal strain at a WZ–ZB interface.86 This makes polytype heterostructures ideal candi- dates for the exploration of the low dimensionality properties of NWQDs. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Regardless of the specic growth technique employed (MBE, MOCVD, Au-assisted, self-catalysed.), the way to dene a crystal phase NWQD depends on the band alignment between WZ and ZB phases, and this is specic to a particular material. For example, in the case of InP, GaAs, and InAs there is a type-II band alignment between WZ and ZB,88 thus leading to the connement of electrons in the ZB phase when it is sandwiched between WZ barriers. Moreover, these type-II structures can support the formation of indirect excitons at the interfaces between crystal phases (Fig. 4). Finally, the main limitation of the crystal phase approach is the lack of exibility in the formation of QDs and the xed and relatively small band oﬀsets. Moreover, there are materials where it is not possible to form crystal phase QDs in a controlled way. This is the case of III-nitrides. 2.2 Crystal phases: zinc blende and wurtzite Before describing the axial growth of crystal phase NWQDs, it is necessary to fully describe the crystal structures to be investi- gated: ZB and WZ. Both crystal phases diﬀer only in their stacking sequence in the close-packed directions (h111i in ZB and h0001i in WZ), as is shown in the top part of Fig. 2. Both structures consist of hexagonally close-packed planes of III–V pairs. The diﬀerence lies in the alignment of these hexagonally close-packed planes with their surrounding planes. For ZB, three sequential layers form a repeating unit (.ABCAB- CABC.), where each letter represents one of the three allowed positions of the pairs of group-III and group-V atomic layers along the specic h111i direction. For the WZ crystal phase, only Fig. 2 The atomic arrangement for the ZB and WZ structures. 2. Crystal phase axial NWQDs The idea behind the growth of NWQDs is relatively simple: a material with a lower band gap should be inserted inside another material with a higher band gap. Thus, forming an axial structure of the style A/B/A, where B shows quantum conne- ment due to the diﬀerences in band gap energy and the relative position of the band edges. The stacking sequence is obtained typically by changing the growth precursors during the axial NW growth. The patterning of the catalysts in VLS controls the position of the nanowire, and the diameter of the catalyst denes the NW and QD diameters. The axial position of the QD and its length are dened by the growth time. Therefore, the combination of patterning together with the VLS bottom-up growth oﬀer full control over the size and position of the QD inside the NW and on the substrate. Moreover, radial growth can be employed either to fully embed the QD inside the NW or to grow an oxide shell, using atomic layer deposition (ALD), to improve the waveguiding properties of the NW.79 Another advantage of axial NWQDs arises when the NW diameter is properly chosen. This way, the emission from the QD can be eﬃciently coupled to the fundamental waveguiding mode of the NW, thus enhancing the extraction eﬃciency. Nowadays, this is possible thanks to a deep understanding and control of the growth process. In addition, axial NWQDs can be electrically injected when contacting the region above and below the QD. In this case, electrons, and holes ow from the top region to the bottom one going through the QD region dened in between. © 2023 The Author(s). Published by the Royal Society of Chemistry 1.1 Top-down fabrication of NWQDs In addition, single electron transport83 and quantum light sources have been demonstrated using crystal phase NWQDs.87 In general, NWs and NWQDs can be grown by diﬀerent techniques ranging from metal–organic vapor-phase epitaxy (MOVPE),2,28,67 to chemical beam epitaxy (CBE),68 and molecular beam epitaxy (MBE).56,69 Moreover, NWs and NWQDs can be in situ characterized by transmission electron microscopy during growth by either MOVPE70,71 or MBE.57 Furthermore, crystal phase heterostructures can be exploited not only with NWs, but also with nano-membranes72,73 and microdisks.74 The area of NWQDs is extremely broad with application examples in several elds such as quantum technologies75,76 and nanophotonics.77,78 Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In III–V semiconductor NWs, the most frequently reported defects are twin planes, stacking faults, and the lack of control over the WZ and ZB formation. For the discussion on how to control the crystal phase, the reader is referred to Section 2.4.2. In general, twin planes and stacking fault defects have a nega- tive impact on the optical and electronic properties of III–V semiconductor NWs by reducing the carrier lifetime, mobility, and quantum eﬃciency.90 On the other hand, stacking faults can have a positive impact on some of the physical properties of NWs as in the case of the eld emission properties of GaN NWs.91,92 It has also been reported that there is an increase in Young's modulus with the increase in stacking faults for GaAs NWs.93 Twin planes are commonly observed in the ZB crystal phase. There, the stacking sequence is oen found to unex- pectedly change from an .ABCABCABC. stacking sequence to an .ABCACBACBA. The stacking sequence still corresponds to the ZB-type sequence aer ‘A’, but before and aer this bilayer the sequences are mirrored in ‘A’. This ‘mirror’ plane (A) is known as a twin plane and the sequences above and below the mirror plane have diﬀerent twin orientations (Fig. 3). Never- theless, the small part of the sequence surrounding the twin plane (.ABCACBACBA.) can be regarded as a WZ-type stack- ing sequence. In this review, we will follow the criteria The diﬀerent stacking sequences for ZB and WZ slightly change the local environment for each atom in the crystal lattice. In particular, the third-nearest-neighbour spacing is shorter in WZ. The shiin cohesive energy ranges from around −45 meV per III–V pair for AlN96 to around 25 meV for GaSb.97 This diﬀerence in bulk cohesive energy between the two phases is the reason why WZ is the preferred crystal structure for III- nitrides and, for example, WZ GaAs is not stable in bulk. Moreover, ZB and WZ crystal structures also have diﬀerent surface terminations, as shown on the right-hand side of Fig. 2. Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In particular, for NWs growing in the h111i/h0001i directions, the surfaces present in the sidewalls of the NWs are diﬀerent for the two structures.98,99 Although there is a wide dispersion in the estimates of what the actual energies of these surfaces are, most of the reports agree that the WZ {1010} surface should have a lower surface energy than the ZB {110}.98–101 2.3 The role of supersaturation Supersaturation is a state of thermodynamic instability and hence the system becomes prone to reduce the supersaturation through the process called precipitation. Therefore, supersatu- ration is the driving force of the epitaxial growth process. Supersaturation can be dened as the diﬀerence in chemical potential between the growth environment and the substrate. Fig. 3 (a) Two diﬀerent twin orientations of the ZB stacking sequence, denoted as ‘I’ and ‘II’. (b) When the two twin orientations of ZB are combined, the common plane A is a twin plane or twin plane. (c) Transmission electron microscopy image of a section of an InAs nanowire exhibiting two diﬀerent orientations of zinc blende stacking (twin segments), separated by a twin plane. In the TEM image, group II and V atoms in adjacent planes cannot be resolved and appear as a single dot. Fig. 2 from Kimberly A Dick et al., 2010, Semicond. Sci. Technol., 25, 024009, reproduced with the permission of IOP Publishing. Dm = mgas −msubs (1) (1) At equilibrium, there is no diﬀerence between the chemical potential, the growth environment, and the chemical potential of the substrate. Therefore, Dm = 0. To achieve crystal growth, a supersaturated growth environment is required. Crystal growth continues as long as the chemical potential of the growth environment is higher than that of the substrate. When the chemical potential of the substrate is higher than that of the growing environment, then the etching of the substrate is promoted over crystal growth. For NWs in the VLS mode, growth occurs at the interface between the catalyst and NW, so this discussion will be focused on the chemical potential diﬀerence between the interface and the supply atoms. Supersaturation is aﬀected by the growth temperature, pressure, and concentration of the growth species at the catalyst–NW interface. The concentration of growth species is directly controlled by the partial pressures of the precursors. However, it is vital to highlight that supersaturation is aﬀected by many other growth parameters. As mentioned Fig. 3 (a) Two diﬀerent twin orientations of the ZB stacking sequence, denoted as ‘I’ and ‘II’. (b) When the two twin orientations of ZB are combined, the common plane A is a twin plane or twin plane. (c) Transmission electron microscopy image of a section of an InAs nanowire exhibiting two diﬀerent orientations of zinc blende stacking (twin segments), separated by a twin plane. © 2023 The Author(s). Published by the Royal Society of Chemistry 2.1 Material composition vs. crystal phase QDs The axial growth of III–V NWQDs relies on the possibility to change the material composition or crystal phase during growth. Two approaches can be diﬀerentiated to realize NWQDs. First, by heterostructuring semiconductor materials with diﬀerent band gaps, or second, by tuning the crystal phase of a single material. For the rst approach, the perfection of the NWQD is limited by the diﬃculty to control the interface abruptness and material composition at the atomic level80–82 which unfortunately are determining factors for the physical properties of QDs as they dene the shape of the potential well.29,83 Recent studies have shown that perfect atomically sharp interfaces can be achieved if the crystal structure is Fig. 2 The atomic arrangement for the ZB and WZ structures. © 2023 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2023, 5, 1890–1909 \| 1893 Review View Article Online View Article Online Nanoscale Advances Review published by Dick and co-workers89 where they exclusively use the terms twin and twin plane for this type of defect or inter- ruption in the stacking sequence. They use WZ only when the WZ-type stacking sequence happens over at least four bilayers. Moreover, the term stacking faults refer to interruptions in the WZ stacking sequence such as .ABABCBABA. where C is the erroneous bilayer creating the stacking fault. Diﬀerent strate- gies have been implemented to successfully reduce the density of stacking faults. Controlling the growth temperature and V/III ratio,38,43 NW diameter94 or the use of growth interruption strategies95 has been demonstrated to be critical in reducing the density of stacking defects. two of the allowed positions are occupied, giving the stacking sequence .ABABAB. Otherwise, each atomic layer can be denoted separately, diﬀerentiating the two diﬀerent atomic species, using lower-case and upper-case letters. Then the ZB and WZ stacking sequences can be written as .AaBbC- cAaBbCc. and .AaBbAaBb., respectively (see the bottom part of Fig. 2). When there is an intermixing of crystal structures and/or diﬀerent faults appear in their stacking sequence, this notation becomes mandatory to fully describe and distinguish between diﬀerent defects and stacking faults in the sequence of these two types of crystal structures.89 Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 (A) High-resolution transmission electron micrograph of an InAs NW with a ZB QD sandwiched between WZ barriers. (B) Sche- matic representation of the crystal structure stacking sequence and the resulting type-II band alignment. Adapted from Fig. 1 by Potts, H., Chen, I., Tsintzis, A. et al., Electrical control of spins and giant g-factors in ring-like coupled quantum dots, Nat. Commun., 10, 5740, (2019), https://doi.org/10.1038/s41467-019-13583-7 Is licensed under CC BY 4.0. For III–V semiconductors, the group III and V elements, two diﬀerent species, must combine to form the growing NW. These two species follow diﬀerent, not always independent, kinetic processes. Both species have diﬀerent adsorption, diﬀusion, and desorption rates, aﬀecting their individual supersatura- tions at the catalyst–NW interface. When molecular precursor species are used, another degree of complexity is added to the system. This is because the two diﬀerent precursor molecules will have to decompose rst to deliver the atomic species to the growth front at a diﬀerent decomposition rate. In addition, precursor molecules may compete for reactants, or react with each other at the surface or in the gas phase generating unde- sired by-products. Determining the optimum ratio of precursor molar fractions required to obtain equal adatom concentrations of the two species at the catalyst–NW interface is not straight- forward. Therefore, the molar fraction ratio between the two species (V/III ratio) is an important parameter for the optimum NW growth. an example of crystal phase NWQD in InAs of the style WZ/ZB/ WZ. If the dimensions of the lower band gap crystal phase are small enough, the corresponding segment should show quantum connement and behave like a QD. Aer that, the growth conditions can be switched to promote radial growth and a shell can be added to nalize the NWQD structure. This can be useful to create 3D structures by growing a shell (or a selectively grown shell) of a second material that replicates the crystal phase of the core.22,104,105 Despite III–V semiconductor NWs typically growing vertically, stochastic processes of growth parameter modulation can lead to a change in the growth direction. This is the kinking phenomenon. For a deeper insight into the kink-type defects, the reader is kindly referred to the review published in 2022 by Vlassov and co- workers.106 © 2023 The Author(s). Published by the Royal Society of Chemistry 2.3 The role of supersaturation In the TEM image, group III and V atoms in adjacent planes cannot be resolved and appear as a single dot. Fig. 2 from Kimberly A Dick et al., 2010, Semicond. Sci. Technol., 25, 024009, reproduced with the permission of IOP Publishing. © 2023 The Author(s). Published by the Royal Society of Chemistry 1894 \| Nanoscale Adv., 2023, 5, 1890–1909 Fig. 4 (A) High-resolution transmission electron micrograph of an InAs NW with a ZB QD sandwiched between WZ barriers. (B) Sche- matic representation of the crystal structure stacking sequence and the resulting type-II band alignment. Adapted from Fig. 1 by Potts, H., Chen, I., Tsintzis, A. et al., Electrical control of spins and giant g-factors in ring-like coupled quantum dots, Nat. Commun., 10, 5740, (2019), https://doi.org/10.1038/s41467-019-13583-7 Is licensed under CC BY 4.0. Nanoscale Advances View Article Online View Article Online Review Nanoscale Advances before, it depends on growth temperature and pressure. In general, the chemical potential diﬀerence between a vapour and its solid phase will decrease with temperature. However, for growth techniques that involve molecular precursors, such as MOVPE, the precursor decomposition rate depends exponen- tially on temperature, which increases the supersaturation for xed precursor molar fractions. Moreover, surface reconstruc- tion of the substrate varies with temperature and vapour molar fractions and directly aﬀects the surface diﬀusion of adatoms. Therefore, the dependence of the supersaturation on the growth parameters is highly complex. NW systems are even more complicated. For example, when growth is diﬀusion limited, i.e., when the density of the NWs is high compared to the supply material, NWs compete for the supply material and the super- saturation per nanowire is therefore reduced. Supersaturation decreases as the NW grows away from the substrate, reducing the amount of material reaching the metallic particle, as re- ported for InP NWs.102 Other factors may include the local variation in precursor decomposition rate, solubility of the seed particle (which can be varied with temperature), trace impuri- ties, and dopants in the system (which may aﬀect adsorption, diﬀusion, and dissolution kinetics). Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2.4 Nanowire growth The crystal phase sequence in the area surrounding the QD is of the style A/B/A, where crystal phase B shows quantum connement. In the VLS method, the material forming the NW is dissolved in the metal droplet and, when reaching supersat- uration, precipitates at the droplet–NW (liquid–solid) interface. Layer by layer, generating the longitudinal growth of the NW. However, multilayer growth has also been demonstrated.103 Aer a certain growth time and for a chosen NW length, the growth conditions are changed to switch to crystal phase B. Note that the growth rate may change. At the desired length of the segment of crystal phase B, the growth conditions are changed to switch back to crystal phase A. Eventually, the growth is stopped, and the result is a NW of a single material containing a crystal phase QD of the style A/B/A. Fig. 4 illustrates 2.4.1 Controlling the nucleation and layer growth. In the VLS growth method, the material forming the NW is dissolved in the metal droplet and, when reaching supersaturation, precipitates at the droplet–NW (liquid–solid) interface, thus, generating the longitudinal growth of the NW layer by layer. Controlling this process is critical to developing nanomaterials with atomic precision, and therefore, to meet the demands of nanophotonics, electronics, and quantum technology applica- tions. During the last decade, in situ experiments have been performed and the NW growth process has been recorded. This has allowed scientists to solve questions regarding growth dynamics.71,107–109 In the growth process of a new layer two steps can be diﬀerentiated: the incubation and the layer growth © 2023 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2023, 5, 1890–1909 \| 1895 Review View Article Online View Article Online Nanoscale Advances Review across the liquid–solid interface.110,111 As the catalyst size is relatively small, the number of atoms needed to reach super- saturation is small, so they can be consumed during the growth of a layer.112 Then, the catalyst droplet needs to accumulate enough material to reach the supersaturation state again and to overcome the nucleation barrier for a new layer. Therefore, the incubation time can be dened as the time interval between the end of the growth of a layer and the beginning of the following one.71,107,113 This interpretation implies that there is a nucleation step during which the critical nucleus forms. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. p y 2.4.2.2 Radius of the NW. Apart from growth temperature, the radius of growing NWs plays a key role in the selection between the ZB and the WZ crystal structure. From an empirical perspective, the radius of the NW is well known to aﬀect the crystal structure selection26,94,119 but from a theoretical point of view the role of the nanowire radius is still under debate. Johansson and co-workers119 presented a theoretical model that explained the observed WZ preference in thin nanowires in terms of the Gibbs–Thomson (GT) eﬀect. The GT eﬀect refers to a reduction of the supersaturation, the driving force for growth when the size of nanowires decreases.120 These results diﬀer from the ones reported by Dubrovskii and co-workers121,122 that consider both, the solid and liquid phases when calculating the GT eﬀect. They concluded that the GT eﬀect does not have an inuence in the choice of crystal structure. Diﬀerent thermo- dynamic stability studies have reported that the WZ structure can become energetically favourable for a suﬃciently small NW radius (with transition radii ranging from a few to a few tens of nanometres).99,123–125 However, pure ZB NWs have been grown with a radius of 5 nm.126 Therefore, in terms of crystal phase selection, the connection between theory and experiment is limited. In 2020 Maliakkal and co-workers114 reported the indepen- dent control of nucleation and layer growth in Au-catalysed GaAs NWs using in situ TEM imaging. They demonstrated that the time scales for the nucleation and layer growth are of similar magnitude but follow diﬀerent trends. According to their ndings, the incubation time can be controlled by tuning the Ga precursor partial pressure, while the layer growth time can be controlled using the ow of the As precursor. They observed a similar order of magnitude for the incubation and layer completion times. The layer completion times range from 0.1 to 0.5 s, and the incubation times range from about 0.2 to 5 s. The underlying reason of why Ga and As control the growth in a diﬀerent way is the ease with which Ga alloys with the metallic catalyst while the As incorporation into the catalyst is comparatively low. Their ndings can be generalized for binary materials where each of the species interacts very diﬀerently with the catalyst. Therefore, oﬀering a mechanism to indepen- dently control the nucleation and layer growth. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. On the one hand, the incubation step is controlled by the atomic species that easily alloys with the catalyst droplet, given the fact that every atom increases the chemical potential of the alloy by small steps. On the other hand, the layer growth is controlled by the other atomic species, that, in comparison, its dissolution into the droplet is much smaller. Therefore, for the other atomic species only very few atoms are required to supersaturate the catalyst droplet. Recently, M˚artensson and co-workers developed a stochastic simulation model127 where the main input parameters are the ows of the growth species and the temperature of the process, allowing a direct transference of results between the simula- tions and the experimental epitaxial process. They use the model to simulate GaAs NW growth with varying As ows, obtaining a good agreement between their simulation results and the trends observed experimentally. In contrast with the previous discussion, multilayer growth has been observed for binary compound NWs.108,115 Moreover, when growing ternary NWs, a third element (group III or V) is incorporated. It is worth mentioning that the layer evolution in ternary NWs can determine and aﬀect the resulting composi- tion.116 Recently, Sjökvist and co-workers reported the multi- layer growth of InGaAs NWs.104 They observed that the uctuations in the ow of growth species in and out the metal nanoparticle inuence the composition of the growing NW. In a later paper,128 they applied the developed model to analyse the eﬀect of the radius on the crystal structure selection of GaAs nanowires. To understand the potential eﬀects of the NW radius, they simulated the growth at diﬀerent As ows and expanded the model to account for the GT eﬀect and the surface diﬀusion length of Ga atoms. First, the GT eﬀect results in a size-dependent eﬀective supersaturation due to the change in the surface area of the seed particle upon nucleation. Second, the incorporation of Ga atoms can occur via surface diﬀusion and direct impingement, which gives rise to a radius-dependent growth rate.122 They concluded that the geometry of the system (i.e. radius of the NW) plays a key role in the dynamics of the 2.4.2 Controlling the crystal phase. The control over the NW crystal structure has been achieved by several research groups via the variation of diﬀerent process parameters: (i) growth temperature; (ii) radius of the NW; (iii) introduction of foreign species; and (iv) nominal V/III-ratio. © 2023 The Author(s). Published by the Royal Society of Chemistry 2.4 Nanowire growth In general, this nucleation step is extremely fast and not visible in experiments. Aer the incubation time, the droplet reaches supersaturation again and a new layer is formed. Therefore, the layer completion time can be dened as the time observed for the completion of each layer. In the literature, the layer completion time is also referred to as the step-ow time. 2.4.2.1 Growth temperature. The growth temperature has been successfully employed for controlling the crystal phase.38,43,55,117 However, using the temperature for controlling the crystal phase increase signicantly the complexity of the process. The timespan for changing the temperature generally ranges between minutes and tenths of minutes, therefore making it extremely challenging, or even impossible, to form sharp interfaces and short segments and thus minimizing two of the main advantages of crystal phase QDs. Moreover, temperature changes aﬀect almost all relevant growth param- eters118 such as chemical potentials, precursor pyrolysis, migration length, eﬀective V/III-ratio, and the total amount of available material, therefore making temperature changes really complicate to be considered theoretically. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. © 2023 The Author(s). Published by the Royal Society of Chemistry 1896 \| Nanoscale Adv., 2023, 5, 1890–1909 Nanoscale Advances View Article Online Nanoscale Advances View Article Online Review seed particle composition throughout the growth. This directly controls the supersaturation, which is of vital importance for VLS NW growth. As the radius of the NW decreases, nucleation takes place at higher values of supersaturation, thus promoting the formation of the WZ crystal structure.110,122 Moreover, when in addition to direct impingement, considering the group-III atoms that reach the seed particle by means of diﬀusion, the V/III ratio becomes radius dependent. Therefore, NWs with smaller radii typically grow with lower V/III ratios, and the V/III ratio is one of the parameters controlling the crystal phase. WZ is promoted when the group-V precursor ow value is about 50–150 times larger than the corresponding ZB. In contrast to temperature variations, this approach allows for fast (in the order of a few seconds) switching between ZB and WZ. It also leaves other process parameters (particle diameter and doping) as degrees of freedom. However, the early studies in crystal structure control showed opposite tendencies for increasing group-V supply in molecular beam epitaxy (MBE) and metalorganic vapour phase epitaxy (MOVPE). More specically, in MBE, the WZ crystal phase is promoted for increasing group-V supply in Au110,136–138 and self-catalysed growth139–141 while ZB is favoured in MOVPE for GaAs,27,85 InAs,27,94,117,133 InP,44,142 and GaP.95,111,143–145 However, few MBE studies reported similar tendencies as in MOVPE for Au-seeded NW growth.146,147 In 2015, Lehmann and co-workers reported135 that the growth regimes (nominal V/III ratio) for crystal phase control in MBE and MOVPE are not mutually exclusive. By increasing the V/III ratio (changing only group-V precursor ow) in Au-catalysed GaAs NW growth it is possible to switch from a regime favourable to the ZB-to-WZ transition, to another regime where the WZ-to-ZB transition is favourable. They grew a set of NWs with increasing V/III-ratio from 0.61 until 240.2 (Fig. 5). All NWs were initiated with a stem grown at a nominal V/III ratio of 2.43 (conditions previously found to yield the WZ structure),44 followed by a second step were only the group-V precursor ow, and consequently, the V/III ratio was varied. They observed that ZB is formed for nominal V/III ratios well below (Fig. 5a) and above (Fig. 5g) the reference value of 2.43 for WZ (Fig. 5c). Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Neverthe- less, twin-free ZB was only formed for a nominal V/III ratio of 240.2 (Fig. 5g). For intermediate V/III ratios a mixed crystal structure (WZ together with both ZB twin orientations) is promoted (Fig. 5b, d–f). A clear transition from WZ to ZB was s Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2.4.2.3 Introduction of foreign species. The term foreign species is employed here to refer to species that are not strictly needed for growth. For example, in GaAs, we are considering foreign species to all other materials diﬀerent from the Ga or As precursor. The introduction of dopants37,129–131 or gases, such as HCl,132 has been employed to control the crystal structure of MOVPE-grown NWs but the eﬀect of foreign species on precursors is rather complex. Hence, it cannot be excluded that several growth parameters are indirectly aﬀected as well. Furthermore, with respect to device fabrication, doping, and diameter should remain free variables rather than a necessary parameter for controlling the crystal phase. 2.5 Selective core/shell growth The diﬀerence in surface energies between the WZ and ZB polytypes enables, under the appropriate growth conditions, a crystal phase selective core/shell growth. The selective shell growth opens new opportunities for the design of NW devices. This selective growth is demonstrated for diﬀerent core/shell combinations, such as GaAs/InAs,148,149 InAs/GaSb,104,150 and InAs/AlSb.151 In the work of G´omez and co-workers,148 they identify the two key factors governing the core/shell selectivity in GaAs/InAs core/shell NWs: shell growth rate and shell growth time. They attributed the occurrence of the selectivity to either a diﬀerence in crystal phase or a diﬀerence in lateral facet termination. Selectivity was found for short growth times and high growth rates. However, for long enough growth times the selectivity was lost, and homogeneous shells were grown, thus, limiting the maximum thickness of the shell growth under selective conditions. To overcome this limitation, they demon- strated a two-step growth formed by a high growth rate step followed by a low growth rate. By doing so, they demonstrate thickness control while keeping the shell selective growth. g In GaAs, at low temperatures, the energy of the WZ and ZB band gaps is very close.31,154 The theoretical calculations of the band oﬀset between WZ-GaAs and ZB-GaAs range from 70 to 150 meV,36,88,155,156 while they have been experimentally measured between 50 and 120 meV.31,155 Typically, the band oﬀset for a type-II band alignment is determined across the type-II interface using the energy (in the photoluminescence, PL, spectrum) of the spatially indirect recombination. In InP, the band gap of the WZ crystal phase is 1.49 eV at low temperatures,157–159 70 meV higher than the band gap of the ZB phase, and is shied to higher energy by the valence band oﬀset.88 Theoretical calculations give a diﬀerence ranging from 45 to 80 meV36,88,156 with experimental values measured between 35 and 70 meV.160 In the WZ–ZB–InP interface it is possible to create a 2DEG free to move in the radial but conned in the axial direction.160 For a deeper insight into the band oﬀsets between WZ and ZB in III–V semiconductors, the reader is kindly referred to the work done by Belabbes88 and Luiz H. Galv˜ao Tizei161 and co-workers. Nanoscale Advances Nanoscale Advances observed for nominal V/III ratios away from the reference for WZ (2.43). Therefore, ZB occurs for decreasing the V/III ratio in the lower range for increasing the V/III ratio in the higher range. In addition, they showed that the rst transition occurs for a V- limited growth regime, while the second transition arises in a III-limited regime, with WZ occurring when the group-III and group-V precursors are balanced. 3. Properties of NWQDs In this section, we give a brief overview on the properties of axial bottom-up crystal phase NWQDs. We are going to focus on their optical and electronic properties. © 2023 The Author(s). Published by the Royal Society of Chemistry Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 This article is licensed under a Creative Commons Attribution 3.0 Unporte 2.4.2.4 Nominal V/III-ratio. Control of crystal structure has also been reported by varying the nominal V/III ratio, and the total precursor ows.27,44,117,133–135 In 2013, Lehmann and co- workers44 reported crystal phase control in GaP, GaAs, InP, and InAs by tuning only the group-V precursor ow, while keeping constant all other growth parameters, promoting the bidirec- tional transition from WZ to ZB. They successfully fabricated sharp WZ–ZB and ZB–WZ interfaces. This is a simple yet powerful approach to control the polytypic behaviour of Au- seeded GaP, GaAs, InP, and InAs NWs by only tuning one growth parameter, the group-V precursor ow. They found that Fig. 5 (a–g) Detailed TEM analysis of dual GaAs nanowires grown with a WZ stem and lower, similar, and higher nominal V/III ratios for the top segment with the respective V/III-ratio given at the top right of each ﬁgure. The subﬁgures in each panel include a TEM bright ﬁeld image, a colour-coded TEM dark ﬁeld composite image (red-WZ, green-ZB twin 1, blue-ZB twin 2), a high-resolution TEM image of the interface area, and a SADP with the colour-coding on the pattern as used for the dark ﬁeld images. The scale bar in the SADP insets is 1 nm−1. Fig. 3 from Sebastian Lehmann et al., 2015, Nanotechnology, 26, 301001, reproduced with the permission of IOP Publishing. Fig. 5 (a–g) Detailed TEM analysis of dual GaAs nanowires grown with a WZ stem and lower, similar, and higher nominal V/III ratios for the top segment with the respective V/III-ratio given at the top right of each ﬁgure. The subﬁgures in each panel include a TEM bright ﬁeld image, a colour-coded TEM dark ﬁeld composite image (red-WZ, green-ZB twin 1, blue-ZB twin 2), a high-resolution TEM image of the interface area, and a SADP with the colour-coding on the pattern as used for the dark ﬁeld images. The scale bar in the SADP insets is 1 nm−1. Fig. 3 from Sebastian Lehmann et al., 2015, Nanotechnology, 26, 301001, reproduced with the permission of IOP Publishing. Nanoscale Adv., 2023, 5, 1890–1909 \| 1897 © 2023 The Author(s). Published by the Royal Society of Chemistry Review View Article Online Review View Article Online View Article Online 3.1 Optical properties Similar results were observed for the crystal phase control in InSb NWs. The eﬀect of group-V and group-III ows on morphology and crystal structure in InSb NW was investigated by Anandan and co-workers.134 They observed that the growth of InSb NWs in group-V limited and/or rich regime promoted the ZB crystal structure, while WZ mixed with twinned ZB is promoted when group-V and group-III precursors are eﬀectively balanced. Besides, the WZ formation window is investigated by precisely controlling the group-V ow. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In general, the WZ–ZB interfaces in III–V semiconductors have a type-II (staggered) band alignment.88 A type-II band alignment occurs when, on one side of the junction both, the conduction and valence bands are shied in the same direction (upwards or downwards) with respect to the other side of the junction. The relative position of the lowest conduction band should be above (in energy) the highest valence band. Otherwise, one should talk about type-III (broken gap band alignment). It is worth high- lighting again that the main advantages related to the crystal phase NWQDs are that perfect atomically sharp interfaces can be achieved28,29,84 and the absence of material intermixing.80–82 2.5 Selective core/shell growth Another opportunity oﬀered by the core/shell growth is the realization of crystal phases which otherwise would be impos- sible to obtain by means of the crystal structure transfer method because the shell replicates the crystal structure of the core. This way a material in a non-stable crystal phase can be stabi- lized when growing on the sidewalls of a crystal structure- controlled NW. For example, the lonsdaleite crystal structure of Si has been demonstrated when growing on WZ GaP NWs.152 Another example is the realization of WZ GaSb on InAs crystal structure-controlled NWs. Generally, in III–V NWs the WZ crystal structure is relatively easy to form. This is not the case for III–Sb NWs, where obtaining the WZ crystal structure is highly challenging. The reasons behind this behaviour can be found in the low ionicity of the III–Sb bond and in the surfactant eﬀect of Sb and its eﬀects on the surface energy of the system.153 Those limitations can be overcome when growing a III–Sb shell on a crystal-structure engineered InAs NW under the appropriate growth conditions. By properly tuning the growth conditions, it is possible to realize WZ AlSb151 or WZ GaSb.150 An example of the excellent properties that can be obtained is illustrated by the work done by Geijselaers and co-workers in 2021.28 They fabricated GaAs NWs with individual crystal phase QDs (Fig. 6). The diameters of the NWs ranged between 15 and 25 nm, thus resulting in radial connement of charge carriers. They included a 20 nm thick AlAs passivation layer covered with a 5 nm AlGaAs shell to prevent oxidation. They employed TEM for the structural characterization and photoluminescence spectroscopy (PL) and photoluminescence excitation spectros- copy (PLE) to conrm a sharp density of states (DOS) in the QDs. The normalized power-dependent PL spectra (grey) of a NW containing a ZB-GaAs QD are shown in Fig. 6a. The QD inspected had a diameter of 16.6 nm and a length of 13 nm, as demonstrated by TEM (Fig. 6b and c). For the highest excitation power densities, the spectrum is dominated by the WZ-GaAs band-to-band emission at 1.547 eV. This indicates a radial connement of about 30 meV, comparable with previous reports with similar NWs.162 The PL signal of the WZ band-to- band emission vanishes at low excitation power densities. © 2023 The Author(s). 2.5 Selective core/shell growth They conrmed the labelling of the emission peaks with the PLE spectrum. A good agreement between measured data and calculations (using a one-band eﬀective mass model in full 3D) was found, thus indicating a 0-dimensional DOS at the WZ–ZB GaAs interface. For the 3D calculations, they approximate the NW as a cylinder of WZ-GaAs (diameter of 16.6 nm) with a ZB-GaAs segment of 13 nm with 10 nm of AlAs coaxial with the NW. The vertical lines in Fig. 6a correspond to the calculated values. The calculations show two energy levels separated by 48 meV in the ZB QD, comparable with the measured value of 29 meV. The emission from the ZB QD and the interface when the excitation energy is varied (increasing from top to bottom, as indicated with arrows in the PLE spectrum) is shown in Fig. 6d. When the excitation energy increases, the emission from both the rst QD level and the interface arises. The emission from the second QD level will appear only aer the excitation energy surpasses the WZ-GaAs conned band gap (Eexc = 1.56 eV, purple line). At this point, the emission from the rst QD energy level is strongly increased. State lling could be the reason behind this behav- iour. Below this energy, the QD is excited resonantly, but charge carriers can diﬀuse from the WZ-GaAs barrier into the QD and recombine there. When the number of generated carriers is higher than the emitted through the rst QD energy level, then the emission from the second QD level emerges. This behaviour is diﬀerent from the one reported by Vainorius and co-workers29 for WZ–ZB-GaAs QWs. There the state lling leads to a shiin the type-II emission. They also showed that for the excitation energy of 1.61 eV (cyan line) the emission from the second QD level, and the emission from the interface, completely disap- pears. However, the mechanism behind this behaviour is still unclear. 2.5 Selective core/shell growth Published by the Royal Society of Chemistry 1898 \| Nanoscale Adv., 2023, 5, 1890–1909 1898 View Article Online Review Nanoscale Advances Nanoscale Advances Fig. 6 (a) The normalized PLE (black) and power-dependent PL spectra with varying EPD over four orders of magnitude (grey). The emission from the WZ-GaAs band gap, the ﬁrst and second Q-dot states, and the WZ–ZB GaAs interface are labelled WZ (blue), 1 and 2 (yellow), and I (red), respectively. The calculated transition energies for the ﬁrst and second Q-dot states, as well as the WZ-GaAs band-to-band emission, are indicated with black lines. (b) Bright-ﬁeld TEM image of the nanowire. (c) High-resolution TEM image of the ZB-GaAs dot. (d) PL spectra of the ﬁrst and second Q-dot states and the interface states acquired at diﬀerent excitation energies. An arrow of identical color in the included PLE spectrum indicates the excitation energy for each spectrum. Fig. 3 from Geijselaers. I, et al., Appl. Phys. Lett., 119, 263102, (2021); https://doi.org/ 10.1063/5.0072151, reproduced with the permission of AIP Publishing. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. f Fig. 6 (a) The normalized PLE (black) and power-dependent PL spectra with varying EPD over four orders of magnitude (grey). The emission from the WZ-GaAs band gap, the ﬁrst and second Q-dot states, and the WZ–ZB GaAs interface are labelled WZ (blue), 1 and 2 (yellow), and I (red), respectively. The calculated transition energies for the ﬁrst and second Q-dot states, as well as the WZ-GaAs band-to-band emission, are indicated with black lines. (b) Bright-ﬁeld TEM image of the nanowire. (c) High-resolution TEM image of the ZB-GaAs dot. (d) PL spectra of the ﬁrst and second Q-dot states and the interface states acquired at diﬀerent excitation energies. An arrow of identical color in the included PLE spectrum indicates the excitation energy for each spectrum. Fig. 3 from Geijselaers. I, et al., Appl. Phys. Lett., 119, 263102, (2021); https://doi.org/ 10.1063/5.0072151, reproduced with the permission of AIP Publishing. Three individual peaks, labelled 1, I, and 2, arise at energies of 1.489, 1.508, and 1.518 eV, with full widths at half maximum (FWHM) of 5.5, 7.7, and 6.1 meV, respectively. They correlated peak 1 with the rst QD level, peak I with the type-II interface, and peak 2 with the second QD level. © 2023 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances A magnication of the crossing (OL, OR) = (2, 3) (highlighted in Fig. 7e), together with the electron population, is shown in Fig. 7f. In conductance maps, for a standard DQD congura- tion, sharp corners are interpreted as no inter-dot tunnelling, as it can be observed for the (2,3) crossing. In contrast, Due to the opportunities opened in the design, the electrical characteristics of crystal phase core–shell structures have been intensively investigated. For example, the InAs-GaSb core–shell system is particularly appropriate for devices such as tunnelling eld eﬀect transistors (TFETs) and diodes due to the type II (staggered) band alignment between InAs and GaSb.173 This material system has been extensively investigated in the axial and radial congurations.146,173–176 Namazi and co-workers explored the electrical properties of WZ core/shell InAs/GaSb NWs, where the WZ-GaSb is enabled by the crystal structure transfer method. They observed ambipolar behaviour at 4.2 K, which is indicative of parallel electron and hole channels. Moreover, they observed higher hole conductance in the WZ InAs/GaSb core/shell structure compared to the ZB-GaSb axial Fig. 7 (a) Transmission electron micrograph of a representative nanowire. The centre ZB segment acts as a quantum well accessed by WZ tunnel barriers. (b) SEM of the studied nanowire, overlaid with the contact design. (c) Schematic of the crystal structure and resulting conduction-band alignment. (d) Side-view illustration of the formation of two parallel-coupled QDs. (e) Conductance G as a function of side-gate voltages (VL, VR), where the orbital numbers (OL, OR) are indicated. (f) Magniﬁcation of the (OL, OR) = (2, 3) crossing. Green, red, and yellow arrows indicate important gate vectors, and (NL, NR) represents the electron population on the left and right QD. (g) Measurement of dI/dVds versus Vds along the green gate vector for B‖ = 0.05 T. The Zeeman splitting of the ground state 2EZ is indicated with arrows in the 1e and 3e regimes. From Fig. 1 by Potts, H., Chen, I., Tsintzis, A. et al., Electrical control of spins and giant g-factors in ring-like coupled quantum dots, Nat. Commun., 10, 5740, (2019), https://doi.org/10.1038/s41467-019-13583-7. Is licensed under CC BY 4.0. Fig. 7 (a) Transmission electron micrograph of a representative nanowire. The centre ZB segment acts as a quantum well accessed by WZ tunnel barriers. (b) SEM of the studied nanowire, overlaid with the contact design. (c) Schematic of the crystal structure and resulting conduction-band alignment. Nanoscale Advances Review InAs QDs were sandwiched between WZ-InAs tunnel barriers.171 Thanks to the intrinsic characteristics of the crystal phase approach, they fabricated extremely thin (∼5 nm) ZB-InAs QDs. They employed three gate electrodes to tune the potential prole to either host one or two QDs. Finally, they demonstrated control over the interdot tunnel coupling and determined two coupling regimes, named weak (0.32 meV) and strong (3.1 meV) coupling. Moreover, single-electron transport has also been demonstrated in InAs NWQDs.83,172 In ref. 72, the length of the ZB-InAs QDs is correlated with the frequency of Coulomb oscillations to conrm the role as tunnel barriers of the WZ- InAs segments. They observed the Coulomb blockade eﬀect over a broad range of gate voltages which provided an estima- tion of around +95 meV of the conduction-band oﬀset between WZ- and ZB-InAs. segment. This is indicative of a positive oﬀset in the valence band edge of WZ compared to ZB in agreement with theoretical predictions. 3.2.1 Quantum rings in ZB-InAs NWQDs. Potts and co- workers76 studied quantum rings that appear inside ZB-InAs crystal phase QWs. The InAs crystal phase QDs are shown in Fig. 7a, having a 5 nm ZB segment sandwiched between two WZ segments of about 30 nm that act as tunnel barriers due to the band alignment between WZ and ZB. They fabricated source-, drain- and gate-electrodes (Fig. 7b) to probe the electronic properties of the QW, and to study transport measurements in a dilution fridge equipped with a vector magnet. Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The electrostatics in the QW were manipulated using two side gates (VL and VR) and a back-gate (VBG). This way, two QDs, coupled to source and drain, form in the ZB-InAs segment (Fig. 7d). The DQD is proven by the honeycomb diagram observed in Fig. 7e. The strong connement observed in the ZB-InAs is key in the electron population and occupancies of each QD and in the tunning of the inter-dot tunnel coupling thanks to the connement provided by the crystal phase approach.171 The rst orbital crossing (OL,R = 1) was probed in previous work of the same research group.171,177,178 However, in ref. 76 they focused on the conductance maps of the interactions of higher orbitals. 3.2 Electronic properties A constant quest for architectures where a quantum state, such as spin, can be eﬀectively controlled has been sparked by emerging theoretical concepts for quantum technologies. QDs provide signicant advantages over actual atoms in the manipulation of their properties using electric and magnetic elds. Compared to other systems,163–169 the crystal phase approach presented in this review allows size control in 3-D with atomically abrupt interfaces22,28,135 as presented in Sections 2 and 3.1. The size control, together with the strong spin–orbit interaction and a large g-factor observed for InAs76 and InSb,170 provides a cleaner system to study quantum phenomena not achievable in other systems.163–169 Associations of two quantum dots, in series or in parallel, are named double quantum dots (DQDs) or articial molecules. DQDs allow the control of charge, spin, and orbital interactions and are important systems to study quantum phenomena. Parallel DQDs have been demonstrated in crystal phase InAs NWQDs, where the ZB- © 2023 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2023, 5, 1890–1909 \| 1899 View Article Online Review View Article Online Nanoscale Advances Nanoscale Advances However, in contrast to CNTs, it is possible to control the g- factor from ∼80 to ∼0 using electric elds for the same charge state. Using perturbation theory, they conrmed that ring-like DQDs appear when one odd and one even orbital from each QD hybridize. Fig. 8 shows the conductance and diﬀerential conductance maps for both type A and type B ring-like QDs. The conduc- tance map as a function of VL,R of the type B sample does not show any honeycomb structure (Fig. 8a). This is an indication of transport through a single QD.185 They found that the level structure and magnetic eld evolution (Fig. 8b) are similar to the observed for CNT QDs. From Fig. 8b, the authors extrac- ted gorb = 290 and an upper limit for jgspinj of approximately 15, results that are consistent with those extracted from Fig. 8c. Moving to the type A structure, the conductance map (Fig. 8d) is explained by transport through a DQD parallel- coupled to source and drain.171,186 The numbering of the orbital crossings is indicated in the schematic in Fig. 8e. The authors highlight that the DQD is not ideal and the gate coupling to some orbitals is not constant over the whole measurement interval. They conclude that thanks to the strong phase coherence of the system, uncommon for non- superconducting rings, they could study theoretical predic- tions on the eﬀects of symmetry and parity of a quantum ring. Quantum rings can also be employed to explore the Ahar- onov–Bohm (AB) eﬀect as reported in 2022 by Debbarma and co-workers.181 The AB eﬀect is a quantum mechanical phenomenon in which an electrically charged particle is aﬀected by an electromagnetic potential, despite being conned to a region in which both the magnetic and electric elds are zero.182 In their work, they investigated two diﬀerent kinds of crystal phase ZB-InAs (4–5 nm thick) ring-like QD structures (A and B). A pure ZB-InAs QD for sample A and the same structure together with a thin InAsSb shell for sample B. Fig. 8 Eﬀects of B-ﬁelds on orbital and spin states in QD rings with diﬀerent symmetries (n). (a) Conductance of a QD ring (sample type B) as a function of VL,R at B = 0 (left) and B‖ = 0.2 T (right) for a back-gate voltage, VBG = 3 V, and source-drain voltage, VSD = 0.1 mV. Nanoscale Advances (d) Side-view illustration of the formation of two parallel-coupled QDs. (e) Conductance G as a function of side-gate voltages (VL, VR), where the orbital numbers (OL, OR) are indicated. (f) Magniﬁcation of the (OL, OR) = (2, 3) crossing. Green, red, and yellow arrows indicate important gate vectors, and (NL, NR) represents the electron population on the left and right QD. (g) Measurement of dI/dVds versus Vds along the green gate vector for B‖ = 0.05 T. The Zeeman splitting of the ground state 2EZ is indicated with arrows in the 1e and 3e regimes. From Fig. 1 by Potts, H., Chen, I., Tsintzis, A. et al., Electrical control of spins and giant g-factors in ring-like coupled quantum dots, Nat. Commun., 10, 5740, (2019), https://doi.org/10.1038/s41467-019-13583-7. Is licensed under CC BY 4.0. © 2023 The Author(s). Published by the Royal Society of Chemistry 1900 \| Nanoscale Adv., 2023, 5, 1890–1909 Nanoscale Advances View Article Online Review rounded corners appear when there is a clear hybridization gap as in the (2,2) crossing. Surface band bending in the InAs and in the thin InAsSb shell is crucial for the appearance of the ring-like QD in InAs183 and InAsSb.184 The strong connement inside the QD produced the absence of lled states, while electrons can accumulate close to the surface.76 However, the resulting ring-like states around the NW surface are sensitive to perturbations, resulting in localized electron states.171 They include two diﬀerent samples to enhance the surface electron accumulation and to screen symmetry-breaking eﬀects to further increase the spin–orbit interaction. The diﬀerential conductance, dI/dVds, is represented in Fig. 7g versus the drain-source voltage (Vds) and the side-gate voltages (VL,R) for B‖ = 0.05 T aligned with the NWQD. Char- acteristic Coulomb diamonds are obtained in the dI/dV plot as a function of Vds and VL,R (Fig. 7g) for the 1, 2, and 3 electron regimes. The eﬀective g-factors obtained (∼64 and 40) were signicantly larger than those for bulk InAs (−14.9).179,180 Open Access Article. Published on 06 March 2023. Downloaded on 10/24/2024 6:25:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Moreover, they found interacting orbitals exhibiting an electronic structure almost identical to carbon nanotube (CNT) QDs (a quantum ring platform with a fourfold orbital- and spin-degeneracy, and anisotropic eﬀective g-factors), with highly anisotropic g-factors varying from 3 to 80. 4. Conclusions In this review article, we make a comprehensive revision of the eld of crystal phase NWQDs. Traditionally NWQDs are formed via heterostructure interfaces between materials with diﬀerent band gaps. However, their perfection is limited by the diﬃculty to control the interface abruptness and material composition at the atomic level, which unfortunately are determining factors for the physical properties of QDs as they dene the shape of the potential well. Here, we discuss the growth of NWQDs in the axial direction varying the crystal phase, instead of the material composition, emphasizing the advantages oﬀered by this approach. As this method employs a change in crystal structure instead of a change in material, then perfectly abrupt interfaces can be achieved. However, the band oﬀset between WZ and ZB is xed and relatively small, limiting the application of crystal phase QDs to liquid-He temperatures and to elds where the purity of the interface is more critical than the band oﬀset. This is the case of the inspection of fundamental optical and quantum eﬀects. We review the concepts of WZ and ZB crystal structures, supersaturation, and the diﬀerent options available to control the crystal structure. For controlling the crystal phase, scientists have developed a method where only the group-V supply should be changed. In addition, diﬀerent crystal pha- ses oﬀer diﬀerent lateral facet terminations and, consequently, diﬀerent surface energies, allowing to grow the shell in a selec- tive fashion. That is, the shell can be grown either on the WZ or on the ZB phase of the core, thus leading to the formation of ring-like structures or enabling the growth of crystal phases that would be otherwise very diﬃcult, if not impossible, to obtain. As an example, we describe the case of WZ-GaSb. Then, in Section 3, we discuss the optical and electronic properties of crystal phase NWQDs. The optical properties are focused on the type II transitions that appear in ZB-GaAs QDs and on the atomically sharp nature of the interface between WZ and ZB. Regarding the electronic properties, we mainly analyse the coupling between two QDs (forming DQDs) and the ring-like behaviour of ZB-InAs parallel-coupled DQDs. We strongly believe that crystal phase NWQDs are an excellent platform to discover new quantum phenomena either to probe theoretical models or to engineer new material architectures by means of the crystal structure transfer method. 3 G. Bulgarini, M. E. Reimer, T. Zehender, M. Hocevar, E. Conﬂicts of interest 12 E. D. Minot, F. Kelkensberg, M. van Kouwen, J. A. van Dam, L. P. Kouwenhoven, V. Zwiller, M. T. Borgström, O. Wunnicke, M. A. Verheijen and E. P. A. M. Bakkers, Single Quantum Dot Nanowire LEDs, Nano Lett., 2007, 7, 367–371. There are no conicts to declare. Nanoscale Advances In this case, the crystal phase approach opens the possibility to engineer new material and crystal phase combinations that could provide an even more eﬃcient manipulation of elec- tronic states in ring-like DQDs. Notes and references 1 W. 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The small inset shows an overlay of data from panel d at the two ﬁelds. From Fig. 2 by Debbarma. R, et al., Nano Lett., 2022, 22, 1, 334– 339, https://doi.org/10.1021/acs.nanolett.1c03882 is licensed under CC BY 4.0. Fig. 8 Eﬀects of B-ﬁelds on orbital and spin states in QD rings with diﬀerent symmetries (n). (a) Conductance of a QD ring (sample type B) as a function of VL,R at B = 0 (left) and B‖ = 0.2 T (right) for a back-gate voltage, VBG = 3 V, and source-drain voltage, VSD = 0.1 mV. (b) Conductance along the red line in panel a vs. Bt and B‖. (c) Diﬀerential conductance vs. B‖ in the one-electron regime along the green bar in panel B. The inset shows schematically how the four nearly degenerate spin and orbital states evolve with B‖, where it is possible to identify EZ and extract gorb from EZ = gorbmBB‖. (d) Conductance of a double QD ring (sample type A) for B = 0 and B‖ = 0.7 T at VBG = 0 V and VSD = 0.5 mV. 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https://openalex.org/W2902755028	https://zenodo.org/record/3746870/files/hono_decomp_v5_CFG.pdf	English	null	Decomposition kinetics for HONO and HNO<sub>2</sub>	Reaction chemistry & engineering	2,019	cc-by	9,421	p g Email address: franklin_goldsmith@brown.edu (C. Franklin Goldsmith) Decomposition Kinetics for HONO and HNO2 Xi Chena,1, Mark E. Fullerb,1, C. Franklin Goldsmithb,∗ ∗Corresponding author: C. Franklin Goldsmith g ( ) 1These authors contributed equally to this work. g ( 1These authors contributed equally to this work. Decomposition Kinetics for HONO and HNO2 Xi Chena,1, Mark E. Fullerb,1, C. Franklin Goldsmithb,∗ aDepartment of Chemistry, Brown University, Providence, RI, 02912, USA bSchool of Engineering, Brown University, Providence, RI, 02912, USA Decomposition Kinetics for HONO and HNO2 Xi Chena,1, Mark E. Fullerb,1, C. Franklin Goldsmithb,∗ aDepartment of Chemistry, Brown University, Providence, RI, 02912, USA bSchool of Engineering, Brown University, Providence, RI, 02912, USA Corresponding author: C. Franklin Goldsmith Preprint submitted to Reaction Chemistry and Engineering ∗Corresponding author: C. Franklin Goldsmith Email address: franklin_goldsmith@brown.edu (C. Franklin Goldsmith) 1 Abstract This work presents a detailed investigation into the isomerization and decom- position of HONO and HNO2. State-of-the-art electronic structure theory is used to compute the HNO2 potential energy surface. Temperature and pressure dependent rate coeﬃcients are computed using microcanonical rate theory and the master equation. The electronic structure theory properties are optimized against the relevant experimental data. A novel strategy was developed to incorporate uncertainty in the minimum energy pathway into the optimized mechanism. The new mechanism is in excellent agreement with all available experimental data for H + NO2 →OH + NO and OH + NO →HONO. The calculations identify OH + NO as the dominant prod- ucts for HNO2, which were neglected from all previous mechanisms in the literature. Keywords: HONO, HNO2, nitrogen chemistry, master equation combustion Preprint submitted to Reaction Chemistry and Engineering October 4, 2018 October 4, 2018 1. Introduction Nitrous acid (HONO) and its isomer nitryl hydride (HNO2) are key in- termediates in many gas-phase systems, from atmospheric chemistry to com- bustion to energetic materials. The most common source of these two species is through H-abstraction or disproportionation involving NO2. Additionally, HONO can be formed via concerted elimination from various NO2-containing functional groups, such as nitroalkanes (C−NO2), alkylnitrates (O−NO2), and nitroamines (N−NO2). In contrast, concerted HNO2 elimination from the analogous nitrite compounds, X−ONO, does not exist as a viable product channel, owing to the enthalpically and entropically more favorable O−NO ﬁssion. Recent developments in advanced engines, such as exhaust gas recircu- lation (EGR) and the use of alkyl-nitrates as reactivity enhancers, create new environments in which NO2 chemistry is increasingly important [1–3]. One way in which NO2 contributes to increased reactivity (e.g. decreased ignition delay times) is through various radical + NO2 reactions that shift the composition from comparatively less reactive radicals to more reactive radicals [4–6]: R + NO2 ⇌RO + NO (R1) RO2 + NO ⇌RO + NO2 (R2) RO →→carbonyl + alkene + H (R3) H + NO2 ⇌OH + NO (R4) R + NO2 ⇌RO + NO (R1) RO2 + NO ⇌RO + NO2 (R2) R + NO2 ⇌RO + NO (R1) (R1) RO2 + NO ⇌RO + NO2 (R2) RO →→carbonyl + alkene + H (R3) RO →→carbonyl + alkene + H (R3) H + NO2 ⇌OH + NO (R4) H + NO2 ⇌OH + NO (R4) 2 An alternative pathway involves the abstraction of H from a fuel molecule: RH + NO2 ⇌R + HONO/HNO2 (R5) HNO2 ⇌HONO (R6) HONO ⇌OH + NO (R7) HNO2 ⇌OH + NO (R8) RH + NO2 ⇌R + HONO/HNO2 (R5) HNO2 ⇌HONO (R6) HONO ⇌OH + NO (R7) HNO2 ⇌OH + NO (R8) (R5) Whereas the reaction sequence (R1-R4), is chain propagating, the reac- tion sequence (R5-R8) is chain branching. Recent experimental and modeling work has highlighted the sensitivity of ignition delay times to this reaction se- quence for H2, CH4, and C3H8 [7–11]. One of the authors recently published rate coeﬃcients for (R5) obtained from transition state theory (TST) calcu- lations for a broach range of fuels.[12] The HNO2 potential energy surface (PES), shown in Figure 1. As seen in Figure 1, two diﬀerent rotational conformers of HONO ex- ist. 1. Introduction The more stable rotamer, anti-HONO (frequently referred to as “trans- HONO” in the literature), is when the HO−NO dihedral angle is 180◦. At present, there is little consistency in the combustion literature on whether both anti-HONO and syn-HONO should be included, or if they should be lumped into a single species. The barrier for torsional rotation between the two conformers is 44 kJ/mol (10.6 kcal/mol), which is large enough that these two species can persist as distinct at elevated temperatures. As demonstrated in Ref. 12, these two rotamers have diﬀerent reactivity. The aim of the present work is to provide an accurate analysis of the 3 3 O N O H O N O H OH + NO H + O N O H energy (kJ/mol) -94 -124 -280 -325 0 -288 -323 O N O 38 Figure 1: Potential energy diagram illustrating the major stationary points on the HNO2 PES, energies relative to the most stable isomer. Figure 1: Potential energy diagram illustrating the major stationary points on the HNO2 PES, energies relative to the most stable isomer. HNO2 potential energy surface and the kinetics of reactions (R4, R6-R8). The approach will be to use state-of-the-art methods in electronic structure theory and computational kinetics to provide temperature- and pressure- dependent rate coeﬃcients. The electronic structure and collisional energy transfer parameters are then optimized against diverse experimental data to provide a rigorously optimized system of rate constants. 4 4 2.1. Electronic Structure Theory The stationary points on the HNO2 potential energy surface (PES) were computed using a compound method that is based upon the ANL1 schema[13]. Brieﬂy, geometry optimization and normal mode analysis were performed using UCCSD(T)/cc-pVQZ. The optimized geometries were followed by a sequence of single-point calculations to correct for: (i) basis set extrapola- tion, using aug-cc-pVQZ (aqz) and aug-cc-pV5Z (a5z) basis sets, ECBS; (ii) higher-order excitations that include perturbative quintuples with cc-pVDZ and cc-pVTZ basis sets, ∆ET/Q; (iii) anharmonic corrections, based upon density functional theory calculations, ∆Eanh; (iv) core-valence interactions, which include the core electrons in the coupled-cluster calculations, with the cc-pcVQZ (cqz) and cc-pcV5Z (c5z) basis sets, ∆Ec.v.; and (v) relativis- tic eﬀects, as estimated by the diﬀerence in the CCSD(T) energy with and without the Douglas-Kroll one-electron integrals with the aug-cc-pcVQZ-DK basis (acQZ-DK), ∆Erel. The zero-point energy, ZPE, is that obtained from the UCCSD(T)/cc-pVQZ normal mode analysis. This compound method should be accurate to within ± 1.3 kJ/mol at 2σ uncertainty for local min- ima; for saddle points, we assume that the uncertainty is twice as high. For a more detailed discussion of these corrections and their contributions to the total energy, the reader is referred to ref. 13. 5 5 EANL1 = ECBS + ZPE + ∆ET/Q + ∆Eanh + ∆Ec.v. + ∆Erel (1) (1) ECBS = EUCCSD(T)/a5z − 54 64 −54 EUCCSD(T)/a5z −EUCCSD(T)/aqz ∆ET/Q = EUCCSDT(Q)/dz −EUCCSD(T)/dz ∆ET/Q = EUCCSDT(Q)/dz −EUCCSD(T)/dz ∆Eanh = ZPEanh B3LYP/6-311++G(d,p) −ZPEharm B3LYP/6-311++G(d,p) ∆Ec.v. = EUCCSD(T,full)/CBS(cqz, c5z) −EUCCSD(T)/CBS(cqz, c5z) ∆Erel = EUCCSD(T)/acQZ-DK −EDK-UCCSD(T)/acQZ-DK As depicted in Figure 1, the H + NO2 and the OH + NO pathways do not have ﬁrst-order saddle points in potential energy. The minimum energy paths that are required for the variational transition state theory were treated us- ing the multi-reference methods CASPT2[14–16] and MRCI+Q[17–19]. The active space in the H + NO2 calculations was 14 electrons in 11 orbitals (14e,11o): 6 orbitals for the delocalized π-system of NO2, 4 orbitals for the two pairs of N−O σ, σ∗in NO2, and 1 orbital for the H atom (this active space is eﬀectively equivalent to the complete valence space for the system, not including the two 2s orbitals for the oxygen atoms). The CASPT2 and MRCI+Q calculations were performed with aug-cc-pVQZ and aug-cc-PV5Z basis sets and extrapolated to the complete basis set limit. 2.1. Electronic Structure Theory To further im- prove the accuracy of the multi-reference calculations, a separate set of po- tentials was computed on a higher spin surface[20–23]. The potential energy of the triplet surface was computed using both the ANL1 method of Equation (1) as well as the CASPT2 and MRCI+Q approaches. The coupled-cluster triplet surface is then added to the multi-reference singlet-triple splitting to obtain a more accurate representation of the minimum energy path on the 6 singlet surface: singlet surface: 1EANL1/MR = 3EANL1 + 1EMR −3EMR (2) (2) where the superscript indicates the spin multiplicity of the surface, and “MR” implies either CASPT2 or MRCI+Q with aug-cc-pV∞Z basis sets. The H + NO2 pathway leading to anti-HONO, despite being a radical- radical reaction, has a well-deﬁned ﬁrst-order saddle point, depicted by the dotted line in Figure 1. This transition still has a strong bi-radical character, and multi-reference methods were necessary. The transition state optimiza- tion was performed using CASPT2(14e,11o)/aug-cc-pVQZ, with the same active space as above. The spin-splitting method of (2) was applied to fur- ther improve the accuracy. This local maximum is a consequence of the nodal planes in the NO2 singularly occupied molecular orbital (SOMO); as the H atom approaches either O atom in the anti conﬁguration, it experi- ences signiﬁcant overlap with the lobes of opposite sign on the N atom on syn conﬁguration of the O atoms[12]. For the OH + NO potentials, the active space was (8e,6o): 4 orbitals for the complete π system in NO and 2 orbitals for the π system in OH. The orbitals in these calculations were averaged over four states to account for the spatial degeneracy in both OH and NO on the singlet surface. Because of the low-lying excited states, single determinant methods were not reliable, and the singlet-triplet splitting approach in Equation (2) could not be applied. All wavefunction-based calculations were performed in Molpro.[24] The density functional theory calculations used for the anharmonic correction were done in Gaussian09[25]. 7 7 2.2. Computational Kinetics 2.2. Computational Kinetics Reactions (R4) and (R6)-(R8) proceed through one or more unimolecular intermediates, and thus the rate constants are a function of pressure. The temperature- and pressure-dependent rate constants are computing using the microcanonical rate theory and the master equation (RRKM/ME) code Mess[26, 27], which is part of the computational kinetics package Papr developed by Argonne National Laboratory[28]. A single exponential was used to model the collisional energy transfer, with a preliminary value of ⟨∆Edown⟩= 200 (T/298[K])0.85 cm−1; the pre-factor will be optimized, as described in the next section. The collision frequency is computed using a Lennard-Jones model with either He or N2 as a collider, with σHe = 2.6 ˚A, σN2 = 3.3 ˚A, σHONO = 5.1 ˚A, ϵHe = 0.06 kJ/mol, ϵN2 = 2.2 kJ/mol, and ϵHONO = 2.0 kJ/mol. For OH and NO, the low-lying excited states that result from spin-orbit splitting were included, with a separate energy levels of 140 cm−1 and 112 cm−1 for OH and NO, respectively.[29] In the time-dependent master equation, the eigenvalues of the transi- tion matrix can be separated into chemically signiﬁcant eigenvalues (CSE) and internal energy relaxation eigenvalues (IERE), with the former typically smaller than the latter by several orders of magnitude[30, 31]. When a CSE approaches the smallest IERE, it indicates that a pair of wells (or a well and bimolecular products) will equilibrate on a timescale that approaches the rate of ro-vibrational energy relaxation. When this merging of timescales occurs, it no longer makes sense to think of these species as being chemically distinct in a macroscopic model[32]. An important feature of Mess is the ability to determine when two wells equilibrate rapidly (e.g. at high temperature) and 8 combine them accordingly[26]. combine them accordingly[26]. This “well-merging” capability is important for the HNO2 reactive sys- tem. At low temperatures, the two HONO rotamers remain chemically dis- tinct. At higher temperatures, however, the chemically signiﬁcant eigenvalue that corresponds to the interconversion between anti-HONO and syn-HONO blends into the IERE continuum. Preliminary calculations suggest that this merging occurs at 650 K and 0.01 bar, and at 800 K and 100 bar. Above these temperatures, they exist only as a single HONO. Below these tem- peratures, when the two rotamers are distinct, the rate constant for their interconversion is large (e.g. 2 × 109 s−1), and they will rapidly equilibrate in the conventional sense. 2.2. Computational Kinetics Precisely how species merging should be handled at high temperatures remains an open challenge in reactive ﬂow simulations, since it represents a reduction in the number of species (and accordingly the state vector)[33]. Since our primary interest is in high-temperature kinetics, we lump these two species into a single HONO isomer. In this two-well model, with HONO and HNO2 as distinct isomers, the interconversion between anti- and syn-HONO is treated as hindered internal rotation; the partition function was computed via summation over the energy levels for the corresponding 1D Schr¨odinger equation. For the two remaining transition states with saddle points, H + NO2 ⇌ anti-HONO and HNO2 ⇌anti-HONO, the microcanonical rate constants were computed using conventional rigid-rotor harmonic-oscillator approxi- mations. The transition states for the barrierless reactions are computed using Variable Reaction Coordinate Transition State Theory (VRC-TST)[34–37], 9 9 as implemented in VaReCoF in Papr[38, 39]. In VRC-TST, the internal degrees of freedom of the transition state are divided into conserved and transitional modes. The conserved modes correspond to the internal degrees of freedom of the two fragments at inﬁnite separation. The transitional modes are the coupled, anharmonic modes that correspond to relative translation and rotation at inﬁnite separatation and are converted into vibrational modes in the equilibrium structures. Note that one of the transitional modes is the reaction coordinate. The transitional modes are large in amplitude (if treated harmonically, they would have very low vibrational frequencies), and so these modes are treated using classical phase space theory representations[40]. The multi-dimensional partition function is evaluated via Monte Carlo sampling; the potential energy is computed on-the-ﬂy by calling Molpro to return the CASPT2/cc-pVTZ energy for a given set of Cartesian coordinates. A sequence of 1D corrections are included to account for geometry re- laxation and basis set extrapolation. First, for a ﬁxed bond distance r (e.g. N−O in OH + NO →HONO), the internal degrees of freedom for the two fragments are kept frozen at their respective equilibrium geometries, and the degrees of freedom for the relative position are relaxed at the CASPT2/cc- pVTZ level, Vconstained(r). In the next set of calculations, the internal degrees of freedom are no longer held rigid, and all degrees of freedom (apart from r) are relaxed, Vrelaxed(r). The diﬀerence in these two V (r) is the 1D geometry relaxation correction. 2.2. Computational Kinetics Additionally, single-point calculations are performed using larger basis sets for the fully relaxed case, which can then be used to provide a basis set correction. As will be detailed below, these 1D correc- tions can be manipulated to explore how the uncertainty in the interaction 10 potential corresponds to uncertainties in the ﬁnal rate constants. For the H + NO2 reactions, the H atom can add to either the N or the O atoms. Within the VRC-TST methodology, the ﬂux to these diﬀerent products (HNO2 and HONO, respectively) must be determined separately. Accordingly, a plane of inﬁnite potential was imposed on the NO2, normal to the plane[39, 41]. For H + NO2 →HNO2, the plane is deﬁned such that bonding to the O atoms is impossible, and for H + NO2 →HONO, the plane is in the same location, but with a sign change, such that bonding to the N atom is impossible. The location of this dividing surface was optimized variationally so as to minimize the total H + NO2 ﬂux. The rate constants are computed at temperatures 200 ≤T ≤2500 K and pressures 0.01 ≤P ≤100 bar. 2.3. Optimization Methodology A key aspect of the present work is the inclusion of uncertainty in each of the model parameters. Each parameter can be adjusted within its upper and lower bound as a means of providing global uncertainty quantiﬁcation. Moreover, this ensemble of models can then be compared to diverse exper- imental data to determine the optimum set of model parameters that best reproduce the data. This form of optimization is similar to the Multiscale Informatics (MSI) of Burke and coworkers[42–45]. Previous implementations of the MSI methodology focused on stationary points on the PES, as well as vibrational frequencies and energy transfer models. The present work is the ﬁrst extension of the MSI framework to include the potential energy surface for barrierless reactions. 11 1.5 2.0 2.5 3.0 3.5 4.0 r [Å] 0.50 0.25 0.00 0.25 0.50 scaling factor, f Figure 2: Scaling factors used to create an array of correction potentials. 1.5 2.0 2.5 3.0 3.5 4.0 r [Å] 0.50 0.25 0.00 0.25 0.50 scaling factor, f Figure 2: Scaling factors used to create an array of correction potentials. To explore how the uncertainty in the minimum energy pathway for bar- rierless reactions could inﬂuence the ﬁnal rate constants, a family of scaling factors, f, were created, Figure 2. Starting from the average value (the hor- izontal, wide-dashed black line at f = 0.0), the minimum energy path can be scaled down (up) to produce a curve that is more (less) attractive than the nominal case. For example, the dotted blue line at f = 0.5 represents an increase in the potential of 50% for all r, and the densely dashed magenta line at f = −0.25 represents a new potential that is more attractive by 25%. More importantly, additional curves are created that will cross the average value: some curves may be less attractive at longer distances and more at- tractive at shorter distances, such has the solid green line, which begins at f = 0.25 at r > 4 and limits to f = −0.5 at r < 1.4. The limits of this 12 switching function were chosen so that the variability is greatest over the region of the reaction coordinate where the dynamical bottleneck is mostly likely to occur; for radical-radical reactions above room temperature, the bottleneck is typically found between −50 ≤V (r) ≤−1 kJ/mol. 2.3. Optimization Methodology The scaled potential is given by: Vnew (r) = Vavg (r) (1 −f (r)). These new potentials are incorporated into the VRC-TST framework as 1D cor- rection factors: ∆E1D = Vnew (r) −Vconstained (r). As depicted in Figure 2, since there are 25 scaling factors f, there will be 25 ∆E1D to consider. In the VaReCoF calculations, a separate variational optimization is per- formed for each of the 25 corrections, thereby providing a unique micro- canonical rate constant for each scaling factor. For H + NO2 →HNO2 and H + NO2 →HONO, the average value is the arithmetic mean of the CASPT2(14e,11o)/aug-cc-PV∞Z and MRCI+Q(14e,11o)/aug-cc-PV∞Z, both with and without the spin correction of Equation (2). For OH + NO → HONO, the average value is the arithmetic mean of the CASPT2(8e,6o)/aug- cc-PV∞Z and MRCI+Q(8e,6o)/aug-cc-PV∞Z. The MSI variables are the eight stationary points in Table 1, with the upper and lower limits as expected by the ANL1 method; the collisional en- ergy transfer pre-factor, 100 ≤∆Edown ≤500 cm−1, and the scaling factors, f, for H + NO2 →HNO2, H + NO2 →HONO, and OH + NO →HONO. Two sets of experimental data are used to constrain the model parame- ters. The ﬁrst set of experiments are for the H + NO2 →OH + NO reaction, which includes ﬂow cell data at lower temperatures[46], high-pressure ﬂow cells at intermediate temperatures[47], and shock tube measurements using two diﬀerent diagnostics at high temperatures[48]. These data primarily The MSI variables are the eight stationary points in Table 1, with the upper and lower limits as expected by the ANL1 method; the collisional en- ergy transfer pre-factor, 100 ≤∆Edown ≤500 cm−1, and the scaling factors, f, for H + NO2 →HNO2, H + NO2 →HONO, and OH + NO →HONO. Two sets of experimental data are used to constrain the model parame- ters. The ﬁrst set of experiments are for the H + NO2 →OH + NO reaction, which includes ﬂow cell data at lower temperatures[46], high-pressure ﬂow cells at intermediate temperatures[47], and shock tube measurements using two diﬀerent diagnostics at high temperatures[48]. These data primarily 13 constrain the two H + NO2 interaction potentials. The second set of ex- perimental data are the high-pressure ﬂow cell work by Troe and coworkers on OH + NO →HONO[49]. These data primarily constrain the OH + NO interaction potential and the collisional energy transfer coeﬃcient, ∆Edown. 3. Results and Discussion relative energy [kJ/mol] Name PW ATcT[50] H + NO2 0.0 0.0 OH + NO -124.4 ± 1.3 -125.0 ± 0.03 HNO2 -288.0 ± 1.3 -290.0 ± 1.40 syn-HONO -323.0 ± 1.3 -324.3 ± 0.28 anti-HONO -324.7 ± 1.3 -325.9 ± 0.05 TS: syn-HONO ⇌anti-HONO -280.4 ± 2.6 TS: HNO2 ⇌anti-HONO -93.9 ± 2.6 TS: anti-HONO ⇌H + NO2 38.4 ± 2.6 Table 1: key stationary points on the HNO2 potential energy surface, relative to H + NO2. relative energy [kJ/mol] Name PW ATcT[50] H + NO2 0.0 0.0 OH + NO -124.4 ± 1.3 -125.0 ± 0.03 HNO2 -288.0 ± 1.3 -290.0 ± 1.40 syn-HONO -323.0 ± 1.3 -324.3 ± 0.28 anti-HONO -324.7 ± 1.3 -325.9 ± 0.05 TS: syn-HONO ⇌anti-HONO -280.4 ± 2.6 TS: HNO2 ⇌anti-HONO -93.9 ± 2.6 TS: anti-HONO ⇌H + NO2 38.4 ± 2.6 Table 1: key stationary points on the HNO2 potential energy surface, relative to H + NO2. Table 1: key stationary points on the HNO2 potential energy surface, relative to H + NO2. The ANL1 results for the stationary points on the PES are presented in Table 1. Also included in the table are the ATcT results. The root mean square deviation between the two methods was 0.7 kJ/mol. 14 3.1. H + NO2 →products 2.0 2.5 3.0 3.5 4.0 50 40 30 20 10 0 V [kJ/mol] (a) aqz a5z a∞z CASPT2 CASPT2 w/ CC MRCI MRCI w/ CC CASPT2 CASPT2 w/ CC MRCI MRCI w/ CC 2.0 2.5 3.0 3.5 4.0 1.5 1.0 0.5 0.0 0.5 1.0 1.5 V - Vavg [kJ/mol] (b) 2.0 2.5 3.0 3.5 4.0 r [Å] 10 5 0 5 10 percent difference (c) 2.0 2.5 3.0 3.5 4.0 50 40 30 20 10 0 V [kJ/mol] (a) aqz a5z a∞z CASPT2 CASPT2 w/ CC MRCI MRCI w/ CC CASPT2 CASPT2 w/ CC MRCI MRCI w/ CC 2.0 2.5 3.0 3.5 4.0 1.5 1.0 0.5 0.0 0.5 1.0 1.5 V - Vavg [kJ/mol] (b) 2.0 2.5 3.0 3.5 4.0 r [Å] 10 5 0 5 10 percent difference (c) Figure 3: (a) minimum energy path between H + NO2 and HNO2. The colors correspo to diﬀerent methods, and the line styles correspond to diﬀerent basis sets. (b) diﬀere between the potential and the average of the four methods at the aug-cc-pV∞Z limit; the percent diﬀerence between the methods and the average. 3. Results and Discussion 15 V - Vavg [kJ/mol] Figure 3: (a) minimum energy path between H + NO2 and HNO2. The colors correspond to diﬀerent methods, and the line styles correspond to diﬀerent basis sets. (b) diﬀerence between the potential and the average of the four methods at the aug-cc-pV∞Z limit; (c) the percent diﬀerence between the methods and the average. 15 The results of the H + NO2 →HNO2 potential are shown in Figure 3. The four colors correspond to the four diﬀerent methods that were used to compute the minimum energy pathway: pure CASPT2(14e,11o), cyan; pure MRCI+Q(14e,11o), magenta; ANL1 on the triplet surface, with CASPT2(14e,11o) singlet-triplet splitting, blue; and ANL1 on the triplet surface, with MRCI+Q(14e,11o) singlet-triplet splitting, red. Each of these four methods was computed using both aug-cc-pVQZ and aug-cc-pV5Z basis sets and then extrapolated to the CBS limit. As can be seen in the ﬁgure, all four methods are well converged with respect to the basis set. Figure 3b plots the diﬀerence between each of the four methods and the geometric mean of the four (at aug-cc-pV∞Z). Of greater signiﬁcance is the percent deviation of the methods from the average, Figure 3c, since the percent deviation is a better reﬂection on the ﬁnal uncertainty of the transition state. Note that all twelve curves agree to within 5%. In principle, it is diﬃcult to say exactly what the “true” value for the potential is. However, given the degree of convergence, we estimate that the average value for the minimum energy path is likely within 25% of the correct value. 16 2.0 2.5 3.0 3.5 4.0 50 40 30 20 10 0 V [kJ/mol] (a) average ±25% ±50% 2.0 2.5 3.0 3.5 4.0 r [Å] 50 40 30 20 10 0 V [kJ/mol] (b) 25 different potentials within estimated uncertainty Figure 4: (a) minimum energy path between H + NO2 and syn-HONO. The solid black line the the average potential of the four diﬀerent methods. The shaded regions correspond to the 25% and 50% uncertainty regime. (b) The 25 scaled potentials generated according to the curves in Figure 2. 3. Results and Discussion 2.0 2.5 3.0 3.5 4.0 50 40 30 20 10 0 V [kJ/mol] (a) average ±25% ±50% (a) 2.0 2.5 3.0 3.5 4.0 r [Å] 50 40 30 20 10 0 V [kJ/mol] (b) 25 different potentials within estimated uncertainty 25 different potentials within estimated uncertainty Figure 4: (a) minimum energy path between H + NO2 and syn-HONO. The solid black line the the average potential of the four diﬀerent methods. The shaded regions correspond to the 25% and 50% uncertainty regime. (b) The 25 scaled potentials generated according to the curves in Figure 2. Figure 4a presents the minimum energy path between H + NO2 →syn- HONO; the blue shaded region represents an uncertainty envelope of 25%, and the yellow region 50% uncertainty in the minimum energy path. The degree of convergence for this potential was the same as for the competing 17 H + NO2 →HNO2 pathway, shown in Figure 3. Figure 4b illustrates the 25 Vnew (r) that are created from the scaling factors f in Figure 2. H + NO2 →HNO2 pathway, shown in Figure 3. Figure 4b illustrates the 25 Vnew (r) that are created from the scaling factors f in Figure 2. 3.2. OH + NO →products 2.0 2.5 3.0 3.5 4.0 4.5 5.0 r [Å] 25 20 15 10 5 0 5 V [kJ/mol] dividing surface at 300 K dividing surface at 400 K 1E0 1E1 1E2 1E3 3E0 3E1 3E2 3E3 3E0 3E1 3E2 3E3 Figure 5: Interaction potentials for OH + NO as a function of the N−O distance. The blue curves correspond to the singlet surface, and the red curves the triplet. The broken lines are for the excited states for each electronic spin. The black hexagons indicate the location of the dividing surface on the ground state singlet as a function of temperature. 3.2. OH + NO →products 2.0 2.5 3.0 3.5 4.0 4.5 5.0 r [Å] 25 20 15 10 5 0 5 V [kJ/mol] dividing surface at 300 K dividing surface at 400 K 1E0 1E1 1E2 1E3 3E0 3E1 3E2 3E3 3E0 3E1 3E2 3E3 Figure 5: Interaction potentials for OH + NO as a function of the N−O distance. The blue curves correspond to the singlet surface, and the red curves the triplet. The broken lines are for the excited states for each electronic spin. 3. Results and Discussion The black hexagons indicate the location of the dividing surface on the ground state singlet as a function of temperature. The results for the OH + NO interaction potential are shown in Figure 5. Proper calculation of the rate constant for OH + NO →HONO is compli- cated by non-adiabatic eﬀects, with four states on the singlet surface (blue curves) and four states on the triplet surface (red). For r ≥3.5 ˚A, all eight curves are attractive, but only the ground state singlet remains attractive 18 for r ≤2.4 ˚A. A detailed analysis of the contribution of these higher states would require non-adiabatic dynamics simulations, which is beyond the scope of the present work. Further complications are spin-orbit eﬀects and rovi- bronic coupling at lower temperatures. Instead, we note that these eﬀects become increasingly irrelevant at higher temperatures. To quantify the temperature at which these eﬀects become negligible, we consider a limiting case in which the crossing from the excited electronic and spin states to the ground state singlet is rapid. In this approximation, the sum of the contributions to the interaction potential should be included prior to the variational optimization of the dividing surface. From the VRC- TST analysis, which used only the singlet ground state (solid blue line in Figure 5), we can obtain the location of the optimum dividing surface as a function of temperature. At 300 K, it is approximately r = 3.6 ˚A, and the contribution of the other surfaces is not insigniﬁcant (nearly a factor of three). As the temperature is increased, the optimum dividing surface shifts to shorter separation distances. By 400 K, the dividing surface is located at r = 2.8 ˚A; under these conditions, the contribution of the seven additional states is a mere 4%. This value likely is an over-estimate, since our strong- coupling assumption may be appropriate for the exited singlet states, but is less likely to be appropriate for the triplet surface. (The other limiting model is to assume that the crossing is slow, in which case the variational analysis is performed on each surface independently. Given that the other surfaces have local minima between −3.8 and −0.5 kJ/mol, their ﬂux through the dividing surface will be comparatively negligible.) For these reasons, we do not include experimental data below 400 K in the optimization procedure, 19 since it would bias the result towards more attractive minimum energy paths. 3. Results and Discussion Instead, we restrict ourselves to the 400 K data of Fulle et al. since it would bias the result towards more attractive minimum e Instead, we restrict ourselves to the 400 K data of Fulle et al. Instead, we restrict ourselves to the 400 K data of Fulle et al. 3.3. Optimized Mechanism 0.5 1.0 1.5 2.0 2.5 1000/T [K−1] 10-10 10-9 k [cm3/molecule-s] ANL (2002) Princeton (2015) Fontijn (1991) ±50% ±25% nominal optimized Figure 6: Rate constants for H + NO2 →OH + NO. The symbols correspond to the experimental data: the blue diamonds are the high-temperature shock tube from ANL [48]; the red squares are the intermediate temperature ﬂow tube from Princeton [47], and the cyan circles are the low-temperature ﬂow cells of Fontjin [46]. The solid black line is the VRC-TST result for the nominal case, and the solid red line is the optimized result. The dashed magenta line is the linear least squares ﬁt to the experimental data. 3.3. Optimized Mechanism 0.5 1.0 1.5 2.0 2.5 1000/T [K−1] 10-10 10-9 k [cm3/molecule-s] ANL (2002) Princeton (2015) Fontijn (1991) ±50% ±25% nominal optimized 3.3. Optimized Mechanism 0.5 1.0 1.5 2.0 2.5 1000/T [K−1] 10-10 10-9 k [cm3/molecule-s] ANL (2002) Princeton (2015) Fontijn (1991) ±50% ±25% nominal optimized 3.3. Optimized Mechanism 3.3. Optimized Mechanism nominal optimized Figure 6: Rate constants for H + NO2 →OH + NO. The symbols correspond to the experimental data: the blue diamonds are the high-temperature shock tube from ANL [48]; the red squares are the intermediate temperature ﬂow tube from Princeton [47], and the cyan circles are the low-temperature ﬂow cells of Fontjin [46]. The solid black line is the VRC-TST result for the nominal case, and the solid red line is the optimized result. The dashed magenta line is the linear least squares ﬁt to the experimental data. Figure 6: Rate constants for H + NO2 →OH + NO. The symbols correspond to the experimental data: the blue diamonds are the high-temperature shock tube from ANL [48]; the red squares are the intermediate temperature ﬂow tube from Princeton [47], and the cyan circles are the low-temperature ﬂow cells of Fontjin [46]. The solid black line is the VRC-TST result for the nominal case, and the solid red line is the optimized result. The dashed magenta line is the linear least squares ﬁt to the experimental data. The optimized results for H + NO2 →OH + NO are shown in Figure 6. The dashed line is a least squares ﬁt to the experimental data (open symbols). The solid lines are the theoretical predictions, which includes the ﬂux through both entrance channels (addition to N and O). The nominal 20 value (black line) corresponds to the unscaled result, which is higher than the least-squares ﬁt by slightly less than a factor of two. As part of the optimization process, the barrier height for HNO2 →anti-HONO was reduced from -93.9 to -96.4 kJ/mol (relative to H + NO2). In terms of the scaling factors for the barrierless entrance channels: for H + NO2 →syn-HONO, the optimized potential was 25% more attractive at close range, but unperturbed at long range (e.g. close-dash black in Figure 2); for H + NO2 →HNO2, the optimized potential was unperturbed at close range, but was 25% less attractive at long range (e.g. wide-dash green in Figure 2). The H + NO2 →OH + NO showed no sensitivity to collisional energy transfer variation, nor to the OH + NO minimum energy path. Note that the optimized result (red line) agrees with the experimental data to within 10%; thus, subtle changes to the curvature of the minimum energy path provide an impressive improvement to the ﬁnal rate constant. 3.3. Optimized Mechanism 21 0 20 40 60 80 100 120 140 160 P (He) [bar] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 k [cm3/molecule-s] 1e 11 Fulle (1998) 250 K 298 K 400 K 250 K 298 K 400 K Figure 7: Rate constants for OH + NO →HONO. The symbols correspond to the data of Fulle et al. [49], and the solid lines are the optimized model predictions. Only the 400 K data (black) was used in the optimization process. 0 20 40 60 80 100 120 140 160 P (He) [bar] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 k [cm3/molecule-s] 1e 11 Fulle (1998) 250 K 298 K 400 K 250 K 298 K 400 K Figure 7: Rate constants for OH + NO →HONO. The symbols correspond to the data of Fulle et al. [49], and the solid lines are the optimized model predictions. Only the 400 K data (black) was used in the optimization process. The optimized results for OH + NO →HONO are shown in Figure 7. The high-pressure data of Fulle [49] are an excellent compliment to the H + NO2 data, since they provide constraints for both collisional energy transfer and OH + NO minimum energy path. The optimized result for the collisional energy transfer prefactor was ⟨∆Edown⟩= 300 (T/298[K])0.85 cm−1. The OH + NO interaction potential was shifted up by a constant of 25% (e.g. dash-dot green in Figure 2). Note that even though only the data from 400 K (black symbols) were included in the optimization process, the lower termperature data are still well reproduced by the optimized model. The optimized rate 22 constant slightly under-predicts the experimental data at 250 and 298 K, which is consistent with our expectation that non-adiabatic eﬀects will be important at lower temperatures. 3.4. Unimolecular kinetics 3.4. Unimolecular kinetics 0.8 1.2 1.6 2.0 1000/T [K−1] 10-8 10-4 100 104 108 k [s−1] a) HONO OH + NO HNO2 0.8 1.2 1.6 2.0 1000/T [K−1] 10-8 10-4 100 104 108 b) HNO2 OH + NO HONO 100 atm 10 atm 1 atm 0.1 atm 100 atm 10 atm 1 atm 0.1 atm Figure 8: Rate constants for unimolecular decomposition (red) and isomerization (blue) for (a) HONO and (b) HNO2. The diﬀerent line styles correspond to diﬀerent pressures. Decomposition to H + NO2 is negligible. 0.8 1.2 1.6 2.0 1000/T [K−1] 10-8 10-4 100 104 108 k [s−1] a) HONO OH + NO HNO2 0.8 1.2 1.6 2.0 1000/T [K−1] 10-8 10-4 100 104 108 b) HNO2 OH + NO HONO 100 atm 10 atm 1 atm 0.1 atm 100 atm 10 atm 1 atm 0.1 atm Figure 8: Rate constants for unimolecular decomposition (red) and isomerization (blue) for (a) HONO and (b) HNO2. The diﬀerent line styles correspond to diﬀerent pressures. Decomposition to H + NO2 is negligible. The results for the decomposition of HONO and HNO2 are shown in Fig- ure 8. For HONO, Figure 8a, the only signiﬁcant product is OH + NO, with isomerization to HNO2 being four orders of magnitude smaller. This result is unsurprising, since, as seen in Figure 1, the OH + NO products are lower in energy than the barrier for isomerization to HNO2, and thus the barrierless 23 decomposition is both enthalpically and entropically favorable at all temper- atures. For HNO2 decomposition, Figure 8b, thermally activated OH + NO also is the dominant product channel, with isomerization approximately two orders of magnitude smaller. For this product channel, as the population of HNO2 isomerizes to HONO, the rate of collisional stabilization cannot compete with bond ﬁssion, and the vibrationally hot HONO intermediate promptly dissociates. Thus, isomerization between HONO and HNO2 is, for all intents and purposes, negligible, with both isomers decomposing to OH + NO. Formation of H + NO2 is negligible under all conditions. 3.5. Comparison with Literature Values 600 1200 1800 10-1 100 101 ratio Dagaut (2008) 100 atm 10 atm 1.0 atm 0.1 atm 600 1200 1800 10-1 100 101 Konnov (2009) 600 1200 1800 10-1 100 101 ratio Mathieu (2016) 600 1200 1800 10-1 100 101 Ahmed (2016) 600 1200 1800 Temperature [K] 10-1 100 101 ratio Zhang (2017) 600 1200 1800 Temperature [K] 10-1 100 101 Glarborg (2017) Figure 9: Ratio of literature values to the present work for the HONO →OH + NO rate constant. The line styles correspond to diﬀerent pressures. Figure 9: Ratio of literature values to the present work for the HONO →OH + NO rate constant. The line styles correspond to diﬀerent pressures. 24 To assess the impact of the newly optimized HONO/HNO2 mechanism on model predictions, we consider six diﬀerent mechanisms that have been published in the past ten years: Daguat et al.[51], Konnov [52], Mathieu et al.[53], Ahmed et al.[54], Zhang et al.[10], and Glarborg et al. [6]. These mechanisms, along with the present work, were imported into Cantera.[55] In all cases, the literature mechanisms represent the reaction in the associa- tion direction, OH + NO →HONO, with values taken from either Tsang and Herron[56] (Dagaut, Konnov, Mathieu) or Fulle et al.[49] (Ahmed, Zhang, Glarborg). In some cases, the Troe broadening factor Fcent[57] was modiﬁed by the authors. The largest source of discrepancy between the literature models, however, is due to the thermochemistry of OH, NO, and HONO, since the rate constant for HONO →OH + NO is computed using the equi- librium constant to maintain thermodynamic consistency. Nonetheless the agreement of the literature models with each other and with the present work is good, typically diﬀering by less than an order of magnitude. The best agreement is with the Glarborg mechanism, which uses the ATcT ther- mochemistry, and is within a factor of two of the present work. 4. Conclusions The HNO2 potential energy surface is computed using post-coupled clus- ter methods, with an uncertainty of ± 1.3 kJ/mol. For the barrierless reactions, the minimum energy paths are computed using diﬀerent multi- reference methods. Temperature- and pressure-dependent rate constants for HONO and HNO2 are computed using microcanonical rate theory and the master equation. A novel strategy was developed to incorporate uncertainty in the minimum energy pathway into the optimized mechanism. The new mechanism is in excellent agreement with all available experimental data for H + NO2 →OH + NO and OH + NO →HONO. This work highlights the importance of well-skipping reactions, with OH + NO being the most impor- tant product channel for HNO2, even though all prior literature mechanisms for HNO2 failed to include this pathway. The resulting HONO and HNO2 mechanism, coupled with the uncertainty analysis, is expected to provide im- portant constraints on future mechanism development for gas-phase nitrogen chemistry. 3.5. Comparison with Literature Values 25 600 1200 1800 10-4 100 104 108 ratio Dagaut (2008) HNO2 not in mechanism 100 atm 10 atm 1.0 atm 0.1 atm 600 1200 1800 10-4 100 104 108 Konnov (2009) HNO2 not in mechanism 600 1200 1800 10-4 100 104 108 ratio Mathieu (2016) 600 1200 1800 10-4 100 104 108 Ahmed (2016) 600 1200 1800 Temperature [K] 10-4 100 104 108 ratio Zhang (2017) 600 1200 1800 Temperature [K] 10-4 100 104 108 Glarborg (2017) Figure 10: Ratio of literature values to the present work for the HNO2 →HONO rate constant. The line styles correspond to diﬀerent pressures. Figure 10: Ratio of literature values to the present work for the HNO2 →HONO rate constant. The line styles correspond to diﬀerent pressures. For HNO2 kinetics, however, the present work is signiﬁcantly diﬀerent from all prior mechanisms. Two of the mechanisms, Dagaut and Konnov, do not include HNO2 as a distinct species. For the other four mechanisms, the only unimolecular reaction for HNO2 is isomerization to HONO. The ratio of the literature values to the present work for HNO2 →HONO are shown in 10. Here the published values diﬀer by up to eight orders of magnitude. As demonstrated in Figure 8, the most important product channel for HNO2 is the well skipping reaction to form OH + NO, but none of the previous mechanisms include this reaction. Instead, they tend to rely on early predic- tions of Dean and Bozzelli for HNO2, which used QRRK and modiﬁed strong collision[5]. Importantly, that earlier work did not include OH + NO as a 26 possible channel and thus overestimated the rate collisional stabilization of HONO from HNO2. Conﬂicts of interest There are no conﬂicts to declare. 27 Acknowledgements The authors would like to thank Stephen Klippenstein, Lawerence Hard- ing, and Ahren Jasper of Argonne National Laboratory for many helpful dis- cussions on variational transition state theory, electronic structure methods, and nonadiabatic dynamics. MEF gratefully acknowledges support by the U.S. National Science Foundation through Award Number CBET-1553366, with Dr. Song-Charng Kong as the program manager. XC gratefully ac- knowledges support by the U.S. Oﬃce of Naval Research through ONR Grant Number N00014-1-61-2054, with Dr. Chad Stoltz as the program manager. CFG gratefully acknowledges the NSF, ONR, and Brown University for sup- port on this project. 28 28 References [1] A. M. Ickes, S. V. Bohac, D. N. Assanis, Eﬀect of 2-ethylhexyl nitrate cetane improver on NOx emissions from premixed low- temperature diesel combustion, Energy & Fuels 23 (10) (2009) 4943– 4948. doi:10.1021/ef900408e. [2] A. B. 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https://openalex.org/W2791483362	https://www.nature.com/articles/s41598-018-21547-y.pdf	English	null	Allocation of photosynthesized carbon in an intensively farmed winter wheat–soil system as revealed by 14CO2 pulse labelling	Scientific reports	2,018	cc-by	7,819	Allocation of photosynthesized carbon in an intensively farmed winter wheat–soil system as revealed by 14CO2 pulse labelling Zhaoan Sun1,QingChen1, Xiao Han1, Roland Bol 2, BoQu1 & Fanqiao Meng 1 Received: 6 November 2017 Accepted: 6 February 2018 Published: xx xx xxxx Zhaoan Sun1, Qing Chen1, Xiao Han1, Roland Bol 2, Bo Qu1 & Fanqiao Meng 1 Understanding the rhizodeposited carbon (C) dynamics of winter wheat (Triticum aestivum L.), is crucial for soil fertility and C sequestration. Pot-grown winter wheat was pulse labelled with 14CO2 at the key growth stages. 14C in the shoots, roots and soil was measured at 5 or 2 days after 14C-labelling (DAL 5/2) at each growth stage and at harvest. The 14C in the shoots increased from 4% of the net 14C recovered (shoots + roots + soil) during tillering to 53% at harvest. Approximately 14–34% of the net 14C recovered was incorporated into the soil. Allocation of photosynthesized C was extrapolated from the pot experiment to field condition, assuming a planting density of 1.8 million plants ha−1. The estimated C input to the soil was 1.7 t C ha−1, and 0.7 t C ha−1 of root residues was retained after wheat harvest; both values were higher than those previously reported (0.6 and 0.4 t C ha−1, respectively). Our findings highlight that C tracing during the entire crop season is necessary to quantify the temporal allocation of photosynthesized C, especially the contribution to soil carbon in intensified farming system. Wheat covers approximately 220 million hectares of farmland worldwide1 and plays an important role in food supply and soil organic carbon (SOC) regulation2. SOC is primarily derived from plants3, such as deposition of both crop straw4 and root litter5,6, as well as rhizodeposits3. Rhizodeposits include root exudates and other root-borne organic substances released into the rhizosphere during plant growth as well as sloughed root hairs and decaying root cells3,7; and it also affects SOC and nutrient cycling8–11. Accurate investigation of rhizodeposits during the entire wheat season is crucial important for the achievement of high crop yield and understanding of its contribution to farmland C sequestration, especially in intensified agricultural region such as northern China12,13.il Types and developmental stage of crop growth significantly influences the distribution of photosynthesized C. Mathew et al.14 found that grasses can store up to 45% of their C stocks in the roots, while cereals show much lower rates with 16% for maize and 23% for wheat. The belowground allocation of photosynthesized C to the soil decreases from approximately 10% at tillering to 5% at grain-filling in wheat and barley (Table 1). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 6 November 2017 Accepted: 6 February 2018 Published: xx xx xxxx Allocation of photosynthesized carbon in an intensively farmed winter wheat–soil system as revealed by 14CO2 pulse labelling Zhaoan Sun1,QingChen1, Xiao Han1, Roland Bol 2, BoQu1 & Fanqiao Meng 1 This decrease might be attributed to differences in photosynthetic capacity and C requirements for root growth at different growth stages3,11. Extrapolating the values from short periods to the whole lifetime of wheat plants (approximately 240 days after emergence in northern China) might overestimate the amount of C allocated to the belowground pool, as young plants exhibit faster root growth and greater sink strength of C allocated to the roots and soil than do older plants15,16. Compared with conventional non-tracing methods, C tracers (13C or 14C) labelling method can distinguish between soil-derived and root-derived C in the soil, determine the C allocation in different crop stages and com- pletely quantify the whole seasonal C rhizodeposition3,17. Continuous labelling18,19 is particularly appropriate for the estimation of total C transferred to the soil and belowground pools. However, highly sophisticated instrumen- tation required for continuous isotopic 14C-labelling and sampling makes field studies difficult17, especially for long-lived crops such as winter wheat (up to eight months). As an alternative, 13CO2 or 14CO2 pulse labelling3,8,9 at different growth stages provides discrete information about the temporal C dynamics associated with specific 1Beijing Key Laboratory of Farmland Soil Pollution Prevention and Remediation, College of Resources and Environmental Sciences, China Agricultural University, Beijing, 100193, China. 2Institute of Bio- and Geosciences, Agrosphere Institute (IBG-3), Forschungszentrum Jülich GmbH, 52425, Jülich, Germany. Correspondence and requests for materials should be addressed to F.M. Allocation of photosynthesized carbon in an intensively farmed winter wheat–soil system as revealed by 14CO2 pulse labelling Zhaoan Sun1,QingChen1, Xiao Han1, Roland Bol 2, BoQu1 & Fanqiao Meng 1 (email: mengfq@cau.edu.cn) SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y 1 www.nature.com/scientificreports/ Plant age (days) Growth stage Shoots Roots Soil Soil/(soil + roots) References Wheat 42 Tillering 70.9 17.6 11.5 39.6 Keith et al.25 Wheat 50 79.3 20.2 0.5 2.3 Gregory and Atwell24 Wheat 43 54.2 26.8 19.0 41.5 Palta and Gregory43 Wheat 49 80.8 19.2 Martin and Kemp44 Wheat 166 54.2 23.7 22.1 48.2 Swinnen et al.29 Wheat 37 53.7 43.0 3.2 6.9 Lodhi et al.45 Barley 49 60.6 37.0 2.4 6.1 Gregory and Atwell24 Barley 34 78.0 13.8 8.2 37.3 Jensen46 Barley 47 56.5 33.7 9.8 22.5 Swinnen et al.29 Averagea 63.4 ± 3.6 27.0 ± 3.4 9.6 ± 2.6 25.5 ± 6.1 Wheat 23 This study 36.4 10.8 52.9 83.0 Wheat 77 Elongation 81.2 11.8 7.0 37.1 Keith et al.25 Wheat 71 75.2 23.4 1.5 5.9 Gregory and Atwell24 Wheat 51 53.7 27.4 18.9 40.9 Palta and Gregory43 Wheat 151 71.4 17.7 10.9 38.2 Swinnen et al.11 Wheat 193 91.3 6.7 1.9 22.2 Swinnen et al.29 Wheat 211 66.0 22.5 11.5 33.9 Qi and Wang47 Wheat 50 53.7 43.0 3.2 6.9 Atwell et al.28 Barley 67 82.4 13.5 4.1 23.4 Swinnen et al.29 Barley 70 89.2 9.9 0.9 8.7 Gregory and Atwell24 Barley 66 69.1 8.3 22.6 73.1 Jensen48 Barley 56 74.3 7.8 17.8 69.4 Jensen46 Barley 25 62.3 15.3 22.4 59.3 Zagel49 Average 74.6 ± 3.2 15.4 ± 2.0 10.0 ± 2.5 34.7 ± 6.8 Wheat 173 This study 67.8 9.2 23.0 71.5 Wheat 133 Anthesis 97.0 1.1 1.8 62.1 Keith et al.25 Wheat 106 94.2 5.7 0.1 1.8 Gregory and Atwell24 Wheat 179 85.3 5.3 9.4 63.9 Swinnen et al.11 Wheat 60 79.0 16.0 5.0 23.8 Martin and Kemp44 Wheat 226 96.5 3.3 0.2 5.9 Qi and Wang47 Wheat 70 84.1 14.7 1.3 8.2 Atwell et al.28 Barley 101 93.1 1.0 5.9 85.3 Jensen48 Barley 86 86.1 4.6 9.3 66.9 Jensen46 Barley 40 66.4 9.5 24.1 71.8 Zagel49 Barley 105 82.3 1.8 15.9 89.7 Gregory and Atwell24 Average 86.4 ± 3.0 6.3 ± 1.7 7.3 ± 2.5 47.9 ± 10.8 Wheat 194 This study 58.7 3.9 37.3 90.5 Wheat 154 Grain-filling 98.9 0.4 0.7 63.6 Keith et al.25 Wheat 120 94.2 5.6 0.2 3.6 Gregory and Atwell24 Wheat 207 90.9 2.1 7.0 77.1 Swinnen et al.11 Wheat 255 95.4 2.5 2.1 45.5 Swinnen et al.29 Wheat 239 86.7 2.0 11.3 84.8 Qi and Wang47 Wheat 86 94.7 4.7 0.7 13.1 Atwell et al.28 Barley 115 97.9 1.1 1.0 47.6 Swinnen et al.29 Barley 126 97.4 2.3 0.2 8.0 Gregory and Atwell24 Barley 124 98.1 0.7 1.1 61.1 Jensen48 Barley 105 88.1 1.9 9.9 83.6 Jensen46 Barley 55 72.6 6.4 21.0 76.5 Zagel49 Average 92.3 ± 2.3 2.7 ± 0.6 5.0 ± 2.0 51.3 ± 9.2 Wheat 208 This study 74.7 1.1 24.2 95.6 Wheat 28 DALb Whole growth period 73.5 22.0 4.4 16.8 Chowdhury et al.22 Wheat 80–90 89.0 2.3 8.4 78.1 Martens et al.34 Wheat 141 66.3 18.6 14.5 43.8 Butterly et al.33 Average 76.5 ± 2.3 14.4 ± 2.3 9.1 ± 2.3 46.1 ± 2.3 Wheat 224 This study 69.0 8.7 22.2 71.8 Table 1. Table 1. Partitioning (%) of pulse-labelled carbon (C) (mean ± SE) at different growth stages of wheat and barley. aData excludes that of Martin and Kemp41. bDAL, days after 14C pulse labelling. Results Bi Biomass C of shoots, roots and whole wheat plants. As the winter wheat plants grew, the biomass C of the shoots, roots and whole plants increased and peaked at the anthesis stage, after which it remained sta- ble, but the biomass C of the roots decreased after the anthesis stage (Fig. 1). The ratios of roots/shoots and shoots/whole wheat plants decreased and increased, respectively, from emergence until the grain-filling stage. The growth rate of the whole wheat plants was quite low (8.8 mg C d−1 pot−1) at the tillering stage but increased to 189.0 and 269.4 mg C d−1 pot−1 at the elongation and anthesis stages, respectively; at the grain-filling stage, the growth rates were negligible. Specific 14C activity of the shoots, roots and soil. Enrichment of labelled 14C was highest in the shoots, followed by the roots and soil for all four labelling events (Fig. 2a–d). The specific 14C activity of the shoots, roots and soil at 5 DAL (DAL 5; DAL 2 at the tillering stage) was in the order of tillering> elongation> anthesis ≈ grain-filling stages. With the prolonged wheat growth duration and the dilution of plant biomass and respiratory release, the specific 14C activities of shoots and roots decreased. For the labelling at the tillering stage, the specific 14C activity of the soil remained stable between DAL 2 and DAL 175 but declined thereafter (Fig. 2a); at the other three labelling stages (Fig. 2b,c,d), the specific 14C activity of the soil was stable after labelling. 14C recovery and allocation within the wheat–soil system. The 14C recovered in the soil was highest for the labelling at the tillering stage, followed by that in the roots and shoots (with the exception of DAL 2 in the roots; Fig. 3a). For the labelling at the other three stages, the 14C recovered always followed the order of shoots> roots> soil (Fig. 3b,c,d).i We quantified the proportion of 14C recovered in the shoots, roots and soil at DAL5/2 and at harvest, and that in the respired losses from DAL 5/2 to harvest. Of the total net 14C recovered (shoots + roots + soil), 36.4%, 67.8%, 58.8% and 74.7% were allocated in the shoots at DAL 5/2 at the tillering, elongation, anthesis and grain-filling stages, respectively; these proportions decreased to 4.0% (tillering), 34.3% (elongation), 49.9% (anthesis) and 52.9% (grain-filling) at harvest (Fig. 4a). www.nature.com/scientificreports/ crop stages9–11. Pulse labelling also has the advantage of being simple and applicable under field conditions and provides seasonal dynamics of assimilate partitioning. A series of 14C-labelling pulses applied at regular intervals during crop growth has been proven to accurately estimate cumulative belowground C inputs3,11, especially in calcareous soils11. It was commonly considered that in conventional pulse-chase labeling studies, the distribu- tion of assimilated C was completed at the time when the losses of labeled CO2 by respiration could no longer be detected11,16. As the plant tissues and soil microorganisms prefer to utilize the labile organic substrates, the decline or disappearance of the labeled CO2 respiration may not necessarily indicate that the transfer of the less labile components (such as root debris and sloughed-off cells) into the soil is also completed20. Therefore, due to the short duration of labelling and chasing periods, most previous conventional pulse–chase labelling studies provides information only on the newly assimilated C3,11.f p y y In current study, we undertook the pulse labelling at different growth stages of the whole winter wheat season and harvested the winter wheat at 5 days after 14C-labelling (DAL) at each growth stage and at the end of the wheat growth season10,15,20,21. The 14C distribution in the wheat biomass and soil pools at the end of the growing season represents the net contribution of photosynthates formed at various stages of wheat15,20,21. We aimed to quantify the temporal 14C distribution in the winter wheat biomass and soil pools throughout the whole wheat season. We hypothesized that the C allocated into belowground (roots and soil) by pulse labelling quantified for the whole wheat season was much lower than that C quantified by extrapolating the C amount from the young stage to the whole wheat season. To validate this hypothesis, we labelled the winter wheat with 14C at key growth stages, i.e., tillering, elongation, anthesis and grain filling stages, and measured the photosynthesized C allocated into shoot, roots and soil pools and lost by respiration. The results from the pot labelling were also estimated to field level in discussion Section, to evaluate the contribution of wheat production to farmland SOC changes under agricultural intensification process. Results Bi The proportion of 14C in the total net 14C recovered in the soil (Fig. 4c) was lower than that in the shoots but higher than that in the roots (Fig. 4b; except at the tillering stage), and this proportion decreased from DAL 5/2 to harvest (Fig. 4b,c). Between DAL 5/2 and harvest, the proportion of 14C via respiration (aboveground and belowground respiration) was highest (63.9%) at the tillering stage but decreased to 46.7%, 13.8% and 22.1% at the elongation, anthesis and grain-filling stages, respectively (Fig. 4d). In absolute values, the amount of respired C was 0.21, 2.47, 0.51 and 0.07 C pot−1 at the tillering, elongation, anthesis and grain-filling stages, respectively. Contribution of photosynthesized C to SOC. At the end of wheat growing season, the contribution of photosynthesized C to SOC at the four growth stages was calculated by multiplying the relative distribution of assimilated 14C in the soil to the increment of wheat biomass C (Eq. (3)). Approximately 3.3 g C pot−1 was respired (i.e., 20% of the total photosynthate C recovered), 9.1 g C pot−1 (55%) remained in the shoots, 1.1 g C pot−1 (7%) was in the roots, and 2.9 g C pot−1 (18%) was translocated to the soil (Fig. 5). Regarding the 2.9 g C pot−1 allocated to the soil, contributions of 42.1% and 48.5% occurred during the elongation and anthesis stages; these percent- ages were significantly higher than those at the tillering (6.4%) and the grain-filling (3.0%) stages. Allocation of photosynthesized carbon in an intensively farmed winter wheat–soil system as revealed by 14CO2 pulse labelling Zhaoan Sun1,QingChen1, Xiao Han1, Roland Bol 2, BoQu1 & Fanqiao Meng 1 Partitioning (%) of pulse-labelled carbon (C) (mean ± SE) at different growth stages of wheat and barley. aData excludes that of Martin and Kemp41. bDAL, days after 14C pulse labelling. SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y 2 www.nature.com/scientificreports/ Discussion Assimilated C is translocated to plant–soil compartments such as shoots and roots, exuded into the soil and lost due to respiration, and this allocation pattern changes with crop growth15,20–22. In the present study, the assimi- lated 14C in the winter wheat shoots at the labelling event of tillering stage was only 36.4%, but increased at the SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y 3 www.nature.com/scientificreports/ Figure 1. Biomass C of (a) shoots, (b) roots, (c) whole plants, and (d) the root/shoot and shoot/whole plant ratios as well as (e) winter wheat growth rates at different growth stages. Vertical bars represent the standard errors of the means (n = 3). Letters (a–c) indicate significant differences (p < 0.05, LSD) for comparison among different stages. Figure 1. Biomass C of (a) shoots, (b) roots, (c) whole plants, and (d) the root/shoot and shoot/whole plant ratios as well as (e) winter wheat growth rates at different growth stages. Vertical bars represent the standard errors of the means (n = 3). Letters (a–c) indicate significant differences (p < 0.05, LSD) for comparison among different stages. later labelling events: i.e., the shoots retained the majority (60–74.7%) of their assimilated 14C at DAL 5 during the elongation, anthesis and grain-filling stages (Fig. 4). Similar results were also reported for rice20,23, wheat, barley22,24,25 and maize16 but not for pasture grasses26. Several reasons could explain the higher proportion of assimilated C allocated belowground in pasture plants than in cereal plants: (i) approximately 80% of pasture plants are perennial and have well-developed roots that are used as for C storage for newly grown plant tissues; (ii) a long history of selective cultivation of cereals has led to the preferential allocation of assimilates to aboveground parts14; and (iii) intensive fertilization of crops significantly decreases the assimilates in roots needed for the uptake of soil nutrients3,27. These findings are consistent with those of other studies (Table 1), which have reported that wheat and barley allocate more than 90% of photosynthates to their shoots during the mature stage in com- parison with approximately 60% during the tillering stage. In a broader context, this indicates that different types of crop have various potentialities of atmospheric C sequestration14. p p p q Photosynthates are actively translocated to belowground parts more during the early establishment of wheat and barley crops than at the mature stage28–30 (Table 1). Discussion The summary for previous labelling studies (Table 1) showed that the C sink in the roots was greater in young plants (27%) than in mature plants (3%) (Table 1). In our study, the percentage of 14C incorporated into the roots also decreased from the tillering to the grain-filling stages (Fig. 4). However, as the wheat growth rate at the anthesis and elongation stages (189−269 mg C d−1 pot−1) was much greater than that at the grain-filling and tillering stages (8.8−15.4 mg C d−1 pot−1), C translocation to the soil also occurred mainly at the anthesis and elongation stages, i.e., 48.5% and 42.1% of the total C was trans- located to the soil, respectively. The majority of labelling studies have been conducted at the early growth stages of wheat, e.g., 60 days after emergence by continuous labelling31,32 and <150 days after emergence by pulse label- ling33,34. Hence, extrapolating the values from early wheat stages to the whole lifetime of wheat plants (approxi- mately 240 days after emergence in northern China) substantially overestimated the amount of C allocated to the belowground pool, as young plants exhibit faster root growth and greater sink strength of C allocated to the roots and soil than do older plants15,16. SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y 4 www.nature.com/scientificreports/ Figure 2. Turnover of specific 14C activity for shoot, root and soil components at different DAL at the (a) tillering, (b) elongation, (c) anthesis and (d) grain-filling stages of winter wheat. Vertical bars represent the standard errors of the means (n = 3). LSD values (p < 0.05) are presented as whisked segments for comparison among different components at the same stage. Figure 2. Turnover of specific 14C activity for shoot, root and soil components at different DAL at the (a) tillering, (b) elongation, (c) anthesis and (d) grain-filling stages of winter wheat. Vertical bars represent the standard errors of the means (n = 3). LSD values (p < 0.05) are presented as whisked segments for comparison among different components at the same stage. Figure 3. Recovered 14C in shoot, root and soil components at different DAL at the (a) tillering, (b) elongation, (c) anthesis and (d) grain-filling stages of winter wheat. Vertical bars represent the standard errors of the means (n = 3). Figure 3. Discussion Recovered 14C in shoot, root and soil components at different DAL at the (a) tillering, (b) elongation, (c) anthesis and (d) grain-filling stages of winter wheat. Vertical bars represent the standard errors of the means (n = 3). SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y 5 5 www.nature.com/scientificreports/ Figure 4. Proportion of 14C allocated at DAL 5/2 and at the end of the growing season (harvest) for (a) shoots, (b) roots, (c) soil, and (d) respiration at harvest for the different labelling events. Vertical bars represent the standard errors of the means (n = 3). Letters (a–c) indicate significant differences (p < 0.05, LSD) for comparison among different stages. Asterisks () denote significant differences (p < 0.05, t-test) between DAL 2/5 and harvest. Figure 4. Proportion of 14C allocated at DAL 5/2 and at the end of the growing season (harvest) for (a) shoots, (b) roots, (c) soil, and (d) respiration at harvest for the different labelling events. Vertical bars represent the standard errors of the means (n = 3). Letters (a–c) indicate significant differences (p < 0.05, LSD) for comparison among different stages. Asterisks () denote significant differences (p < 0.05, t-test) between DAL 2/5 and harvest. Figure 5. Total carbon distribution in the wheat-soil pot system. Figure 5. Total carbon distribution in the wheat-soil pot system. Using a field planting density of 1.8 million plants ha−1 in northern China35, we found that the assimilated C input to the soil, excluding the C allocated to the roots (690 kg C ha−1), was approximately 1730 kg C ha−1 throughout the whole winter wheat season. This estimate is higher than reported 300 kg C ha−1 for maize16, 460–822 kg C ha−1 for rice23, and 710–1020 kg C ha−1 for wheat36. If the C retained as root residue after harvest (690 kg C ha−1) was also included in the total C input to the soil, the total C input belowground by winter wheat was 2420 kg C ha−1, which is significantly higher than both the values (992 kg C ha−1) summarized in Table 2 and the rough estimates (1500 kg C ha−1) by Kuzyakov and Domanski3 based on C tracer studies. The differences between our study and other studies might be related to the higher root biomass C resulting from agricultural intensification in our study than the quantity defined using tracer techniques (690 in our study vs. Experimental setup. Winter wheat (Triticum aestivum L. cv. Jingdong 8) seeds were surface-sterilized in 30% hydrogen peroxide for 30 min, after which they were soaked for 6 h in saturated copper sulphate solution and rinsed with deionized water. This pretreatment effectively sterilized seeds and improved germination rates38. After disinfection, the seeds were placed into glass Petri dishes lined with wet filter paper and germinated for 2 days at 22 °C in dark- ness. Six healthy germinated winter wheat seeds were transplanted to an individual pot (2-cm depth below the soil). One week after wheat germination, three vigorous seedlings were kept in each pot, i.e., equivalent to a field density of 1.8 million plants per ha, which was estimated from local wheat fields. In accordance with local farm- ing practices, the soil was premixed with urea, diammonium phosphate and potassium chloride at rates of 0.15 g N, 0.09 g P and 0.25 g K kg−1 soil as a base fertilizer, respectively. Urea was top-dressed at the elongation stage (mid-March of the following year) at a rate of 0.15 g N kg−1 soil.h ( g y ) g g The pots with transplanted winter wheat were placed inside a greenhouse to provide light and temperature conditions similar to those in the field. The soil water content of each pot was controlled gravimetrically to sim- ulate local wheat production and was adjusted daily to 65% (during the seedling stage), 70% (tillering), 80% (elongation), 80% (anthesis) and 70–75% (grain-filling) of field water-holding capacity. When the daily average air temperature dropped to 4–5 °C during the winter season, the pots with wheat plants were placed underground (50-cm depth) to prevent cold damage. The total wheat growing period was 230 days, and six different growth stages were recognized in terms of days after sowing (DAS): (i) seeding (1–17 DAS); (ii) tillering (18–38 DAS); (iii) wintering (39–150 DAS); (iv) elongation (151–179 DAS); (v) anthesis (180–193 DAS); (vi) grain-filling (194– 214 DAS); and (vii) harvest (215–230 DAS). 14C pulse labelling. 14CO2 labelling was performed at the tillering, elongation, anthesis, and grain-filling stages (i.e., 21, 168, 189 and 203 DAS, respectively). Discussion 402 kg C ha−1 in Table 2). In addition, the ratio of net rhizodeposited C into the soil to root was reported to be 25% by Kuzyakov and Domanski3 and was therefore 2.5-fold lower than that in our study. This result is because most of the esti- mates of Kuzyakov and Domanski3 for wheat labelling have been carried out at young plant stages, during which translocation is relatively higher than that during subsequent stages of growth. Again, this finding validates our hypothesis and highlights that labelling and sampling throughout the entire growing season of winter wheat (for instance> 200 days in our study) are necessary to accurately quantify the C budget within the crop–soil system. 6 SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y www.nature.com/scientificreports/ Plant age (days) Roots (kg C ha−1) Soil (kg C ha−1) Roots + soil (kg C ha−1) References Wheat 167 327 16 343 Gregory and Atwell24 Wheat 63 231 560 791 Martin and Merckx50 Wheat 288 350 730 1080 Swinnen et al.29 Wheat 239 840 526 1366 Qi and Wang47 Barley 167 324 24 348 Gregory and Atwell24 Barley 127 449 818 1267 Jensen48 Barley 148 430 880 1310 Swinnen et al.29 Barley 95 264 1171 1435 Jensen46 Average 402 ± 68 591 ± 143 992 ± 158 Wheat 224 690 1730 2420 This study Table 2. Amount of carbon (C) translocated belowground (mean ± SE) for wheat and barley quantified by C pulse labelling methods. Table 2. Amount of carbon (C) translocated belowground (mean ± SE) for wheat and barley quantified by C pulse labelling methods. Agricultural intensification has not only increased crop productivity but also contributed to increased SOC in northern China12. Smith et al.37 reported that inputs of crop residues lead to higher rates of C sequestration (0.7 Mg C ha yr−1) than does mineral nitrogen (N) fertilizer (0.2 Mg C ha yr−1). The estimated wheat C inputs in our study were 2420 kg C ha−1 (soil + root) and 1730 kg C ha−1 (soil only), which were two-fold greater than those of comparable studies (Table 2). The high amount of C allocated to belowground plant parts in this calcareous soil might explain the rapid increase in SOC in northern China12. Conclusions During a growing season, the estimated photosynthesized C input from wheat to the soil was 1.7 t C ha−1 as rhizodeposits. Wheat plant also produced 0.7 t C ha−1 of root residues that was retained in the soil after harvest. Therefore, approximately 2.4 t C ha−1 of atmospheric CO2 was fixed as a relatively stable form in the soil after the wheat season; this value is approximately twice that of other comparable studies. Of the wheat C input to the soil, 90.6% was photoassimilated between the elongation and anthesis stages. This information will be critical in the construction of predictive models of C dynamics in wheat–soil systems if the amount of plant C transferred to the soil requires estimation. Materials and Methods Experimental setup. Soil samples were collected from the ploughing layer (0–30 cm) of crop fields at the Quzhou Experimental Station of China Agricultural University in Hebei Province, China (36˚52′N; 115˚01′E). The main soil properties were as follows: 17.1 g kg−1 SOC, 7.8 g kg−1 soil inorganic C, 1.6 g kg−1 total N, pH 8.0 (soil/water = 1/2.5), 148 mg kg−1 available potassium (K), and 9.9 mg kg−1 Olsen phosphorus (P). The soil was sieved (5 mm), after which each polyvinylchloride (PVC) plastic pot was filled (height: 50 cm, inner diameter: 10 cm; 5.6 kg soil pot–1 with calculated bulk density of 1.42 g cm−3) and rewetted to 65% of the water-holding capacity (0.21 g water g−1 dry soil). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5 and 35 (harvest) after labelling; and six pots were labelled at the grain-filling stage (203 DAS) for destructive sampling at days 5 and 21 (harvest) after labelling.h p g yt g The labelling system adapted from Cheng39 consisted of a labelling chamber and a pot containing transplanted wheat. Each pot was closed at the bottom with a rubber stopper and had an air inlet and air outlet. The soil surface was covered with a PVC board and sealed with silicon, including around the winter wheat stems. A flask contain- ing Ba14CO3 with a 14C activity of 713 kBq was placed inside the labelling chamber. The chamber was then closed, and 14CO2 was released into the chamber by carefully adding an excess volume of 4 M HClO4 to the Ba14CO3 solution with a syringe to ensure complete evolution of the 14CO2 into the labelling chamber atmosphere. To help guarantee a uniform distribution of 14CO2, an electric fan was used to homogenize the gases inside the chamber. g 2, g g CO2 depletion under identical conditions in another chamber supplied with unlabelled CO2 was monitored using an infrared gas analyser (GXH305, Beijing Analytical Equipment Co., China)40,41. This system was used because 14CO2 could not be directly monitored, as the infrared range was set for 12CO2 and only slightly over- lapped with the range for 14CO2 40,41. If the speed of CO2 concentration decrease slowed considerably (less than 200 μL L−1), 12CO2 was released into the chamber until the 14CO2 + 12CO2 concentrations increased to approximately 400 μL L−1 by adding with a syringe an aliquot of 4 M HClO4 to an unlabelled NaHCO3 solution. The wheat plants were labelled for 1.5 h to facilitate the assimilation of 14CO2. After Ba14CO3 labelling, the air within the labelling chamber was pumped for 0.5 h through 50 mL of 1 M NaOH solution to remove unassimilated 14CO2 before the labelling chamber was opened. Plant and soil sampling. Destructive sampling of wheat and soil samples was carried out for the C and 14C analyses. The shoots were cut at the base of the wheat plants, and all the soil was removed from the pots. The roots were separated from the soil manually and then washed with 125 mL of deionized water to remove the soil adhering to the roots42. The soil samples were placed onto thick paper (0.5 cm) and divided into 100 groups. www.nature.com/scientificreports/ Approximately 1 g per group was sampled with a spoon and mixed to obtain a representative soil sample38. The shoots, roots and soil samples were oven-dried at 65 °C to a constant weight. Sample analysis. The plant and soil samples were ground (<500 µm) using a ball mill (Restol MM2000, Retsch, Haan, Germany) prior to analysing 14C content and determining total organic C. To determine the 14C content of SOC, carbonates were removed from the soil samples using a 0.5 M HCl solution for 6 h4. The soil was then washed using deionized water and centrifuged three to four times to remove the HCl. The 14C content of the plant (approximately 0.2 g) and soil (approximately 1 g) samples was measured after combustion with an oxidizer unit. The evolved 14CO2 was directly trapped in a scintillation cocktail, followed by liquid scintillation counting (FJ-2101, Xi’an Analytical Equipment Co., Xi’an, China). The 14C counting efficiency was approximately 94%. Calculations and statistical analysis. At 2 DAL (DAL 2) at the tillering stage or 5 DAL (DAL 5) at the elongation, anthesis and grain-filling stages, we assumed that the allocation of 14C photosynthesized within the wheat–soil system represented the initial photosynthate distribution in the shoot, root and soil compart- ments15,20,21. At each sampling, the amount of net 14C recovered (Fig. 3) was calculated by multiplying the specific 14C activity (Fig. 2) by the corresponding C amount in the shoots, roots or soil (Fig. 1). The net 14C recovered at DAL 5 or 2 (DAL 5/2) served as the basis for the calculation and comparison of photosynthesized C distributed in different wheat–soil compartments during the later days of wheat growth and at the harvest period. Experimental setup. In total, 33 pots of wheat were labelled: 12 pots were labelled at the tillering stage (21 DAS) for destructive sampling at days 2, 154, 175 and 203 (harvest) after labelling (3 pots per sampling); nine pots were labelled at the elongation stage (168 DAS) for destructive sampling at days 5, 28 and 56 (harvest) after labelling; six pots were labelled at the anthesis stage (189 DAS) for destructive sampling at days SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y 7 www.nature.com/scientificreports/ f g y g The (Net 14C recovered)DAL5/2 of winter wheat was estimated by summing the 14C amount in the shoots 4Cshoot), roots (14Croot) and soil (14Csoil) measured at DAL 5/2 at each stage16–18: = + + (Net C recovered) ( C C C ) (1) 14 DAL 5/2 shoot 14 root 14 soil 14 DAL 5/2 (1) The per cent distribution of 14C recovered (Distribution%) either at DAL 5/2 or at the end of the wheat grow- ing season was calculated as: = Distribution% C (Net C recovered) (2) sample 14 14 DAL 5/2 (2) where 14Csample is the 14C amount in the measured pools, i.e., the shoots, roots, and soil and the respired CO2 aboveground and belowground) at DAL 5/2 or at the end of the growing season.h g g g g The whole wheat seasonal contribution (g C pot−1 soil) of photosynthesized C to the soil formed during the four growth stages of winter wheat was calculated by the relative distribution of 14C recovered in the soil to the accumulation of plant biomass C as follows: = × × − Contribution (C pot soil) ( C ) (Net C recovered) WPGR D (3) soil 1 14 end 14 DAL5/2 (3) where (14Csoil)end is the amount of 14C in the soil at the end of wheat growing season; (net 14C recovered)DAL 5/2 is calculated by Eq. (1); WPGR is the wheat plant growth rate (mg C d−1 pot−1; Fig. 1e); and D represents the num- ber of days in each growth stage. Seventeen days of the seeding stage was considered to encompass tillering, i.e., a total of 38 days at the tillering stage. Within 111 days of the wintering period, wheat stopped growing, and its growth rate was considered 0 mg C d−1 pot−1. The duration of the elongation, anthesis and grain-filling stages was 29, 14 and 21 days, respectively. 8 SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y www.nature.com/scientificreports/ The respired C assimilation (from the shoots, roots and soil) was estimated from the accumulation of plant biomass C and from the 14C distribution by respiratory losses at the end of the wheat growing season. www.nature.com/scientificreports/ The amount of respired C (aboveground and belowground) was calculated as follows: = × × − ( ) Respiratory C (C pot ) C (Net C recovered) WPGR D (4) 1 total respiration 14 end 14 DAL 5/2 (4) where (14Ctotal respiration)end indicates 14C-CO2 losses from total respiration calculated by the difference between the net 14C recovered at DAL 5/2 and the amount of 14C remaining in the wheat–soil system at the end of the growing season. Statistical analysis. Winter wheat growth stage was the experimental factor in this study. 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Arch. Agron. Soil Sci. 63, 362–374 (2017).flf g 2. Cheng, W. Acknowledgements g This work received financial support from the National Key R&D Project (No. 2017YFD0800605) and the National Natural Science Foundation (Nos 31370527 and 30870414). References Effect of grazing on carbon stocks and assimilate partitioning in a Tibetan montane pasture revealed by 13CO2 pulse labeling. Global Change Biol. 18, 528–538 (2012).h 28. Atwell, B. J., Fillery, I. R. P., McInnes, K. J. & Smucker, A. J. M. The fate of carbon and fertiliser nitrogen when dryland wheat is grown in monoliths of duplex soil. Plant Soil. 241, 259–269 (2002).l 29. Swinnen, J., Van Veen, J. A. & Merckx, R. Carbon fluxes in the rhizosphere of winter wheat and spring barley with conventional vs integrated farming. Soil Biol. Biochem. 27, 811–820 (1995).i l integrated farming. Soil Biol. Biochem. 27, 811–820 (1995). g g ( ) 0. Swinnen, J., Van Veen, J. A. & Merckx, R. 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Carbon and nitrogen partitioning of wheat and field pea grown with two nitrogen levels under elevated CO2. Plant Soil. 391, 367–382 (2015). SCIentIFIC REPOrtS \| (2018) 8:3160 \| DOI:10.1038/s41598-018-21547-y 9 © The Author(s) 2018 Additional Informationh Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W4385244142	https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/41/e3sconf_eepes2023_02004.pdf	English	null	A model of a cyber-physical installation for smart greenhouse agriculture	E3S web of conferences	2,023	cc-by	3,809	* Corresponding author: m.temelkova@utp.bg © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). A model of a cyber-physical installation for smart greenhouse agriculture Miglena Temelkova* and Nikola Bakalov University of Telecommunications and Posts, Faculty of Telecommunications and management, 1 Acad. Stefan Mladenov Str., 1000 Sofia, Bulgaria Abstract. The article reviews the main aspects, parameters and effects integrated by smart agriculture. An analysis of the main definitions of the “smart agriculture” concept is drawn, and, a cyber-physical installation for smart greenhouse agriculture is modeled on that basis. The specific benefits from the cyber-physical systems in the agricultural production process are also defined by taking into account the actual prerequisites, factors and indicators related to smart-based agriculture. The main restrictive conditions are generated, as well as the managing parameters and the criterion for optimality of the model of a cyber-physical installation for smart greenhouse agriculture. At the same time, the architecture of a cyber-physical installation for smart greenhouse agriculture is described, while there are the specific mathematical variables integrated in it from the optimization task set in the model. Highlighted are the specific activities performed by each structural element of a cyber- physical installation for smart greenhouse agriculture, where their practical-and-applied benefits and efficiency are also defined, which ultimately leads to achieving the set optimization criterion – maximum return of the invested financial resources. E3S Web of Conferences 404, 02004 (2023) EEPES 2023 E3S Web of Conferences 404, 02004 (2023) EEPES 2023 https://doi.org/10.1051/e3sconf/202340402004 1 Introduction The relevance of the Concept for smart agriculture nowadays is related primarily to a basis of cumulatively available fundamental global needs – supplying the growing world population with foodstuff, efficient use and preservation of the global natural assets, climatic and nature-sustainable development of agriculture, meeting the requirements of Industry 4.0 for digitalization and smart-based development of agriculture as a sector of economy. A number of authors have researched the meaning of the “smart agriculture” concept as a starting point for reforming and restructuring the agricultural industry. According to some authors [1], innovative technologies, such as artificial intelligence, the Internet of things and mobile internet provide realistic solutions for a number of challenges in the agricultural sector. Many authors [2, 3, 4, 5, 6, 7, 8, 9] assume that smart agriculture is based on the integration of information and communication technologies into machines, equipment and sensors related to agricultural production. On this basis, the combined application of information-and-communication technological solutions, such as E3S Web of Conferences 404, 02004 (2023) EEPES 2023 https://doi.org/10.1051/e3sconf/202340402004 accurate equipment, the Internet of things, blockchain, cloud technologies, sensors and driving mechanisms, geopositioning systems, large bulks of data, unmanned aircrafts and robotics are a prerequisite for achieving more efficient, more productive and more- sustainable agriculture. This accurate-based and resource efficient approach is influenced by the parameters of Industry 4.0 and enables the implementation of the cyber-physical systems in agriculture as a prerequisite for smart-based planning, management, resource return and yield efficiency. In some publications [3], smart-based agriculture is determined as the latest agricultural revolution, which, through the Internet of things, connects smart machiness and sensors in farms, and makes the sustainable agricultural practices data-based. A number of publications [10] assume that a smart farm should be based on a blockchain security framework, which efficiently checks the status of the device and the sensor disorders, and mitigates threats. The researched authors also point out that a key point in smart agriculture is “optimization“, which is aimed at optimizing each variable and input during the production stage [3]. The sustainability of agriculture using information technologies is reduced to increasing the quantity and quality of the manufactured production, and, at the same time – keeping the environmental balance, preserving the natural resources and avoiding their irreversible depletion. 1 Introduction According to some other authors [11], the digitalization of agriculture facilitates monitoring, marketing, technologies and accelerates the production process through smart and critical application of digital models and technologies. Smart agriculture is a management concept focused on providing to the agricultural industry an infrastructure for using advanced technologies – including big data, cloud computing and the Internet of things – for following up, monitoring, automation and analyzing operations. [4] The technologies, which are most often mentioned in literature [3, 4], and are applicable in smart agriculture, are the following:  sensors for scanning the soil and managing irrigation, light, humidity and temperature;  telecommunication technologies, such as GPS and GIS;  hardware and software for specialized applications;  digital tools for taking online decisions;  smart forecasting;  smart planning;  smart management;  robotics;  automation. It can be summarized on the basis of the carried out theoretical research of “smart agriculture” that it resolves the main problems related to sustainable development and environment preservation through optimal, but also environment-friendly and nature- sparing use of the soil, water and energy [12, 13]. In that sense, the main task in implementing cyber-physical systems in greenhouse production is observing the parameters of the core resources involved in manufacturing agricultural production:  management of maintaining a healthy soil;  irrigation management;  pesticide use management;  energy source management;  management of the provided light;  management of biodiversity in the soil and air;  production process management. 2 2.1 Parameters ensuring the optimality of the model The integration of the cyber-physical system within the Concept for smart greenhouse agriculture requires the formulation of the main target parameters. Undoubtedly, the introduction of the philosophy of the cyber-physical systems in greenhouse production is aimed at achieving higher added value based on a more efficient decision making (in respect to expedience, resource consumption and profitability) and more efficient production operations (in respect to resource consumption and effectiveness), related to the exploitation of resources, data gathering and analysis in the course of the production process, the cultivation of the farm lands and harvesting the crop. That means to largely reduce the conventional manufacturing and labour processes on account of the automated, smart-based and smart-managed production-and-technological operations. The restrictive conditions in the model of a cyber-physical installation for smart greenhouse agriculture result from the need to reduce resource consumption in the agricultural production process. The main production resources in agriculture are time (as duration of the entire production process), water (for irrigation), chemicals ensuring healthy and high quality production (fertilizers, spraying chemicals, etc.), energy (used for heating and air-conditioning), labour (in terms of costs for human resource remunerations), depreciation of the used technical equipment (in cash equivalent), penalty fines related to breaching the ecological and biological equilibrium (if that is the case). The aforementioned resources have their value expression, and, in general, determine the cost price for manufacturing 1 kg of agricultural greenhouse production. The managing parameters in the model of a cyber-physical installation for smart greenhouse agriculture should be related to preliminary set borderline values in respect to the qualitative and quantitative characteristics of the soil, humidity, heat, illumination, biodiversity, as well as in respect to the borderline qualitative parameters for the content of pesticides in the agricultural production. On the background of the defined restrictive conditions and managing parameters in the model of a cyber-physical installation for smart greenhouse agriculture, there could also be determined the optimality criterion – maximum return of the invested financial resources. 2 E3S Web of Conferences 404, 02004 (2023) EEPES 2023 https://doi.org/10.1051/e3sconf/202340402004 The researches by a number of theoretical sources show that the sustainability of smart agriculture is a complex, multilayer concept, which sets as a goal achieving in parallel profitability and preservation of the global natural assets and ecological balance for the future generations. 2.2 Structural parameters of the model The determined target parameters of the model of a cyber-physical installation for smart greenhouse agriculture have their substantial contribution also for structuring the cyber- physical installation itself. It has a six-degree hierarchical structure, where each structural element is interrelated and determinating in respect to all the others. On this basis, the cyber-physical installation for smart greenhouse agriculture has the following structure (Figure 1):  a greenhouse agricultural system, comprised of its two main subsystems – production and management one, where the physical manufacturing process takes place in a particular temporal and spatial aspect;  a greenhouse agricultural system, comprised of its two main subsystems – production and management one, where the physical manufacturing process takes place in a particular temporal and spatial aspect; 3 E3S Web of Conferences 404, 02004 (2023) EEPES 2023 https://doi.org/10.1051/e3sconf/202340402004  a sensor-smart system designed for gathering data from the real-life physical farm land through sensors, controlling apparatuses, technologies, management systems, which through the Internet of things maintain connection both with the real-life physical environment, and with the virtual one;  a sensor-smart system designed for gathering data from the real-life physical farm land through sensors, controlling apparatuses, technologies, management systems, which through the Internet of things maintain connection both with the real-life physical environment, and with the virtual one;  an information system, where the signals gathered by the sensor-smart system are transformed into semantically rationalized information;  an information system, where the signals gathered by the sensor-smart system are transformed into semantically rationalized information;  a cyber system functioning as a central information centre, comprised of a network of cyber devices, which store a large bulk of information for different time periods, and with different situational variables, which analyze algorithms, models and instruments via specific methodologies that have been programmed in them, and generate, on this basis, alternative options for a choice of management decisions;  a smart system generating in-depth knowledge about the physical greenhouse agricultural system based on expert systems, whereby a certain decision is analyzed and informationally supported;  a smart system generating in-depth knowledge about the physical greenhouse agricultural system based on expert systems, whereby a certain decision is analyzed and informationally supported;  a management and feedback system, which implements the decision chosen by the system and configures the feedback from the cyber space to the physical greenhouse agricultural system. 2.2 Structural parameters of the model  a management and feedback system, which implements the decision chosen by the system and configures the feedback from the cyber space to the physical greenhouse agricultural system. Fig. 1. Architectural structure of a cyber-physical installation for smart greenhouse agriculture. Fig. 1. Architectural structure of a cyber-physical installation for smart greenhouse agriculture. At the same time, the described smart greenhouse agriculture is based on information and communication technologies. Thus, the model of a cyber-physical installation for smart greenhouse agriculture should include in its structure three interrelated and interdependent technological areas:  management information systems;  smart-based agriculture;  process automation;  robotized performance of certain activities. The management information systems are associated with data gathering, processing, storing, analyzing and using by its designation on the basis of sensors, transducers and other technological solutions. It is these systems that allow adequate smart-based forecasting, planning, coordinating, controlling and managing the agricultural production processes. The management information systems draw their physical data from the soil, the air, from plants’ root and foliar systems. This data is gathered by the sensor-smart system of the cyber-physical installation and is processed and transformed into digital information (by the information system of the cyber-physical installation), which is compared to the 4 4 4 E3S Web of Conferences 404, 02004 (2023) EEPES 2023 https://doi.org/10.1051/e3sconf/202340402004 determined borderline values in the restrictive conditions defined in the model (by the cyber system of the cyber-physical installation). If necessary (when the measured physical data exceeds the indicated limits), in a smart-based way (through the management and feedback system of the cyber-physical installation) specific production-and-technological processes are activated, such as irrigation, heating or cooling, or ventilating, opening and closing the roof of the greenhouse, nourishing the soil with certain substances, treating the foliage against pests, etc. determined borderline values in the restrictive conditions defined in the model (by the cyber system of the cyber-physical installation). If necessary (when the measured physical data exceeds the indicated limits), in a smart-based way (through the management and feedback system of the cyber-physical installation) specific production-and-technological processes are activated, such as irrigation, heating or cooling, or ventilating, opening and closing the roof of the greenhouse, nourishing the soil with certain substances, treating the foliage against pests, etc. 2.2 Structural parameters of the model Smart agriculture concerns managing the spatial and temporal variability to ensure the return of the invested financial resources with coordinated observation of no impact, or at least, reducing the impact on the environment. Critically important are the systems facilitating the decision making in view of ensuring optimality in respect to the return of the invested resources. The automation of the agricultural production processes issues from the commands given by the smart system to the main robotized and automated elements of the cyber- physical installation. Robotics (unmanned aircrafts for treatment, drones, etc.) ensures maintaining the parameters, which are set via the restrictive conditions of the model, and, on this basis, achieving the optimality criterion set as a goal. 3 A model of a cyber-physical installation for smart greenhouse agriculture At the same time, on the basis of the integrated therein managing parameters of the model of a cyber-physical installation for smart greenhouse agriculture, it defines accurately and precisely:  the quantities of water necessary for irrigation and the duration of the irrigation process;  the quantities of water necessary for irrigation and the duration of the irrigation process;  the quantities of fertilizers or chemicals, whereby soil is enriched (if such a need is defined by the cyber system);  the quantity of energy necessary for reaching particular temperature of the air (heating or cooling);  the quantity of water, which has to be sprayed into the air in the greenhouse, or the time necessary for the greenhouse roof to remain opened (on the basis of information from a sensor in the indoor greenhouse environment about humidity outside of the greenhouse) until reaching the required air humidity;  the extent of the opening of the roof or of other parts of the greenhouse in view of ensuring the necessary circulation of the air and illumination, providing for the respective period of the crops growth;  the type and quantity of the substance necessary for treating plants’ foliage or root system in the greenhouse. Upon diagnosing the needs of the real-life physical agricultural environment, the smart system sends data about those needs also to the information system, which generates a new move to the cyber system, in order to check the result, which would have been generated when carrying out the activities determined by the smart system of the cyber-physical installation for smart greenhouse agriculture. Thus, the cycle of the cyber-physical installation is turned again, but solely and only in respect to the relation “information system – cyber system – smart system”. 3 A model of a cyber-physical installation for smart greenhouse agriculture The model of a cyber-physical installation for smart greenhouse agriculture is related to a particular process organization, which is based on the parameters ensuring the optimality of the model and of its structural parameters. (Figure 2). Fig. 2. A model of a cyber-physical installation for smart greenhouse agriculture. Author’s own elaboration. Fig. 2. A model of a cyber-physical installation for smart greenhouse agriculture. Author’s own elaboration. The sensor-smart system of a cyber-physical installation for smart greenhouse agriculture gathers via sensors and transducers data from the real-life physical greenhouse agricultural system about:  the soil humidity;  the minerals in the soil;  the temperature of the air;  the air humidity;  the air circulation; 5 5 E3S Web of Conferences 404, 02004 (2023) EEPES 2023 https://doi.org/10.1051/e3sconf/202340402004  plants’ illumination;  plants’ illumination;  plants’ illumination;  h di i f l ’ f li i i f h f i f p ;  the condition of plants’ foliage in view of the presence of a certain type of pests;  the condition of plants’ root system foliage in view of the presence of a certain type of pests. The data gathered via the sensors and transducers is transmitted into the information system, where it is turned into semantically meaningful and relevant information. The synthesized information is transmitted from the information system into the cyber one, where a comparison is made with a big database, as well as an analysis of the condition of the physical greenhouse agricultural system. On the basis of the integrated restrictive conditions of the model in respect to the resources, the cyber system provides recommendations for the necessity of carrying out certain operational activities, such as fertilizing, irrigating, spraying against pests, heating or cooling the air, ventilating and other agricultural activities related to the normal development of the cultivated crops. The smart system of the cyber-physical installation for smart greenhouse agriculture is based on the alternative options for a decision generated by the cyber system, where the latter provides approval for carrying out certain operational activities. 1. S. Mohamed, A. Belal, S. K. Abd-Elmabod, M. A. El-Shirbeny, Egyptian Journal of Remote Sensing and Space Science, 24(4), (2021) 4 Conclusion Modelling a cyber-physical installation for smart greenhouse agriculture in an abstract and synthesized way summarizes the main parameters of a real-life example for a smart-based agricultural system. The precise definition of the restrictive conditions, the managing parameters and the criterion for optimality of the agricultural production, as well as the integration of those mathematical variables into the real-life architecture of a cyber-physical system adapted for the needs of the smart-based greenhouse agriculture, leads to achieving several major effects:  reduced duration of the communication, management, planning and forecasting processes related to the functioning of the greenhouse; elated to the functioning of the greenhouse; p g g  reduced time for carrying out particular agricultural activi  reduced loss of resources when performing certain agricultural activities, such as fertilizing, spraying, irrigating, heating, cooling, etc.;  efficient growing of agricultural crops is ensured on the basis of accurate, precise and correct adherence to the technological processes and norms in agriculture. The model of a cyber-physical installation for smart greenhouse agriculture leads to the following synergic effects, which provide grounds for the model to be upgraded and further improved: The model of a cyber-physical installation for smart greenhouse agriculture leads to the following synergic effects, which provide grounds for the model to be upgraded and further improved: p  a rational production organization enabling the optimal capacity loading of both facilities and staff;  optimal production costs ensuring the cost price per kilogram or generated yield   efficient production, technological and social added value ensuring the ecological and biological equilibrium, the preservation of the natural resources and the sustainability of production in a long-term period of time. The aforementioned effects irrevocably determine also the achievement of the criterion for optimality set in the model of a cyber-physical installation for smart greenhouse agriculture – maximum return of the incurred financial expenses. 3 A model of a cyber-physical installation for smart greenhouse agriculture The management and feedback system, after completing the verification of the efficiency of the activities envisaged to be performed (the verification is on the basis of going through the cycle “information system – cyber system – smart system”, ordered by the smart system), commands the automated and robotized systems and technologies to perform certain operational activities, such as:  programming and starting the drip irrigation systems;  programming and starting the robotized fertilizing equipment;  programming and starting the air-conditioning units;  programming and starting the installations for air humidification or opening to a certain extent the roof and/or other parts of the greenhouse in view of ensuring the 6 6 E3S Web of Conferences 404, 02004 (2023) EEPES 2023 https://doi.org/10.1051/e3sconf/202340402004 necessary humidity of the air on the basis of information from the external environment of the greenhouse; necessary humidity of the air on the basis of information from the external environment of the greenhouse;  programming and starting the air circulation installations or opening to a certain extent the roof and/or other parts of the greenhouse in view of ensuring the necessary air flow on the basis of information from the external environment of the greenhouse;  programming of the opening to a certain extent of the roof and/or other parts of the greenhouse and starting this process in view of ensuring the necessary and illumination of the crops;  programming of the opening to a certain extent of the roof and/or other parts of the greenhouse and starting this process in view of ensuring the necessary and illumination of the crops;  programming and starting drones for spraying the plants and/or their root system in view of protecting them from certain pests.  programming and starting drones for spraying the plants and/or their root system in view of protecting them from certain pests. 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https://openalex.org/W4393868389	https://ojs.unimal.ac.id/galenical/article/download/10256/pdf	Indonesian	null	Leimyosarcoma	Galenical	2,024	cc-by-sa	2,280	GALENICAL: Jurnal Kedokteran dan Kesehatan Mahasiswa Malikussaleh Vol.3 No.2 April 2024 AL: Jurnal Kedokteran dan Kesehatan Mahasiswa Malikussaleh Vol.3 No.2 April 2024 Laporan Kasus Laporan Kasus Laporan Kasus GALENICAL: Jurnal Kedokteran dan Kesehatan Mahasiswa Malikussaleh Vol.3 No.2 April 2024 Leimyosarcoma Muhammad Sayuti 1, Nurul Afni 2 Muhammad Sayuti 1, Nurul Afni 2 1Departemen Bedah Fakultas Kedokteran, Universitas Malikussaleh/RSUD Cut Meutia, Aceh Utara Lhokseumawe, 24351, Indonesias 2Mahasiswa Program Studi Kedokteran Fakultas Kedokteran Universitas Malikussaleh, Lhokseumawe, 24351, Indonesia 1Departemen Bedah Fakultas Kedokteran, Universitas Malikussaleh/RSUD Cut Meutia, Aceh Utara Lhokseumawe, 24351, Indonesias 2Mahasiswa Program Studi Kedokteran Fakultas Kedokteran Universitas Malikussaleh, Lhokseumawe, 24351, Indonesia 1Departemen Bedah Fakultas Kedokteran, Universitas Malikussaleh/RSUD Cut Meutia, Aceh Utara Lhokseumawe, 24351, Indonesias 2Mahasiswa Program Studi Kedokteran Fakultas Kedokteran Universitas Malikussaleh, Lhokseumawe, 24351, Indonesia JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH\| 42 Galenical is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License Abstrak Sarkoma jaringan lunak atau soft-tissue sarcoma (STS) muncul dari mesoderm embrionik. STS adalah keganasan langka yang mengenai kurang dari 1% semua kanker pada orang dewasa. Leiomyosarcoma (LMS) adalah salah satu subtipe yang lebih umum dari STS, yaitu sekitar 25% dari semua sarkoma. Secara klasik, LMS berasal langsung dari sel otot polos atau dari sel punca mesenkim prekursor yang pada akhirnya akan berdiferensiasi menjadi sel otot polos. Meskipun sel-sel ini ada di mana-mana, LMS menunjukkan kecenderungan terjadi di jaringan dan organ abdominopelvic, dibandingkan dengan ekstremitas. Pasien yang masuk dari poli RSU Cut Meutia dengan keluhan nyeri perut, mual yang memberat kurang lebih 3 hari sebelum masuk RS dan ukuran perut yang semakin membesar yang dirasakan selama 19 bulan terakhir dan pada inspeksi didapatkan abdomen asimetris, pada palpasi di dapatkan nyeri tekan dan massa teraba keras, pada perkusi didapatkan redup. Penatalaksanaan yang dilakukan pada pasien berupa laparotomy eksplorasi dengan surgical resection, serta dilakukan pemeriksaan penunjang berupa patologi anatomi. Kata Kunci: Leiomyosarcoma,Sarkoma jaringan lunak, saluran pencernaan Kata Kunci: Leiomyosarcoma,Sarkoma jaringan lunak, saluran pencernaan Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 Keywords : Leiomyosarcoma, soft-tissue sarcoma (STS), digestive tract Keywords : Leiomyosarcoma, soft-tissue sarcoma (STS), digestive tract Abstract Soft-tissue sarcoma (STS) arises from embryonic mesoderm. STS is a rare malignancy that accounts for less than 1% of all cancers in adults. Leiomyosarcoma (LMS) is one of the more common subtypes of STS, accounting for about 25% of all sarcomas. Classically, LMS are derived directly from smooth muscle cells or from precursor mesenchymal peunca cells that will eventually differentiate into smooth muscle cells. Although these cells are ubiquitous, LMS shows a tendency to occur in the abdominopelvic tissues and organs, as compared to the extremities. Patients who were admitted from the Cut Meutia Hospital with complaints of abdominal pain, nausea that worsened approximately 3 days before admission to the hospital and an increasingly enlarged abdomen that had been felt for the last 19 months and on inspection found an asymmetrical abdomen, on palpation there was tenderness and mass. palpable hard, on percussion found dim. Management carried out on the patient in the form of exploratory laparotomy with surgical resection, as well as supporting examinations in the form of anatomical pathology. 1. PENDAHULUAN Sekitar 80% sarkoma berasal dari jaringan lunak, sedangkan sisanya berasal dari tulang. Dalam hal ini, World Health Organization (WHO) mengklasifikasikan sebagian besar neoplasma jaringan lunak sesuai jaringan asal, diantaranya berupa liposarcoma, sarkoma sinovial, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, dan angiosarkoma.1 Sarkoma jaringan lunak atau soft-tissue sarcoma (STS) muncul terutama dari mesoderm embrionik dengan beberapa kontribusi dari neuroektoderm. Ini merupakan kelompok tumor yang sangat heterogen dengan lebih dari 70 subtipe molekuler. Leiomyosarcoma (LMS) adalah salah satu subtipe yang lebih umum dari STS, terdiri hingga 25% dari semua sarkoma. Secara klasik, LMS berasal dari sel otot polos atau dari sel punca mesenkim prekursor yang pada akhirnya akan berdiferensiasi menjadi sel otot polos. Meskipun sel-sel ini ada di mana-mana, LMS menunjukkan kecenderungan terjadi dijaringan dan organ abdominopelvic, dibandingkan dengan ekstremitas. Kelainan genetik pada LMS sangat kompleks dan membuatnya cukup sensitif terhadap kemoterapi. Meskipun diperdebatkan dalam literatur, perilaku LMS dan sensitivitas untuk pengobatan tampaknya tergantung pada organ asal. Pendekatan interprofesional dianggap sangat penting dilakukan untuk pengobatan LMS.3 JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 43 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 LAPORAN KASUS Pasien perempuan usia 33 tahun, datang ke Poli RS Cut Meutia dengan keluhan perutnya keras dan bertambah besar yang dirasakan sejak 19 bulan sebelum masuk RS, setelah melahirkan secara sectio caesar anak ke-3, sejak 3 hari terakhir pasien mengeluhkan nyeri perut dan mual yang semakin memberat, disertai haid tidak teratur, sering BAK, dan penurunan berat badan sebanyak 15kg. Pasien menyangkal memiliki penyakit diabetes mellitus dan hipertensi di sangkal. Pasien juga menyatakan bahwa sebelum di bawa ke RS, pasien telah mengkomsumsi obat-obat herbal dan tidak ada perubahan. Pada pemeriksaan fisik didapatkan bahwa kesadaran compos mentis, tekanan darah 110/70 mmHg, nadi 100 x/m regular, frekuensi pernapasan 16x/menit, suhu 36°C. Pada inspeksi didapatkan abdomen asimetris, pada palpasi di dapatkan nyeri tekan dan benjolan teraba keras di regio lumbal kanan, pada perkusi didapatkan redup dan pada auskultasi didapatkan peristaltis usus normal. Pada pemeriksaan penunjang darah lengkap didapatkan Hb 11,40 g/dl, eritrosit 4,38 Juta/uL, Hematokrit 32,91%, MCV 75,19 fl, MCH 26,05 Pg, MCHC 36,64 g/dl, leukosit 5,10 ribu/uL, RDW-CV 11,24 %, bleeding time 2’ menit, Clothing time 8’ menit, ureum 22 mg/dl, kreatinin 0,60 mg/dl, dan KGDs 120 mg/dl. Gambar 1. Ct-Scan abdomen Gambar 1. Ct-Scan abdomen Pada pemeriksaan Ct-Scan didapatkan Massa solid ukuran +/-16.7 x 16.5 x 11,84 cm di cavum abdomen atas dengan infiltrasi musculusrectus abdominis dan JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 44 Gambar 1. Ct-Scan abdomen Gambar 1. Ct-Scan abdomen Pada pemeriksaan Ct-Scan didapatkan Massa solid ukuran +/-16.7 x 16.5 x 11,84 cm di cavum abdomen atas dengan infiltrasi musculusrectus abdominis dan JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 44 JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 44 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) y ( y ) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 menempel dengan dinding bowel disertai pendesakan bowel ke lateral. Inferior dan pembesaran kelenjar getah bening (KGB) multiple di parailiaca, inguinal kanan kiri dengan ukuran subcentimeter mengarah gambaran gastrointestinal stromal tumors (GIST), Hidronefrosis ringan dextra. Pasien ini didiagnosis dengan Tumor intra abdomen dan susp. GIST dengan diagnosis banding berupa Leimyosarcoma, Fibrosarcoma, dan Angiosarkom. LAPORAN KASUS Tatalaksana yang dilakukan pada pasien berupa tatalaksana non farmakologi yang terdiri dari istirahat dan diet yang cukup, sedangkan tatalaksana farmakologi terdiri dari pemberian IVFD RL 20gtt/I, Inj.Ketorolac 1amp/8j, Inj. Ranitidin 1amp/12j, Inj.Ceftriakson 1gr pre operasi. Tatalaksana bedah dilakukan laparotomy explorasi dengan surgical resection. Pasien dirawat selama 6 hari, hari ke-4 post operasi dipulangkan dengan keadaan umum sangat baik. Hasil pemeriksaan histopatologi didapatkan bahwa massa tumor merupakan suatu sarcoma dd leimyosarcoma dd fibrosarcoma. Gambar 2. Lyeimyosarcoma Gambar 2. Lyeimyosarcoma PEMBAHASAN Gambar 2. Lyeimyosarcoma PEMBAHASAN Pasien datang dengan keluhan utama ukuran perut yang semakin membesar dan taraba keras, nyeri perut, dan mual yang semakin memberat, haid tidak teratur, sering BAK, dan penurunan berat badan sebanyak 15 kg. Hal ini sesuai dengan gejala leimyosarcoma yaitu berupa sakit perut, mual sampai muntah, dan benjolan JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 45 \| 45 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 y ( y ) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 perut merupakan keluhan yang paling umum terjadi.10 Leiomyosarcoma (LMS) adalah salah satu subtipe yang paling umum dari sarkoma jaringan lunak pada orang dewasa dan dapat terjadi di hampir semua bagian tubuh.4 Tumor mungkin tidak menimbulkan gejala apa pun untuk jangka waktu yang lama, sampai tumbuh menjadi ukuran besar dan menekan organ-organ yang berdekatan di perut. 7 Tanda dan gejala Leiomyosarcoma Retroperitoneal meliputi perasaan penuh yang tidak biasa di daerah panggul atau perut (rongga retroperitoneal), nyeri di daerah panggul atau perut, sering buang air kecil, penurunan berat badan, kelemahan, serta demam. Penting untuk dicatat bahwa tidak ada gambaran klinis spesifik yang dapat digunakan untuk mengidentifikasi leiomyosarcoma dari sarkoma jaringan lunak panggul lainnya. Biopsi diperlukan untuk memastikan suatu leiomyosarcoma. 7 Leiomyosarcoma adalah jenis sarkoma kedua yang paling umum terlihat di retroperitoneum, terhitung 15% dari semua RPS (Retroperitoneal Soft Tissue Sarcomas). Tumor ini muncul dari sel otot polos terutama ditemukan di dalam pembuluh darah, khususnya vena besar retroperitoneum (misalnya, vena cava inferior, vena ginjal, vena iliaka, dan vena gonad), meskipun mereka juga dapat berkembang dari vena kecil yang tidak disebutkan namanya. Faktor risiko utama untuk Leiomyosarcoma adalah riwayat genetik keluarga, faktor kesuburan, riwayat menstruasi, menyusui, indeks tinggi badan dan massa tubuh, kontrasepsi, olahraga, gaya hidup, diet, penyakit ginekologis, faktor psikologis, dan terapi hormonal.11 Tidak ada faktor pasti yang dapat diidentifikasi sebagai faktor penyebab LMS. Riwayat radioterapi (RT) sebelumnya, yang merupakan salah satu faktor risiko paling signifikan untuk memunculkan STS, juga dapat menyebabkan perkembangan LMS. Pasien dengan sindrom genetik seperti retinoblastoma herediter (penghapusan gen RB1) dan sindrom Li-Fraumeni (mutasi pada gen TP53) beresiko tinggi menderita LMS dari pada STS lainnya. Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) PEMBAHASAN Insidensi LMS pada pasien immunocompromised datanya sangat terbatas (penerima transplantasi organ yang memiliki mutasi bawaan). Epstein Barr virus berkorelasi dengan LMS.4 JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 46 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 Untuk RPS, pemindaian computed tomography (CT) adalah modalitas pencitraan yang lebih disukai, berbeda dengan sarkoma trunkal dan ekstremitas, di mana MRI dianggap sebagai modalitas pencitraan standar. Pencitraan penampang perut dan panggul, biasanya dengan CT scan, digunakan untuk menentukan sejauh mana penyebaran tumor dan invasi ke organ visera dan struktur vaskular yang berdekatan, yang penting untuk perencanaan bedah. Hati dan peritoneum juga harus dievaluasi untuk mencari bukti adanya metastasis. Mengingat bahwa 14% pasien dengan RPS derajat tinggi (misalnya, liposarkoma terdiferensiasi dan leiomiosarkoma) biasanya datang dengan metastasis paru sehingga harus dilakukan pemeriksaan tambahan berupa penambahan CT scan thoraks.5 KESIMPULAN Telah dilaporkan pasien yang datang ke Poli RS Cut Meutia dengan keluhan nyeri perut dan mual yang semakin memberat sejak 3 hari sebelum masuk RS serta ukuran perut yang semakin membesar selama kurang lebih 19 bulan terakhir, keluhan pertama dirasakan setelah 2 bulan melahirkan secara sectio caesarea anak ke-3, yaitu perutnya semakin membesar dan keras. Berdasarkan anamnesis, pemeriksaan fisik dan pemeriksaan penunjang pada pasien didiagnosis dengan leimyosarcoma. Pasien telah dilakukan tindakan Laparotomy explorasi dengan surgical resection. Pasien pulang pada hari ke empat post operasi dengan keadaan umum sangat baik. DAFTAR PUSTAKA 1. Tay F. Local Recurrence of Primary Cardiac Leiomyosarcoma After Resection : A Rare Case. Indones J cancer. 2021;15:223–5. 1. Tay F. Local Recurrence of Primary Cardiac Leiomyosarcoma After Resection : A Rare Case. Indones J cancer. 2021;15:223–5. 2. Mestiri S, Elghali MA, Bourigua R, Abdessayed N, Nasri S, Amine BA, et al. rare tumors Soft tissue leiomyosarcoma — diagnostics , management , and prognosis : Data of the registry cancer of the center of Tunisia. J sage pub. 2019;11:1–7. 2. Mestiri S, Elghali MA, Bourigua R, Abdessayed N, Nasri S, Amine BA, et al. rare tumors Soft tissue leiomyosarcoma — diagnostics , management , and prognosis : Data of the registry cancer of the center of Tunisia. J sage pub. 2019;11:1–7. 3. Mangla A, Yadav U, Health CC. Cancer , Leiomyosarcoma Cancer , Leiomyosarcoma. NCBI J. 2019;(November). 3. Mangla A, Yadav U, Health CC. Cancer , Leiomyosarcoma Cancer , Leiomyosarcoma. NCBI J. 2019;(November). JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 47 Leimyosarcoma… (Muhammad Sayuti, Nurul Afni) GALENICAL Volume 3 Nomor 2. Bulan April, Tahun 2024. Hal. 74-82 4. George S, Hensley ML, Ray-coquard I. Soft Tissue and Uterine Leiomyosarcoma. J Am Soc Clin Oncol. 2019;36(2):144–51. 5. Turgeon MK. S o f t T i s s u e Tu m o r s o f t h e Abdomen and R e t ro p e r i t o n e u m. Surg Clin NA. 2020;1–19. 6. Roberts ME, Aynardi JT, Chu CS. Gynecologic Oncology Uterine leiomyosarcoma : A review of the literature and update on management options. Gynecol Oncol [Internet]. 20187. Kumar V, Kumar A, Chand G, Johri G. of Cancer Cancer Case Case Reports Reports American Leiomyosarcoma of Colon Presented as Retroperitoneal Mass : A Rare Case Report. Am Cancer Case Reports. 2017;5(1):1–6. 8. 8. Wang W, Soslow R, Hensley M, Asad H, Zannoni GF, Nictolis M De, et al. Histopathologic Prognostic Factors in Stage I Leiomyosarcoma of the Uterus : A Detailed Analysis of 27 Cases. J Surg Pathol. 2018;35(4):522–9. 9. Soslow RA, Nonaka D, Olshen AB, Ph D, Aghajanian C, Sabbatini P, et al. of Estrogen , Progesterone , and Androgen Receptors in Uterine Leiomyomata and Leiomyosarcoma. J Am Cancer Soc. 2004;(July). 10. Luthra M, Kumar C. Surgical Management of Adnexal Masses in the Pediatric and Adolescent Age Group: Our Experience. J Indian Assoc Pediatr Surg. 2021;26(1):70–98. 11. DAFTAR PUSTAKA Hu X, Li D, Liang Z, Liao Y, Yang L, Wang R, et al. Indirect comparison of the diagnostic performance of 18 F-FDG PET / CT and MRI in differentiating benign and malignant ovarian or adnexal tumors : a systematic review and meta-analysis. J BMC Cancer. 2021;1–12. 12. Byar KL, Fredericks T. Uterine Leiomyosarcoma. J Adv Pract Oncol. 2022;13(1):1–17. 13. Folpe AL. Fibrosarcoma : A Review and Update. J Histopatology. 2013;1–3 JURNAL KEDOKTERAN DAN KESEHATAN MAHASISWA MALIKUSSALEH \| 48
https://openalex.org/W3034420755	https://repositorio.uam.es/bitstream/10486/694799/1/dynamic_travieso_FP_2020.pdf	English	null	Dynamic Touch as Common Ground for Enactivism and Ecological Psychology	Frontiers in psychology	2,020	cc-by	9,603	Dynamic Touch as Common Ground for Enactivism and Ecological Psychology David Travieso1, Lorena Lobo2,3, Carlos de Paz1, Thijme E. Langelaar4, Jorge Ibáñez-Gijón1* and David M. Jacobs1 1 Facultad de Psicología, Universidad Autónoma de Madrid, Madrid, Spain, 2 Facultad de Ciencias de la Salud y de la Educación, Universidad a Distancia de Madrid, Villalba, Spain, 3 Embodied Cognitive Science Unit, Okinawa Institute of Science and Technology, Okinawa, Japan, 4 Faculty of Medical Sciences, University of Groningen, Groningen, Netherlands The main purpose of this article is to show that enactivism and ecological psychology share more aspects than is often recognized. Rather than debating about differences, commonalities between the approaches are illustrated with the example of dynamic touch. Dynamic touch is a form of touch that implies muscles and tendons and that allows the perception of hand-held objects that are wielded but not seen. Given that perceivers perform the wielding movements with effort, dynamic touch necessarily implies active exploration. The strength of dynamic touch as an example lies in the fact that it has been formalized and analyzed in detail at the level of the laws that govern the organism-environment system. The example provides empirically supported instantiations of sensorimotor contingencies, in enactivist terms, and of intentional exploration and information detection, in ecological terms. Moreover, dynamic touch is a practical example of the enactivist concepts of bringing-forth the world and sense- making. As a second purpose, we use the example of dynamic touch to clarify key concepts of the ecological approach. Speciﬁcally, we analyze the concepts of invariance and affordance, indicating the crucial difference between perceiving and actualizing affordances, and highlighting the importance of these concepts for the dialogue between enactivism and ecological psychology. HYPOTHESIS AND THEORY published: 10 June 2020 doi: 10.3389/fpsyg.2020.01257 ENACTIVISM AND ECOLOGICAL PSYCHOLOGY SHOULD BRIDGE THE UNCANNY VALLEY Received: 26 February 2020 Accepted: 14 May 2020 Published: 10 June 2020 Keywords: ecological psychology, dynamic touch, enactivism, intentionality, postcognitivism Edited by: Edited by: Ezequiel A. Di Paolo, IKERBASQUE Basque Foundation for Science, Spain Reviewed by: Fernando Bermejo, National University of Córdoba, Argentina Carlos Avilés, Complutense University of Madrid, Spain Correspondence: Jorge Ibáñez-Gijón jorge.ibannez@uam.es Correspondence: Jorge Ibáñez-Gijón jorge.ibannez@uam.es Specialty section: This article was submitted to Theoretical and Philosophical Psychology, a section of the journal Frontiers in Psychology Keywords: ecological psychology, dynamic touch, enactivism, intentionality, postcognitivism WHAT IS DYNAMIC TOUCH? As far as we are aware, the study of dynamic touch can be traced back to Gibson’s article Observations on Active Touch (Gibson, 1962) and his seminal book The Senses Considered as Perceptual Systems (Gibson, 1966). In the article, Gibson questioned the everyday relevance of touch as conceived as a passive sensory channel. Instead, he highlighted the active nature of touch and the prominent role of exploration. He did so by comparing passive and active conditions of haptic perception in a series of experiments. In his words: This is one of the reasons for the admittedly rather quick dismissal of ecological psychology by Varela et al. (1991), who saw it only capable of providing a theory of cognition on the side of the environment. In later years, there have been many attempts at bringing the two traditions closer to each other. (Footnote 3, p. 18) As indicated in the previous quote, recently there have been a number of attempts to relate enactivism and ecological psychology, giving room to at least three main positions (Higueras-Herbada et al., 2019). The ﬁrst position considers the approaches irreconcilable (Cariani, 2016) or with substantial diﬀerences (Mossio and Taraborelli, 2008). The second position considers the approaches complementary, focusing on diﬀerent levels of analysis. Thus, whereas the ecological theory of perception focuses on the ecological level of analysis, enactivism focuses on autonomous agency or on a subpersonal level of analysis (Heras-Escribano, 2016, 2019a,b; McGann, 2016). A third position, as formulated by Stapleton (2016) in her commentary on Fultot et al. (2016), holds that there are already examples that joint crucial concepts of the considered approaches: Active touch refers to what is ordinarily called touching. This ought to be distinguished from passive touch, or being touched. In one case the impression on the skin is brought about by the perceiver himself and in the other case by some outside agency . . . . Active touch is an exploratory rather than a merely receptive sense. When a person touches something with his ﬁngers he produces the stimulation as it were. More exactly, variations in skin stimulation are caused by variation in his motor activity. . . . Such movements are not the ordinary kind usually thought of as responses. They do not modify the environment but only the stimuli coming from the environment. (Gibson, 1962, p. Citation: The enactive and ecological approaches are two disciplines concerned with understanding cognitive systems using a perspective that avoids scientiﬁc constructs such as internal representations or mental states, focusing instead on the interaction between the agent and its environment. Despite these strong commonalities, confrontation between enactive and ecological approaches has been the norm since the early days of enactivism. In the introduction of their book, Sensorimotor Life, Di Paolo et al. (2017) consider the relation between the enactive approach to cognition and the ecological approach to perception and action. In their words: Travieso D, Lobo L, de Paz C, Langelaar TE, Ibáñez-Gijón J and Jacobs DM (2020) Dynamic Touch as Common Ground for Enactivism and Ecological Psychology. Front. Psychol. 11:1257. doi: 10.3389/fpsyg.2020.01257 June 2020 \| Volume 11 \| Article 1257 1 Frontiers in Psychology \| www.frontiersin.org Travieso et al. Enactivism and Ecological Psychology are valuably incorporated by enactivists to ﬂesh out a full framework of life and mind. Likewise, ecological psychology can beneﬁt from the depth of the enactivist enterprise. (p. 327) The relation between the schools of thought is one of strange familiarity, as if their respective practitioners were staring at each other across an uncanny valley. It is true that both approaches overlap in their rejection of representationalism, but this does not mean they are necessarily rejecting the same thing. As we will discuss further in this chapter and demonstrate in the following ones, the enactive perspective rejects a functionalist general approach to cognition, whereas ecological psychology rejects the assumption of the poverty of environmental information. These are not the same thing. For Gibsonians, perception is still about information pickup, but not for enactivists, who conceive of perception as an aspect of sense-making, a concept that is explicitly grounded in the notion of autonomous agency. (Footnote 3, p. 18) We believe that theoretical papers concerning the enactive and ecological approaches should be understood as being engaged in dialogical discussions (Voloshinov and Bachtin, 1986). In such discussions, main concepts are treated as dialogemes, which is to say, as voices where the content of the terms has to be colonized by a tradition. In such a process, diﬀerences between approaches are often stressed, even subtle ones, concealing strong commonalities. In order to avoid such detrimental drift, the present article takes the opposite direction and aims to stress commonalities. Citation: In this article, we unfold the concepts that, according to Di Paolo et al. (2017), divide the traditions. We consider what information pick-up is for ecological psychology and whether it is compatible with the ideas of sense-making and autonomous agency from the enactivist approach. Just above the quote that opened this article, Di Paolo et al. (2017) state: As proof of how close the approaches can be in concrete cases, we make use throughout this book of work originating in the Gibsonian tradition (Gibson, 1979/1986). This tradition usually supplies some of the clearest examples of how dynamical engagements and bodily synergies can be explanatorily powerful. (Footnote 3, p.18) As discussed in our previous work (Higueras-Herbada et al., 2019), the so-called 4E approaches (Gonzalez-Grandon and Froese, 2018; cf. Calvo and Gomila, 2008) – which include enactivism – only turn an eye to the much older ecological approach occasionally. As do the 4E approaches, the ecological approach has criticized the cognitivist approach from its very beginning (see Fultot et al., 2016, and the commentaries on that article; see also Lobo et al., 2018, for a historical overview of the ecological approach). The diﬀerent origins and foci of enactivism and ecological psychology, however, have at times prevented that the commonalities between the approaches are appropriately reﬂected in the writings of scholars of the respective approaches (cf. Baggs, 2018). As Di Paolo et al. (2017) state: In this article, we focus on what we consider the best example to indicate points of coincidence of the approaches, and to describe classic ecological concepts such as aﬀordance, invariant, and information pick-up through intentional (goal- driven) exploration. This example is the one of dynamic touch. Frontiers in Psychology \| www.frontiersin.org THE LAWS THAT GOVERN THE ORGANISM-ENVIRONMENT INTERACTION IN DYNAMIC TOUCH Measuring the force exerted by the rod is one of the ways that allows the detection of the mechanical property mass. To facilitate the presentation of less well-known properties later in this section, it may be helpful to note that the mass (M) of an object can approximated by: Having the sensation of softness consists in being aware that one can exercise certain practical skills with respect to the sponge: one can, for example, press it, and it will yield under the pressure. The experience of softness of the sponge is characterized by a variety of such possible patterns of interaction with the sponge, and the laws that describe these sensorimotor interactions we call, following MacKay (1962), laws of sensorimotor contingency (O’Regan and Noë, 2001). (p. 56) M = N X i=1 mi, (1) (1) in which the mis represent the point-masses of the object at N diﬀerent points (integral-form versions of this equation and of Eqs 2 and 3 can be found in Jacobs et al., 2009). As captured by Newton’s second law of motion (force = mass × acceleration), mass can also be deﬁned as the resistance of the object to linear accelerations. This means that moving a rod in a direction that is aligned with its longitudinal axis may reveal mass as the invariant that relates force and acceleration. In this regard it is curious to note that, when Gibson developed his intuitions about the functioning of dynamic touch, he cited several studies on the perception of softness, much in line with the writings of enactivist scholars. In Gibson’s (1966) words: The equation that deﬁnes mass does not include a term that relates to the length of the object. This means that one cannot diﬀerentiate rods with equal mass and diﬀerent lengths with linear movements along the longitudinal axis. Analogously, an object can be attached to ropes of diﬀerent lengths and if one holds the rope vertically, one can detect mass, but not the length of the rope. If, however, all the rods that one encounters are of the same material and diameter (for instance in a laboratory situation), then length and mass co-vary perfectly. This means The ﬁrmness or softness of a material substance is a property of the substance that is registered when forces are exerted on it by the hand. THE LAWS THAT GOVERN THE ORGANISM-ENVIRONMENT INTERACTION IN DYNAMIC TOUCH Manipulating objects, such as a tennis racket, produces perceptions that cannot be explained appealing exclusively to the stimulation of the skin. In Gibson’s (1966) words: Early research on dynamic touch analyzed the mechanical properties that govern the perception of the length of hand- held rods. In their pioneering study, Solomon and Turvey (1988) reported a linear relation between actual and perceived length and they argued that the observed pattern of results was consistent with the claim that the length judgments were based on the mechanical property that is referred to as ﬁrst principal moment of inertia (cf. Kreifeldt and Chuang, 1979). Although this claim has been supported by later studies (e.g., Fitzpatrick et al., 1994), it has also been questioned (e.g., Kingma et al., 2004). Kingma et al. demonstrated the importance of a diﬀerent mechanical property, referred to as static moment (cf. van de Langenberg et al., 2006). In the present section, we deﬁne such mechanical properties and provide intuitions about how these properties determine the relations between exerted forces and resulting movements. The passive skin can be stimulated by an object resting on it, the amount of pressure (that is, skin deformation) being proportional to the weight of the object, but in this case discrimination is rather poor. It is much better when the object is lifted. (p. 127) The diﬀerent amounts of stretching and contracting of muscles, as well as the forces exerted by and on the muscles and on the tendons, change depending on the movements that are performed and on the form, size, and mass distribution of the object. Those variations form the main sensory basis of dynamic touch (Carello and Turvey, 2015, 2017). The pivotal elements of the ecological conception of dynamic touch are the laws that connect motor components (forces exerted by muscles) and sensory components (in muscles and tendons). In this regard, the ecological portrayal of dynamic touch shows remarkable similarities with key concepts of the enactive tradition. Consider the example of the softness of a sponge that ﬁgures importantly in the writings of enactive scholars. According to O’Regan et al. (2005): Consider a rod that is loosely attached to a support at one of its ends so that the force of gravity orients the rod with its longitudinal axis toward the ground. WHAT IS DYNAMIC TOUCH? 477) Gibson referred for the ﬁrst time to dynamic touch (dynamic touching in the original) in the two chapters about haptics in his 1966 book (Gibson, 1966). In these chapters, Gibson recognized that part of the kinesthetic system allows a muscle- based perception of properties of external objects (Gibson, 1966, It seems to me that much of the research developed in the ecological psychology approach, and the conceptual tools used, June 2020 \| Volume 11 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 2 Enactivism and Ecological Psychology Travieso et al. p.109). This is why dynamic touch, on occasions, is referred to as muscle-based perception. p.109). This is why dynamic touch, on occasions, is referred to as muscle-based perception. Gibson (1966) further suggested that the invariant involved in the perception of softness relates to the ratio of the force exerted and the amount of depression of the surface (cf. Harper and Stevens, 1964). In the case of dynamic touch, the relevant invariants relate forces to movements. Let us now introduce the physics of these invariants. In a practical sense, dynamic touch concerns the everyday haptic perception of natural and manufactured objects that we hold in our hands. Just to name a few examples, this includes the perception of how far we can throw objects through hefting (Bingham et al., 1989; Zhu and Bingham, 2008, 2010), if objects can be used for hammering (Wagman and Carello, 2001), the perception of shape (Burton et al., 1990), tool use (Michaels et al., 2007), the perception of heaviness (Shockley et al., 2001), and even an explanation of the size-weight illusion (Amazeen and Turvey, 1996). Frontiers in Psychology \| www.frontiersin.org June 2020 \| Volume 11 \| Article 1257 THE LAWS THAT GOVERN THE ORGANISM-ENVIRONMENT INTERACTION IN DYNAMIC TOUCH Scott-Blair and Coppen (Harper, 1952) investigated the perception of the ﬁrmness-softness of industrial substances (rubber, bitumen) by having the observer squeeze a cylindrical sample of the material to be graded. The “feel” of the material was quite clear at the end of this dynamic action. They concluded that the perception “had the nature of a Gestalt,” but I would suggest instead that an invariant was isolated. (p. 128) June 2020 \| Volume 11 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 3 Enactivism and Ecological Psychology Travieso et al. that, for such a particular set of rods, exploratory movements that allow the detection of mass also allow the detection of length. diﬀerent axes, leading to diﬀerent amounts of resistance. A full description of the 3-dimensional resistance to rotation that is oﬀered by an object is provided by the inertia tensor, which is a symmetric 3 × 3 matrix with nine inertial components. The inertia tensor, I, relates three-dimensional forces to three- dimensional accelerations according to the formula: τ = I × α, in which the use of boldface indicates the fact that the symbols stand for vector and matrix properties. If one diagonalizes the inertia tensor, the eigenvalues that one obtains are the mechanical properties referred to as the ﬁrst, second, and third principal moments of inertia (I1 to I3). These moments can also be described as the resistance against rotation around three orthogonal axes that are ordered from the highest amount of resistance to rotation to the lowest amount of resistance. For a symmetric rod, I1 and I2 are identical and correspond to rotation around axes that are orthogonal to the longitudinal axis of the rod, and I3 corresponds to rotation around the longitudinal axis. Now consider a rod that is held by its end and maintained with its longitudinal axis horizontal to the ground. The rotational force that is exerted by the rod at the point of rotation corresponds to the mechanical property called static moment. Extending Equation 1, static moment (SM) can be deﬁned as: SM = N X i=1 midi, (2) (2) in which each di is the distance of the corresponding point- mass mi to the axis of rotation. For rods with the same mass, homogeneously distributed over the length of the rod, the static moment is higher for longer rods. THE LAWS THAT GOVERN THE ORGANISM-ENVIRONMENT INTERACTION IN DYNAMIC TOUCH I = N X i=1 mid2 i . (3) (3) In analogy to Newton’s second law for linear motion, the moment of inertia determines the acceleration that is obtained with a rotational force (i.e., τ = I × α), meaning that the moment of inertia can be measured as the invariant relation between rotational acceleration, α, and force, τ. As a ﬁnal step in our introduction to the relevant mechanical properties and laws, one should note that, so far, we have only considered one-dimensional rotations. Even if one considers a single point of rotation, however, objects can be rotated around THE LAWS THAT GOVERN THE ORGANISM-ENVIRONMENT INTERACTION IN DYNAMIC TOUCH Therefore, under certain laboratory circumstances, statically maintaining a rod as described above may allow one to diﬀerentiate the lengths of objects with the same mass. It is easy to intuitively experiment with some of these mechanical properties and laws. To do so, we ask you to hold a small rod between the thumb and index ﬁnger (for instance a wooden ruler or any other elongated object). The rod will move until aligned longitudinally with gravity. Move it up and down, so that you can feel its mass. But, what about its length? Then, keep it in a horizontal position. Does the eﬀort change? What about mass and length perception? Finally, grip the object from the center and rotate it like a seesaw, and do the same while holding it by the end (rotations with low and high I1, respectively). This small experiment provides an intuitive demonstration that the relation between self-produced forces and resulting accelerations allows us to detect properties of wielded objects. As a next step to introduce the mechanical properties and laws that are most relevant to dynamic touch, we illustrate how rods can be modiﬁed without changing their mass or static moment. Consider Figure 1. The rods have the same mass. They also have the same static moment, and thus exert the same rotational force when held still horizontally. However, if one actively rotates the rods, the lower one oﬀers more resistance. This is so because the resistance to rotation oﬀered by mass is a squared function of distance; hence, whereas moving M2 away from the axis of rotation is compensated by moving M1 nearer for static moment, this is not the case for the resistance against rotation. More precisely, for one-dimensional rotations, the moment of inertia (I) can be deﬁned as: To summarize this section, during wielding, diﬀerent types of forces stand in diﬀerent invariant relations to the resulting accelerations. An enactivist may call these relations sensorimotor laws. Being sensitive to these relations, or sensorimotor laws, allows the perceiver to detect the mechanical properties that underlie, or determine, the invariant relations. The starting point of perceiving through dynamic touch, however, is not formed by the mechanical properties that underlie the agent- environment interactions. Instead, the starting point is formed by the exploratory movements exerted by an intentional agent. The next section, therefore, analyzes the exploratory movements and the role of intention as modiﬁer of these movements. concepts for the body of research on dynamic touch. In their words: A common portrayal of the ecological approach from enactive scholars considers that action is reduced to mere movement, thus leaving intentionality out of the explanations. For example, Di Paolo (2016) states: In our eﬀort to stress commonalities between the enactivist and ecological approaches, we should note the similarities between the intentional exploration in dynamic touch and the enactivist concept of bringing-forth the world. The mechanical properties deﬁned in the previous section (mass, static moment, inertia tensor) are intrinsic properties that can be detected only by acting upon them. Although the inertia tensor of an object can physically be described independently of a perceiver, in order to detect or measure the property, perceivers apply forces on the object and identify relational aspects of the emerging interaction. Burton and Turvey (1990) performed experiments on the perception of length through dynamic touch with unrestricted movements as well as with the instruction not to move the rod. With instructional restrictions on the movements, static moment predicted the length judgments better than the ﬁrst principal moment of inertia, whereas this was the other way around for unrestricted movements. Relatedly, in their study on length perception through dynamic touch, Lobo and Travieso (2012) systematically restricted the exploratory movements in six movement conditions with diﬀerent amplitudes and frequencies. With faster movements, more accurate judgments were obtained, supporting the idea that, in unrestricted conditions, length perception relies on the ﬁrst principal moment of inertia. The fact that, in dynamic touch, the perceiver chooses which mechanical properties to act upon, or bring forth, brings us to the concept of intentionality. A common portrayal of the ecological approach from enactive scholars considers that action is reduced to mere movement, thus leaving intentionality out of the explanations. For example, Di Paolo (2016) states: In addition to a deep understanding of the environment, we need a theory that pays attention to the perceiver as an active agent and her capacity to engage her world meaningfully. “Active” here does not mean simply “moving” (this is well-covered in ecological psychology). It means engaged in a regulated coupling with the environment, generating goals and pursuing them, moving in ways that alter the constraints that link the agent and the environment as coupled systems. (p. 327) p p p p Similar conclusions concerning the importance of exploratory movements were obtained by Harrison et al. (2011). concepts for the body of research on dynamic touch. In their words: These authors also showed that the intention to detect diﬀerent aﬀordances (e.g., holdability, rotatability) aﬀected length perception. Their results “revealed perception to be constrained by (a) the moments of mass distribution of the hand-tool system, (b) the qualities of exploratory wielding movements, and (c) the intention to perceive each speciﬁc property” (Harrison et al., 2011, p. 193). A further example of how intentions aﬀect the detected mechanical properties as well as the exploratory movements can be found in Arzamarski et al. (2010; cf. Riley et al., 2002). These authors manipulated the intention of participants by asking them to report either the length or the width of hand-held objects. When participants intended to perceive length, the exploratory movements showed relatively more rotation around the anterior-posterior axis (as deﬁned in Figure 2), and the judgments were more closely related to I1 than to I3. On the contrary, when the intention was to perceive width, participants performed relatively more rotations around the twist axis and estimations were more closely related to I3 than to I1. The example of dynamic touch, however, shows the central role of actions and intentions in the ecological approach to perception. In the ecological portrayal of dynamic touch, it is particularly clear that exploratory movements are performed because the perceiver intentionally, or purposefully, aims to reveal certain mechanical properties of the object while ignoring others. This notion of the role of action and intention in perception is shared with enactivists researchers. A prominent example can be found in the book Action in Perception (Noë, 2004): Think of a blind person tip-tapping his or her way around a cluttered space, perceiving that space by touch, not all at once, but through time, by skillful probing and movement. This is, or at least ought to be, our paradigm of what perceiving is. The world makes itself available to the perceiver through physical movement and interaction. (p. 1) Rather than thinking about relatively simple exploratory movements such as pure rotations or twists, one may prefer to think about more complex ones. It is interesting to indicate in this regard that Stephen et al. Frontiers in Psychology \| www.frontiersin.org INTENTIONAL EXPLORATION: BRINGING-FORTH THE WORLD THROUGH DYNAMIC TOUCH FIGURE 1 \| Two rods with equal mass and static moment but with different principal moments of inertia. The concept of bringing-forth the world may be attributed to Varela et al. (1991), who write: “Cognition in its most encompassing sense consists in the enactment or a bringing forth of a world [emphasis added] by a viable history of structural coupling.” (p. 205). Elaborating on the concept, Proulx (2008) states: FIGURE 1 \| Two rods with equal mass and static moment but with different principal moments of inertia. It is my structure that allows me to “see” or perceive things in the physical world, and so my structure allows me to give June 2020 \| Volume 11 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 4 Enactivism and Ecological Psychology Travieso et al. concepts for the body of research on dynamic touch. In their words: concepts for the body of research on dynamic touch. In their words: meaning to the attributes of the physical world. I – my structure – allow the physical world to be brought forth. If these attributes of the physical world are outside of my structure, outside of my capacity to make sense of them, I cannot distinguish them and cannot perceive them. In other words, they cannot “trigger” anything in me. Hence, I bring forth the physical world’s attributes when I give/create meaning to it – I acknowledge their physical “presence” by bringing them forth. If I do not bring them forth, the physical world’s attributes will still be “there,” but they will remain unnoticed, not made sense of and kept “in the dark.” It is in this sense that the physical attributes themselves are brought forth by my interaction with them (if I perceive them). In some sense, I make the physical world emerge. (p. 21–22) With a haptic subsystem, intentionality identiﬁes the goal to be attained. On the basis of the intention of the subject/performer, the parts of the haptic perceptual system are assembled into a special-purpose machine capable of attaining the desired goal .... when the muscles and other tissues are assembled into a subsystem, a functional unit, the behavior of the subsystem is conjugate to the properties of the object under exploration. (Solomon and Turvey, 1988, p. 405) Later in this section we describe empirical research that demonstrates how diﬀerent intentions lead to diﬀerent exploratory movements. First, however, we describe empirical research that indicates how diﬀerent exploratory movements determine the mechanical properties that are detected. In our eﬀort to stress commonalities between the enactivist and ecological approaches, we should note the similarities between the intentional exploration in dynamic touch and the enactivist concept of bringing-forth the world. The mechanical properties deﬁned in the previous section (mass, static moment, inertia tensor) are intrinsic properties that can be detected only by acting upon them. Although the inertia tensor of an object can physically be described independently of a perceiver, in order to detect or measure the property, perceivers apply forces on the object and identify relational aspects of the emerging interaction. The fact that, in dynamic touch, the perceiver chooses which mechanical properties to act upon, or bring forth, brings us to the concept of intentionality. AFFORDANCES AND INVARIANTS IN DYNAMIC TOUCH FIGURE 2 \| Type of objects used in the experiments by Arzamarski et al. (2010) with the relevant axes of rotation. Figure 8 from Arzamarski et al. (2010). Our next goal is to illustrate and hopefully clarify two key concepts from the ecological approach: aﬀordances and invariants. Instead of providing canonical deﬁnitions of these terms, we consider them using the example of dynamic touch. With respect to aﬀordances, we focus on the distinction between perceiving and actualizing, which, we believe, is often under- emphasized or misunderstood in the writings of scholars that compare enactivism and ecological psychology. When Gibson (1966) introduced the concept of invariant in dynamic touch, he stated the following: FIGURE 2 \| Type of objects used in the experiments by Arzamarski et al. (2010) with the relevant axes of rotation. Figure 8 from Arzamarski et al. (2010). The mass of an object can be judged, in fact, by wielding it in any of a variety of ways, such as tossing and catching, or shaking it from side to side. One can only conclude that the judgment is based on information, not on the sensations. The stimulus information from wielding can only be an invariant of the changing ﬂux of stimulation in the muscles and tendons, an exterospeciﬁc invariant in this play of forces. Whatever speciﬁes the mass of the object presumably can be isolated from the change, and the wielding of the object has the function of separating oﬀthe permanent component from the changes. The merely propriospeciﬁc information can thus be ﬁltered out, as it were, leaving pure information about the object. This process takes time, for an invariant can only emerge from a series of transformations over time. (p. 127) Indeed, even the miniscule musculoskeletal ﬂuctuations incident to static holding have been claimed to inform haptic judgments (Burton and Turvey, 1990). Whereas static holding is simply the maintenance of balance among muscular and gravitational forces, wielding signiﬁes a strongly intentional excursion from balance that is meant to produce new or richer information about the inertial properties of the object. Of course, active forces in wielding movements will still interact with reactive forces: The inertial properties of the object still exert eﬀects on the trajectory of the limb that are not explicitly prescribed by the muscle forces exerted during wielding. (p. AFFORDANCES AND INVARIANTS IN DYNAMIC TOUCH 2) When we wield a tennis racket, for example, rotational hand and arm forces lead to diﬀerent movements. Those movements depend on the original position of the hand-arm system and the racket, and on the dynamics of the whole system in inertial terms. The invariants that reside in the ﬂux of change of forces and movements are speciﬁc to intrinsic properties of the wielded objects. However, in contrast to the interpretation of ecological psychology as a physicalist approach, the information that is used for perception through dynamic touch is not the speciﬁcation of such mechanical properties in discrete stimuli that are internalized, or values that perceivers infer from stimulation that is imposed on them. Quite the opposite, the invariants exist because of the laws that govern the organism-environment system only when these laws are combined with the intentional exploration that creates the necessary change for the invariants to come into existence. Perceiving, then, is the detection of the invariants in the organism-environment interaction that specify possibilities of action. As ecological psychologists would say: we detect invariants and we perceive aﬀordances. As an aside, the idea of multi-scale behavior is reminiscent to an ecological theory of learning, referred to as direct learning (Jacobs and Michaels, 2007), which holds that phenomena at the time-scale of learning can be explained with the ecological principles developed for the time-scale of perceiving and acting. The above-mentioned study of Arzamarski et al. (2010) is related to this theory, as well as other learning studies in the ﬁeld of dynamic touch (Michaels et al., 2008; Jacobs et al., 2009; Withagen and van Wermeskerken, 2009). We refer the reader to Higueras-Herbada et al. (2019) for a description of that theory and its relevance to the dialogue between the enactive and ecological approaches. To summarize the gist of this section, the type of exploratory movements that are performed determine which mechanical properties become determinants of the agent-environment interaction and which mechanical properties are irrelevant for that interaction (e.g., twisting movements are aﬀected by I3 but not by I1). The ecological portrayal of dynamic touch, therefore, starts with the intention to perceive some property. This intention leads to exploratory behavior that is determined by, and hence reveals in the interaction, those mechanical properties that relate to the property that the agent intends to perceive. concepts for the body of research on dynamic touch. In their words: (2010) argue that fractal ﬂuctuations in exploration reveal the detection of information at many time-scales: The concept of intentionality was, in fact, one of the key concepts in the pioneering research on dynamic touch by Solomon and Turvey (1988), and thereby one of the foundational June 2020 \| Volume 11 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 5 Travieso et al. Enactivism and Ecological Psychology Frontiers in Psychology \| www.frontiersin.org AFFORDANCES AND INVARIANTS IN DYNAMIC TOUCH This portrayal is aligned with the writing of many enactivists, who may say that the purposeful, autonomous agent brings forth those mechanical properties that are relevant to her goals, leaving in the dark other properties. As Carello and Turvey (2017) remind us, Gibson (1966) ﬁrst introduced the ideas behind the concept of aﬀordance in his reﬂections on dynamic touch: Although he did not develop his notion of aﬀordance until his 1979/1986 book it is, in fact, mentioned in Gibson (1966) in one of the chapters on the haptic system. In arguing against the tradition of imposing discrete stimuli on passive participants who were to report “awareness of the impression, not the object making it” (p. 98), he recommended that an active observer obtaining patterns of stimulation should be “allowed to report what he perceives, June 2020 \| Volume 11 \| Article 1257 6 Enactivism and Ecological Psychology Travieso et al. including what it aﬀords or might be used for” (p. 99). (Carello and Turvey, 2017, p. 97) including what it aﬀords or might be used for” (p. 99). (Carello and Turvey, 2017, p. 97) in the ﬂux of forces and movements that is created during the exploration, via the mechanical properties of the wielded object, to the actual aﬀordance, as well as vice versa. If an aﬀordance is not speciﬁed in detectable information, it cannot be perceived. Ecological psychologists are interested in the perception of aﬀordances, for which the speciﬁcation of aﬀordances is indispensable, as well as in the actualization of aﬀordances, which may be analyzed with less concern about what is speciﬁed by the information that guides the movement (cf. Michaels et al., 2001). For rods, examples of aﬀordances may be throwability, holdability, rotatability, whether other objects can be reached with the rods, or whether the rods are suitable for certain hitting actions. In contrast to other properties (e.g., the length or shape of rods), aﬀordances are deﬁned with respect to the organism’s purposeful actions. This means taking the organism- environment system as the unit of explanation. Thus, when wielding an object and producing rotations, perceivers detect the relation between exerted forces and resulting movements, perceiving aﬀordances that allow them to prospectively control further movements (e.g., Bongers et al., 2004). Varela et al. (1991), in their treatment of the ecological approach, had in mind the actualization of aﬀordances when they describe their own position. AFFORDANCES AND INVARIANTS IN DYNAMIC TOUCH This is clear, for example, when they write: “The overall concern of an enactive approach to perception is ... to determine the common principles or lawful linkages between sensory and motor systems that explain how action can be perceptually guided” (p. 173). In this regard, the enactive approach is more closely related to the part of the ecological approach about the actualization of aﬀordances than to the part about the perception of aﬀordances. Rather than noting existing commonalities with ecological research about the actualization of aﬀordances, however, Varela et al. (1991) distanciate themselves from the ecological approach by referring to ecological ideas concerning the perception of aﬀordances: “In Gibson’s view ... perceptually guided action consists in ‘picking up’ or ‘attending to’ invariances in the ambient light that directly specify their environmental source” (p. 203). This quote seems to concern the actualization of aﬀordances (“perceptually guided action”) in its ﬁrst part but changes its emphasis toward the perception of aﬀordances (“invariants that specify their source”) in the latter part. To summarize, rather than searching for commonalities with theoretical and empirical research from the ecological tradition about the actualization of aﬀordances, enactivist scholars often choose to stress diﬀerences by focusing on parts of the ecological approach that concern the perception of aﬀordances – an issue that, one way or another, any approach has to address. We believe that it is crucial for the dialogue between enactivism and ecological psychology to clarify that perceiving an aﬀordance is not the same as actualizing an aﬀordance (i.e., performing the action that is aﬀorded; Michaels et al., 2001). For instance, perceiving that a stone aﬀords throwing (Bingham et al., 1989) is not the same as throwing the stone, or perceiving that a ball can be caught is not the same as actually catching the ball (Jacobs and Michaels, 2006). Consider a further example. During the night, a person wakes up and wants to go to the bathroom without turning the lights on. Instead, she uses the light of her phone to illuminate her peripersonal space (the space immediately close to her). With this light, she sees one slipper under the bed, but, unfortunately, a bit far, so she cannot reach it with her arm. She stares at the slipper as she taps nearby objects in an attempt to ﬁnd something that allows her to reach the slipper. AFFORDANCES AND INVARIANTS IN DYNAMIC TOUCH She grabs an object and wields it, but rapidly discards it, as it is too short. Then she grabs another object, does the same quick wielding, and ﬁnally uses it, knowing that it will be long enough to reach the slipper. Using the ecological vocabulary, one may say that the sequence of actions started with the intention to get the missing slipper. Using dynamic touch, in the ﬁrst try with an object she did not perceive the aﬀordance of reaching the slipper. In the second try, she did perceive the aﬀordance. After perceiving the aﬀordance, she actualized the aﬀordance by actually reaching toward the slipper. Apart from the confusion concerning perception and actualization, the concept of aﬀordance is often claimed to be well suited to the enactivist approach (Varela et al., 1991) and is mentioned ubiquitously by enactivists (e.g., Noë, 2004; Di Paolo et al., 2017; Gallagher, 2017). This is so because aﬀordances are related to the bringing-forth the world concept of enactivism and to sense-making. We brieﬂy address the concept of sense-making before our concluding section. When actualizing an aﬀordance, then, such as throwing a stone, catching a ball, or reaching for a slipper, information is used that allows the actor to control the relevant forces. The action itself emerges from the information-action couplings and the dynamics of the agent-environment system. This means that, for the actualization of aﬀordances, one does not need environmental information that speciﬁes the aﬀordance. Hence, in line with what is often claimed by enactivists (e.g., Varela et al., 1991), for a theory of perceptually-guided action (i.e., a theory about actualizing aﬀordances), one does not need environmental information that speciﬁes aﬀordances. Frontiers in Psychology \| www.frontiersin.org SENSE-MAKING AGENCY IN DYNAMIC TOUCH Sense-making agency refers to a conception of agency by which the organism builds up its agency through meaningful interactions with the environment. In this context, perception is not a blind contact with the environment that imprints its eﬀect on the sensory surfaces, which is afterward processed searching for regularities. Using the words of Thompson and Stapleton (2009): In order to veridically perceive an aﬀordance, however, often done before actualizing the aﬀordance, a relation of speciﬁcity must be maintained between the agent and the environment (Jacobs and Michaels, 2002). In the case of dynamic touch, this implies a lawful chain of speciﬁcity that goes from the intention to perceive the aﬀordance, to invariants June 2020 \| Volume 11 \| Article 1257 7 Enactivism and Ecological Psychology Travieso et al. Even the simplest organisms regulate their interactions with the world in such a way that they transform the world into a place of salience, meaning, and value – into an environment (Umwelt) in the proper biological sense of the term. This transformation of the world into an environment happens through the organism’s sense-making activity. (p. 25) Even the simplest organisms regulate their interactions with the world in such a way that they transform the world into a place of salience, meaning, and value – into an environment (Umwelt) in the proper biological sense of the term. This transformation of the world into an environment happens through the organism’s sense-making activity. (p. 25) theorizing about the environment, as noted by McGann (2014). Does this mean that such theorizing is unwanted or that it could not ﬁt well with other enactive ideas? I do not think so. It is more a case that it has not been done yet, and what better encouragement to do this than to engage in ongoing dialogues with ecological psychologists. (p. 329) The concept of sense-making refers to the particular behavior of the autonomous organism that is determined by its structure (for example, only animals with eyes can see), which makes available certain properties of the physical world that constitute the environment for her. Moreover, enactivists remain skeptical of those animal-environment relations in which speciﬁcation does not imply an intentional action on the part of the organism. A clear example is the detection of time-to-contact (Lee, 1976). CONCLUSION In this article we have aimed to reveal commonalities rather than emphasizing diﬀerences between enactivism and ecological psychology, using dynamic touch as example. We have argued that more similarities can be identiﬁed than is often indicated in the literature. In this sense, we have shown how dynamic touch can provide an excellent test bed for enactivist ideas concerning sensorimotor laws, bringing forth the world, goal-directed agency, and sense- making activity. In particular, the active and intentional information detection put forward by ecological psychologists implies that ecological information is not instructive, meaning that the information does not determine what is perceived or acted upon. Meaning is not just something that pops up in the relation between organism and environment. It necessitates a particular kind of activity on the part of the agent to emerge, i.e., sense- making. (p. 328) Given that not all perceptual tasks reveal such an obvious intentional exploration as dynamic touch, however, one may wonder whether dynamic touch can be considered a generalizable example. That is, would the enactivist and ecological approaches show as much similarity if analyzed with another example? In this regard, it is interesting to note that both enactivists and ecological psychologists tend to indicate similarities between vision and touch. To illustrate this for ecological psychology, consider two quotes from Gibson’s (1962) seminal article on active touch. Concerning passive touch and vision, he states: In the paradigmatic case of dynamic touch, the history of interaction with the object consists of the exploratory wielding, where forces exerted by the agent produce changes in the position of the objects. The objects that are wielded during dynamic touch, and the mechanical properties that we described earlier in this article, therefore form part of the meaningful environment of the perceiving agent. An enactivist may claim that dynamic touch is the sense-making activity that makes that the rods and the relevant mechanical properties form part of the meaningful environment of the agent. The ecological example of dynamic touch, therefore, as well as the concept of aﬀordance, can easily be related to enactivist concerns about sense-making. For further debate about the notion of sense-making we refer the reader to Fultot et al. (2016). In passive touch the individual makes no voluntary movements. Similarly, in passive vision he makes no eye movements, which means that he must voluntarily ﬁxate his eyes on a point speciﬁed by the experimenter. SENSE-MAKING AGENCY IN DYNAMIC TOUCH Despite the fact that the original analysis concerned the active control of movement by the perceiver (i.e., the control of braking by a driver), the optical speciﬁcation of time-to-contact can be demonstrated for the case where the perceiver actively approaches a static object as well as for the case where an object approaches a static perceiver. Thus, Di Paolo (2016) states that for enactivists: CONCLUSION Neither state is natural to an individual. (Gibson, 1962, p. 489) From our perspective, at the same time that the sense-making activity determines the ecological niche for the organism – only animals with eyes can see – the environment exerts selective pressure on the organisms, establishing constraints on the structure of the organism – eyes are adaptive only in an environment with light that obeys the laws of optics. Therefore, we can consider that the sense-making activity is determined by the structure of the organism, and the laws governing the sensorimotor activity are those describing the physical world in which they are embedded. The strength of the dynamic touch example is that, together with the sense- making activity, we have a detailed description of the laws governing the physical interaction. Borrowing, again, the words of Di Paolo (2016): Concerning active touch and vision, in contrast, he states: REFERENCES (1990). Can shape be perceived by dynamic touch? Percept. Psychophys. 48, 477–487. doi: 10.3758/bf03211592 Calvo, P., and Gomila, T. (2008). Handbook of Cognitive Science: An Embodied Approach. London: Elsevier. Kingma, I., van de Langenberg, R., and Beek, P. J. (2004). Which mechanical invariants are associated with the perception of length and heaviness of a nonvisible handheld rod? Testing the inertia tensor hypothesis. J. Exp. Psychol. 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(Gibson, 1962, p. 490) To illustrate that similarities between vision and touch are also indicated in the work of enactive scholars, reﬂecting a notion of perception in which the enactive and ecological approaches can meet, we conclude with a quote from a prominent predecessor of the enactive approach, Maurice Merleau-Ponty: “Vision is a palpation with the look” (cited in Noë, 2004, p. 35). Despite the accent on agency as always situated in an environment, it is correct that there is a dearth of enactive June 2020 \| Volume 11 \| Article 1257 Frontiers in Psychology \| www.frontiersin.org 8 Enactivism and Ecological Psychology Travieso et al. FUNDING JI-G was supported by the Talent Attraction contract funded by the Autonomous Region of Madrid (2016-T2/ SOC1976). DT wrote the initial draft of the manuscript. All authors contributed to the planiﬁcation, writing, and correction of the manuscript. DT wrote the initial draft of the manuscript. 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https://openalex.org/W4206964653	https://www.zora.uzh.ch/id/eprint/219103/1/Growth.pdf	English	null	Growth rate mediates hidden developmental plasticity of female yellow dung fly reproductive morphology in response to environmental stressors	Evolution & development	2,022	cc-by	10,475	R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Richard J. Walters1,2 \| David Berger1,3 \| Wolf U. Blanckenhorn1 \| Luc F. Bussière1,4,5 \| Patrick T. Rohner1,6 \| Ralf Jochmann1 \| Karin Thüler1 \| Martin A. Schäfer1 1Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland Correspondence Correspondence Wolf U. Blanckenhorn, Department of Evolutionary Biology and Environmental Studies, University of Zurich, Winterthurerstrasse 190, CH‐8057 Zurich, Switzerland. Email: wolf blanckenhorn@ieu uzh ch This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Evolution & Development Published by Wiley Periodicals LLC Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2022 Growth rate mediates hidden developmental plasticity of female yellow dung fly reproductive morphology in response to environmental stressors DOI: https://doi.org/10.1111/ede.12396 DOI: https://doi.org/10.1111/ede.12396 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-219103 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-219103 Journal Article Published Version ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Walters, Richard J; Berger, David; Blanckenhorn, Wolf U; Bussière, Luc F; Rohner, Patrick T; Jochmann, Ralf; Thüler, Karin; Schäfer, Martin A (2022). Growth rate mediates hidden developmental plasticity of female yellow dung fly reproductive morphology in response to environmental stressors. Evolution Development, 24(1-2):3-15. DOI: https://doi.org/10.1111/ede.12396 Received: 14 July 2021 \| Accepted: 24 November 2021 DOI: 10.1111/ede.12396 Received: 14 July 2021 \| Accepted: 24 November 2021 DOI: 10.1111/ede.12396 Evolution & Development. 2022;24:3–15. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract Understanding how environmental variation influences even cryptic traits is important to clarify the roles of selection and developmental constraints in past evolutionary divergence and to predict future adaptation under en- vironmental change. Female yellow dung flies (Scathophaga stercoraria) ty- pically have three sperm storage compartments (3S), but occasionally four (4S). More spermathecae are thought to be a female adaptation facilitating sperm sorting after mating, but the phenotype is very rare in nature. We manipulated the flies' developmental environment by food restriction, pesti- cides, and hot temperatures to investigate the nature and extent of develop- mental plasticity of this trait, and whether spermatheca expression correlates with measures of performance and developmental stability, as would be ex- pected if 4S expression is a developmental aberration. The spermathecal polymorphism of yellow dung fly females is heritable, but also highly devel- opmentally plastic, varying strongly with rearing conditions. 4S expression is tightly linked to growth rate, and weakly positively correlated with fluctuating asymmetry of wings and legs, suggesting that the production of a fourth spermatheca could be a nonadaptive developmental aberration. However, spermathecal plasticity is opposite in the closely related and ecologically si- milar Scathophaga suilla, demonstrating that overexpression of spermathecae under developmental stress is not universal. At the same time, we found overall mortality costs as well as benefits of 4S pheno‐ and genotypes (also affecting male siblings), suggesting that a life history trade‐off may potentially moderate 4S expression. We conclude that the release of cryptic genetic var- iation in spermatheca number in the face of strong environmental variation may expose hidden traits (here reproductive morphology) to natural selection (here under climate warming or food augmentation). Once exposed, 2Centre for Environmental and Climate Research, Lund University, Lund, 2Centre for Environmental and Climate Research, Lund University, Lund, Sweden 3Evolutionary Biology Centre, University of Uppsala, Uppsala, Sweden 4Biological and Environmental Sciences, University of Stirling, Stirling, Scotland, UK 5Biology and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden 6Department of Biology, Indiana 6Department of Biology, Indiana University, Bloomington, Indiana, USA University, Bloomington, Indiana, USA Growth rate mediates hidden developmental plasticity of female yellow dung fly reproductive morphology in response to environmental stressors Richard J. Walters1,2 \| David Berger1,3 \| Wolf U. Blanckenhorn1 \| Luc F. Bussière1,4,5 \| Patrick T. Rohner1,6 \| Ralf Jochmann1 \| Karin Thüler1 \| Martin A. Schäfer1 1 \| INTRODUCTION Simmons, 2001). As a prominent example, female sperm storage compartments have long been known to vary within and across various insect species (e.g., Dybas & Dybas, 1981; Jones & Ludlam, 1965; Kamimura, 2004; Minder et al., 2005; Pitnick et al., 1999; Presgraves et al., 1999; Puniamoorthy et al., 2010; Singh et al., 2006; Sturtevant, 1926). Spermathecae are sclerotized com- partments of many female arthropods for storing sperm over extended periods of time, which tend to covary with other female but also male reproductive structures (Dybas & Dybas, 1981; Minder et al., 2005; Pitnick et al., 1999; Thüler et al., 2011). As one of the best‐ studied species in the context of sexual selection (Simmons et al., 2020), female yellow dung flies Scatho- phaga stercoraria (Diptera: Scathophagidae) typically possess three (3S; one singlet and one doublet), but sometimes four spermathecae (4S; two doublets; Berger et al., 2011; Demont et al., 2021; Schäfer et al., 2013; Simmons et al., 1999; Ward et al., 2008; Ward, 2000). Intermediate phenotypes with bifurcated spermathecae and shared spermathecal ducts document the develop- mental transition between these alternative reproductive phenotypes (Ward et al., 2008; Figure 1). Sperm compe- tition experiments provided evidence that multiple sperm storage organs can be advantageous during post‐ copulatory sexual selection, and differential sperm sto- rage and use has been repeatedly demonstrated in S. stercoraria and other insects using phenotypic or molecular markers (Bussière et al., 2010; Demont et al., 2012, 2021; Firman et al., 2017; Hellriegel & Bernasconi, 2000; Hellriegel & Ward, 1998; Lüpold et al., 2016; Manier et al., 2013; Otronen et al., 1997; Simmons et al., 2020; Snow & Andrade, 2005; Ward, 1998, 2000, 2007). As an additional spermatheca tends to reduce female fecundity, a trade‐off between any genetic benefits through female choice and life‐history costs has been suggested to maintain the spermathecal dimorphism within natural populations (Ward, 2007; Ward et al., 2008). However, the relative importance of neutral versus selective mechanisms for the evolution of this structure still remains unclear, as the 4S phenotype is rarely expressed in nature and therefore remains Trait evolution occurs through the interplay between neutral and selective processes to ultimately produce morphological diversity by modifying underlying devel- opmental mechanisms (West‐Eberhard, 2003). K E Y W O R D S artificial selection, body size, developmental stability, Diptera, fluctuating asymmetry, growth rate, morphology, mortality, phenotypic plasticity, post‐copulatory sexual selection, spermatheca, survival, temperature Funding information Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, Grant/Award Number: multiple wileyonlinelibrary.com/journal/ede 4 \| 4 WALTERS ET AL. hidden traits can potentially undergo rapid genetic assimilation, even in cases when trait changes are first triggered by random errors that destabilize developmental processes. \| 5 5 WALTERS ET AL. 5 FIGURE 1 Various stages in the expression of 4S phenotypes: (a) normal singlet spermatheca (b) slightly invaginated, (c) fully split doublet with one duct, and (d) 4S phenotype with separate ducts FIGURE 1 Various stages in the expression of 4S phenotypes: (a) normal singlet spermatheca (b) slightly invaginated, (c) fully split doublet with one duct, and (d) 4S phenotype with separate ducts (Floate & Coughlin, 2010; Hosken et al., 2000; Palmer, 2000; Palmer & Strobeck, 1986; Parsons, 1992; Polak, 2003; Polak & Tomkins, 2013; Van Valen, 1962; Vøllestad et al., 1999; Whitlock, 1996). This specifically addresses the hypothesis that 4S (over)expression in yellow dung flies may represent a genetic aberration (as suggested for Drosophila melanogaster: Hadorn & Garber, 1944; Mather & Harrison, 1949; Singh et al., 2006; Wexelson, 1928), that is, a stochastic, nonadaptive consequence of sys- temic developmental instability, rather than an (ancestral) adaptation mediated by life‐history trade‐offs. We further present comparative data for the closely related and ecolo- gically similar S. suilla (see Bernasconi et al., 2001) to demonstrate that spermathecal overexpression is no neces- sary consequence of accelerated growth, and that this type of (mostly hidden) developmental plasticity has evolved differ- ently at least in two (but likely more) related taxa (cf. Minder et al., 2005). largely hidden from selection (Berger et al., 2011; cf. McGuigan & Sgrò, 2009), and growth rate strongly influences spermathecal expression (Schäfer et al., 2013). Genetic variation underlying the 4S phenotype can thus be cryptic, and alternatively this variation may, at least initially, merely represent stochastic developmental er- rors linked to rapid immature growth and decanalized development (Berger et al., 2011; Hadorn & Garber, 1944; Schäfer et al., 2013; Singh et al., 2006; Wexelson, 1928), which later upon exposure to selection may or may not become an adaptation in particular environments. Here, we comprehensively investigate the plasticity of 4S expression in yellow dung flies exposed to a range of stressful environments during development to elucidate whether the production of a fourth spermatheca could be adaptive in the above sense or not. We first present hi- therto unpublished results of laboratory spermathecal selection lines to document the extent of heritable versus plastic effects in spermathecal expression (Thüler, 2009; cf. Berger et al., 2011). \| 5 By manipulating the flies' devel- opmental environment in a variety of ways (by food (=dung) restriction, parasiticides, or hot temperatures) to impede or facilitate larval resource acquisition, develop- ment, and growth, we broadly assessed the nature and extent of variation in 4S expression (cf. McGuigan & Sgrò, 2009). We additionally monitored further life history and fitness traits (Dmitriew, 2011; Roff, 1997), notably pupal and total egg‐to‐adult survival, plus a common indicator of developmental instability, fluctu- ating asymmetry (FA) of paired morphological traits FIGURE 1 Various stages in the expression of 4S phenotypes: (a) normal singlet spermatheca (b) slightly invaginated, (c) fully split doublet with one duct, and (d) 4S phenotype with separate ducts 1 \| INTRODUCTION Tradi- tionally, trait divergence was viewed to be mainly caused by (disruptive) natural selection followed by reinforce- ment upon secondary contact (Dobzhansky, 1937; Mayr, 1963), although recently the role of sexual selec- tion has received more attention, especially for traits related to reproduction (Arnqvist & Rowe, 2005; Birkhead & Møller, 1998; Eberhard, 1996; Panhuis et al., 2001; Simmons, 2001). However, traits also evolve neutrally. Sex‐specific traits tend to accumulate more deleterious mutations under selection‐mutation balance because they are exposed to purging selection only half as often as the traits expressed in both sexes (De Visser et al., 2003; Mank & Ellegren, 2009; Van Dyken & Wade, 2010). Moreover, genetic correlations mediated by functional constraints and/or genetic linkage regularly shape evolutionary trajectories (Dobzhansky & Holz, 1943; Eberhard, 1996; Mayr, 1963; Moczek, 2009). Reproductive organs may thus be less evolutionarily stable and more susceptible to diversification via genetic drift (Civetta & Singh, 1998; De Visser et al., 2003; Van Dyken & Wade, 2010). Developmental plasticity can play a pivotal role for adaptation and divergence in novel environments, but can also constrain adaptive evolution by biasing trait expression towards certain developmental tra- jectories in favor of others (Chevin et al., 2010; Lande, 2009; Pfennig et al., 2010; Uller et al., 2018). Understanding pre- cisely how environmental variation influences even cryptic internal morphological traits is therefore important both to clarify the roles of selection and developmental constraints in past evolutionary divergence and to predict future adaptation under environmental change. f Intraspecific polymorphisms are particularly well suited for studying the evolutionary and developmental processes underlying evolutionary change. Reproductive traits of insects are very well studied in this regard (e.g., Arnqvist & Rowe, 2005; Parker, 1970; Pitnick et al., 1999; 2.3 \| Experimental treatments to manipulate fly growth, development, morphology, and survival We retroactively (see above) only forwarded offspring with the appropriate number of spermathecae (3S or 4S) to set up the next generation, spread as equi- tably as possible across all mothers of the previous generation. Offspring groups were always reared in full‐ sibling groups in plastic containers with overabundant dung (see Section 2.3 and Blanckenhorn et al. [2010] for more detailed standard rearing and holding methods). At the end of artificial selection, males and females from the eighth generation were dissected to take pictures of their genital tracts with a microscope‐mounted camera (cf. Figure 1). Following an earlier, similar selection regime (Ward, 2000), we in parallel held unselected flies from the same population in our laboratory, whose sper- mathecae were however not monitored regularly, as we already knew that the 4S phenotype remains roughly stable across generations under laboratory conditions (see also Berger et al., 2011). There is hence no formal control treatment to report. We exploited data from three separate S. stercoraria la- boratory studies conducted at various temperatures to maximize their range of temperature exposure. In all cases, the adult parental flies originally stemmed from the same farm in Fehraltorf (cf. above) that were subse- quently bred for 2–4 generations in the laboratory using standard methods (see Blanckenhorn et al., 2010, 2018, for details). In general, full‐sib family groups of 5–15 (mainly 10) offspring of laboratory‐mated females were reared in replicate plastic containers with (typically) overabundant (>2 g per larva; Amano, 1983) homo- genized and previously frozen cow dung, thus minimiz- ing larval competition. While two of the original experimental designs (splitting broods among mainly temperature environments) served to assess quantitative genetic variation, we here mainly considered phenotypic effects. One previously reported common garden rearing that also included S. suilla was performed at (always constant) 12, 18, and 24°C (Bauerfeind et al., 2018; Blanckenhorn et al., 2018; Schäfer et al., 2018), and ad- ditionally 26°C. A further, previously unpublished phe- notypic laboratory study raised flies in half‐degree intervals at high temperatures from 24 to 26.5°C to ap- proach the upper lethal limit of S. stercoraria (with no flies surviving beyond this temperature; cf. Blanckenhorn et al., 2014; Ward & Simmons, 1990). 2.3 \| Experimental treatments to manipulate fly growth, development, morphology, and survival A more encom- passing third study included a total of six treatments, to maximize the resulting range of growth rates: two tem- peratures (15 and 23°C) crossed with two food treatments (limited = 5 g dung for 10 larvae; overabundant = 20 g dung for 10 larvae; cf. Amano, 1983), plus an ivermectin treatment at each temperature and overabundant food treatment combination (a common cattle parasiticide, at concentrations of 6.57 µg/kg dung wet weight; cf. Floate et al., 2016; Römbke et al., 2009). (For the purposes here, ivermectin was merely used as yet another environ- mental stressor affecting fly life history and reproductive parameters, to supplement our standard food and tem- perature manipulations.) 2.3 \| Experimental treatments to manipulate fly growth, development, morphology, and survival dissection of the mother after having produced a clutch of eggs (see below). We randomly subdivided the sub- sequent (laboratory) generation 2 in each spermathecal category into three replicate lines (for a total of six lines). All adult flies were maintained individually in the la- boratory under standard conditions in 100 ml glass bot- tles with ad libitum sugar, water, and Drosophila spp. as prey, at population densities of ca. 25–50 females (i.e., families) per generation per line. To produce the next generation, we randomly paired males and females within replicate lines by avoiding sibling matings, and raised a random subset of ca. 10–15 offspring (on average half of them being females) from the first clutch of each mother. We retroactively (see above) only forwarded offspring with the appropriate number of spermathecae (3S or 4S) to set up the next generation, spread as equi- tably as possible across all mothers of the previous generation. Offspring groups were always reared in full‐ sibling groups in plastic containers with overabundant dung (see Section 2.3 and Blanckenhorn et al. [2010] for more detailed standard rearing and holding methods). At the end of artificial selection, males and females from the eighth generation were dissected to take pictures of their genital tracts with a microscope‐mounted camera (cf. Figure 1). Following an earlier, similar selection regime (Ward, 2000), we in parallel held unselected flies from the same population in our laboratory, whose sper- mathecae were however not monitored regularly, as we already knew that the 4S phenotype remains roughly stable across generations under laboratory conditions (see also Berger et al., 2011). There is hence no formal control treatment to report. dissection of the mother after having produced a clutch of eggs (see below). We randomly subdivided the sub- sequent (laboratory) generation 2 in each spermathecal category into three replicate lines (for a total of six lines). All adult flies were maintained individually in the la- boratory under standard conditions in 100 ml glass bot- tles with ad libitum sugar, water, and Drosophila spp. as prey, at population densities of ca. 25–50 females (i.e., families) per generation per line. To produce the next generation, we randomly paired males and females within replicate lines by avoiding sibling matings, and raised a random subset of ca. 10–15 offspring (on average half of them being females) from the first clutch of each mother. 2.1 \| Artificial selection From 65 yellow dung fly pairs originally collected at our experimental farm in Fehraltorf, Switzerland (N47°23′, E8°44′; the parental generation 1), selection lines were established by assigning emerged female flies to one of two regimes depending on whether they had three or four spermathecae, as determined retrospectively by 6 WALTERS ET AL. 6 2.4 \| Assessment of wing and leg fluctuating asymmetry Wing shape was analyzed using 15 landmarks extracted from images photographed by a Leica DM105 light mi- croscope (see Figure 5; cf. Schäfer et al., 2018). Land- marks were digitized using version 2.14 of the software tpsDig2 (Rohlf, 2009), and landmark coordinates were subjected to a full Procrustes analysis using the function gpagen() of the R‐package geomorph, version 3.1.1 (Adams & Otárola‐Castillo, 2013). As a measure of asymmetry for wing shape, we computed the Procrustes distance between the coordinates of the left and the right wing of each individual. That is, we computed the square root of the summed squared distances between corre- sponding landmarks. This distance represents a measure of asymmetry that is equivalent to the magnitude of a vector that describes the shape differences between an individual's left and right wing (Klingenberg, 2015). generation 1 was only 2% (cf. Berger et al. (2011), who found 3% 4S females in the field), but jumped instantly to ca. 25% in the first laboratory generation 2, a plastic effect presumably mediated by unconstrained growth condi- tions in the laboratory, to reach roughly 50% after eight generations of artificial selection in the 4S lines (a genetic response; Figure 2). In the 3S lines the percentage of four spermathecae also first jumped up but then diminished toward 1% in generation 8 (Figure 2). The estimated realized heritability of spermathecal expression in the 4S lines across generations 2–8 was h2 = 0.637 (p < .001; Roff, 1997; cf. Ward et al., 2008). After eight generations of artificial selection, genetically correlated responses of various other female reproductive traits (mainly internal morphological structures) were evi- dent (depicted in Figure 1 of Thüler et al. [2011]). These traits were separately analyzed using ANCOVAs with se- lection regime (3S vs. 4S) as fixed factor, lines within selec- tion regime as nested random factor, and body size as single (within‐individual) covariate for most traits (total N = 289 females in generation 8, roughly equally distributed among the six replicate lines). Females from 4S lines (mean ± SE spermathecal area 0.08329 ± 0.00074 mm2) had more but smaller spermathecae than females from 3S lines (0.08776 ± 0.00046 mm2), indicating a size‐number trade‐off (selection line effect: F1,4 = 11.46, p = .021), although the total spermathecal storage capacity of 4S females was of course greater. 2.2 \| Dissections Previously frozen females were dissected by carefully removing the posterior portion of the female re- productive tract from the body of the female by grasping the genital valves with forceps and tearing them from the abdomen. We could so separate the various internal reproductive female structures (bursa copulatrix, com- mon oviduct, spermathecae and their ducts, accessory glands and their ducts, etc.) under a binocular micro- scope (Leica MZ‐12, Leica Microsystems GmbH) to photograph them (cf. Thüler et al., 2011). Using these photographs, we measured various internal structures of interest (spermatheca area/volume, spermatheca duct length, accessory gland area, and accessory gland duct length) as well as female hind‐tibia length as an index of body size, using ImageJ (Schneider et al., 2012; http:// rsb.info.nih.gov/ij/). In all these studies, we measured egg‐to‐adult devel- opment time of all emerged flies of both sexes, their left and right hind tibia lengths as a structural index of body size and to calculate FA (as described below), and we photographed both wings to later score landmarks, wing (centroid) size, and wing FA. In the elaborate study with various treatments at 15 and 23°C, we additionally mea- sured teneral (fresh) body mass of all flies upon emer- gence. We finally scored the number of spermathecae \| 7 WALTERS ET AL. FIGURE 2 Expression percentage of four spermathecae in the three 4S lines (reddish) and the three 3S lines (greenish) from generation 1 (field situation), via generation 2 (first laboratory‐ reared generation), to generation 8 in response to artificial selection in both directions. No unselected control was scored. Bold lines denote the overall average. 4S expression plastically jumps spontaneously from 2% in generation 1 (limited natural field conditions) to ca. 25% in generation 2 (laboratory growth conditions with ample resources), and after eight generations of selection it reaches close to 50% in 4S lines and nearly 0% in 3S lines. Simple line graphs are depicted \| 7 expressed by all females as a function of their juvenile treatment (after dissection; see Section 2.2). Our experiments further yielded full‐sib, family‐wise egg‐to‐adult survival rates as the number of adults emerged divided by the number of eggs entered per rearing container. These survival data served to test for a global phenotypic relationship between familial sper- mathecal expression and juvenile mortality. 2.2 \| Dissections In the rear- ings at 24–26.5°C, we embedded an additional pupal mortality assessment to specifically investigate individual effects of 3S versus 4S expression and pupal volume on the probability of individual female emergence (i.e., survival) at extremely high and stressful temperatures (involving opening dead pupae to assess their sper- mathecal phenotype; see Polak & Tomkins, 2013). FIGURE 2 Expression percentage of four spermathecae in the three 4S lines (reddish) and the three 3S lines (greenish) from generation 1 (field situation), via generation 2 (first laboratory‐ reared generation), to generation 8 in response to artificial selection in both directions. No unselected control was scored. Bold lines denote the overall average. 4S expression plastically jumps spontaneously from 2% in generation 1 (limited natural field conditions) to ca. 25% in generation 2 (laboratory growth conditions with ample resources), and after eight generations of selection it reaches close to 50% in 4S lines and nearly 0% in 3S lines. Simple line graphs are depicted 2.4 \| Assessment of wing and leg fluctuating asymmetry Spermathecal area was positively related to female hind tibia length (overall r = .33 based on all females, p < .001). At the same time, spermathecal duct length Following our earlier FA studies of this species (Blanckenhorn & Hosken, 2003; Hosken et al., 2000), we additionally computed FA of their hind legs in standard ways following Palmer and Strobeck (1986) as: signed FA as (L – R), unsigned FA as (\|L – R\|) (both in mm), and unsigned, size‐corrected FA as (\|L – R\|)/mean(L, R). While 4S females were larger than average also within a rearing treatment, body size overall was not a predictor of 4S expression across treatments (Figure 3), as flies became larger when grown in abundant dung but smaller at higher temperatures despite both factors increasing growth rate, as is the case for most ectotherms (Berrigan FIGURE 3 (a) Proportion of females expressing 4 versus the standard 3 spermathecae as a function of growth rate (expressed as fresh body mass/development time ± individual SD; y = 0.743x −0.195, R2 = .965, p < .001, total N = 1487 females; descriptive regression line based on means). (b) Comparison of age and size (mass) at maturity for females with 4 versus 3 spermathecae (dark vs. light symbols, respectively). Symbol color indicates rearing temperature: blue = 15°C, red = 23°C; symbol shape indicates dung treatment: large square = ad libitum, small square = d l d l b decreased markedly in 4S (0.537 ± 0.0085 mm) relative to 3S lines (0.600 ± 0.0054 mm; selection line effect: F1,4 = 58.26, p < .001; overall correlation with body size r = .13, p = .046). There were no differences after selection between 3S and 4S lines in female body size (2.68 ± 0.01 mm vs. 2.64 ± 0.01 mm), accessory gland area (0.244 ± 0.0028 mm2 vs. 0.238 ± 0.0044 mm2), or accessory gland duct length (0.477 ± 0.0050 mm2 vs. 0.482 ± 0.0081 mm2; all p > .2). However, fecundity (first clutch size) of 4S line females (57.0 ± 1.88) tended to be higher than that of 3S females (48.3 ± 1.13; p ≈.1), and clutch size overall slightly decreased with spermathecal size in all lines (partial, size‐corrected r = −0.06, p = .1). 3.2 \| Spermathecal expression in relation to growth rate Our most elaborate experiment at 15 and 23°C with two dung treatments (limited & abundant) and ivermectin nicely stretched the range of growth rates observed. Across all females (total N = 1487), and independent of the precise treatment, the expression of a fourth sper- matheca was tightly associated with an increase in growth rate (Figure 3a). Augmented growth of 4S females almost entirely resulted in larger flies, with merely minor variation in development time (Figure 3b). While 4S females were larger than average also within a rearing treatment, body size overall was not a predictor of 4S expression across treatments (Figure 3), as flies became larger when grown in abundant dung but smaller at higher temperatures despite both factors increasing growth rate, as is the case for most ectotherms (Berrigan & Charnov, 1994). FIGURE 3 (a) Proportion of females expressing 4 versus the FIGURE 3 (a) Proportion of females expressing 4 versus the standard 3 spermathecae as a function of growth rate (expressed as fresh body mass/development time ± individual SD; y = 0.743x −0.195, R2 = .965, p < .001, total N = 1487 females; descriptive regression line based on means). (b) Comparison of age and size (mass) at maturity for females with 4 versus 3 spermathecae (dark vs. light symbols, respectively). Symbol color indicates rearing temperature: blue = 15°C, red = 23°C; symbol shape indicates dung treatment: large square = ad libitum, small square = restricted, triangle = ad libitum + ivermectin 3.1 \| Response to artificial selection The transition from three to four spermathecae occurs in stages: the singlet spermatheca first tends to become heart‐shaped, and successively subdivides to finally form four spermathecae with four fully separated ducts (Figure 1). The percentage of 4S females in the field at WALTERS ET AL. 8 8 FIGURE 3 (a) Proportion of females expressing 4 versus the standard 3 spermathecae as a function of growth rate (expressed as fresh body mass/development time ± individual SD; y = 0.743x −0.195, R2 = .965, p < .001, total N = 1487 females; descriptive regression line based on means). (b) Comparison of age and size (mass) at maturity for females with 4 versus 3 spermathecae (dark vs. light symbols, respectively). Symbol color indicates rearing temperature: blue = 15°C, red = 23°C; symbol shape indicates dung treatment: large square = ad libitum, small square = restricted, triangle = ad libitum + ivermectin decreased markedly in 4S (0.537 ± 0.0085 mm) relative to 3S lines (0.600 ± 0.0054 mm; selection line effect: F1,4 = 58.26, p < .001; overall correlation with body size r = .13, p = .046). There were no differences after selection between 3S and 4S lines in female body size (2.68 ± 0.01 mm vs. 2.64 ± 0.01 mm), accessory gland area (0.244 ± 0.0028 mm2 vs. 0.238 ± 0.0044 mm2), or accessory gland duct length (0.477 ± 0.0050 mm2 vs. 0.482 ± 0.0081 mm2; all p > .2). However, fecundity (first clutch size) of 4S line females (57.0 ± 1.88) tended to be higher than that of 3S females (48.3 ± 1.13; p ≈.1), and clutch size overall slightly decreased with spermathecal size in all lines (partial, size‐corrected r = −0.06, p = .1). 3.2 \| Spermathecal expression in relation to growth rate Our most elaborate experiment at 15 and 23°C with two dung treatments (limited & abundant) and ivermectin nicely stretched the range of growth rates observed. Across all females (total N = 1487), and independent of the precise treatment, the expression of a fourth sper- matheca was tightly associated with an increase in growth rate (Figure 3a). Augmented growth of 4S females almost entirely resulted in larger flies, with merely minor variation in development time (Figure 3b). 3.3 \| Survival costs of spermathecal expression Crucially here, wing FA (F1,871 = 4.18; p = .041), but not tibia FA (F1,871 = .33; p = .569), was increased in females expressing four sper- mathecae (simple GLM comparison of N = 638 3S with FIGURE 4 (a) Family‐wise survival rate at any temperature is negatively correlated with the mean frequency of female 4S expression in those families, whether families with only 3S females are excluded (r = −.374, p < .01; N = 192 families) or included (r = −.210, p < .05; N = 507 families). (b) Pupal mortality of 3S versus 4S females at stressfully hot temperatures. Non‐parametric smoothed splines are depicted for description only WALTERS ET AL. \| 9 (hot temperatures, ivermectin, low food; all flies pooled regardless of treatment). Nevertheless, both tibia and wing FA were positively correlated with growth rate in males (partial r = .124 and .111, respectively, both p < .001) and, more weakly so, in females (partial r = .031, p > .1 and r = .109, p < .001, respectively). Crucially here, wing FA (F1,871 = 4.18; p = .041), but not tibia FA (F1,871 = .33; p = .569), was increased in females expressing four sper- mathecae (simple GLM comparison of N = 638 3S with N = 235 4S females raised under various conditions that were ignored in the analysis; Figure 5). (hot temperatures, ivermectin, low food; all flies pooled regardless of treatment). Nevertheless, both tibia and wing FA were positively correlated with growth rate in males (partial r = .124 and .111, respectively, both p < .001) and, more weakly so, in females (partial r = .031, p > .1 and r = .109, p < .001, respectively). Crucially here, wing FA (F1,871 = 4.18; p = .041), but not tibia FA (F1,871 = .33; p = .569), was increased in females expressing four sper- mathecae (simple GLM comparison of N = 638 3S with N = 235 4S females raised under various conditions that were ignored in the analysis; Figure 5). effect: χ2 = 23.13, p < .001; interaction: χ2 = 9.46, p = .002; Figure 4b; N = 73 female pupae). 3.4 \| Spermathecal expression and fluctuating asymmetry as indicators of developmental instability Based on a subset of ca. 800 flies of various treatments measured twice, our tibia and wing vein measurements were highly repeatable, as was our FA measurement (R > 0.9), which could always be discerned from mea- surement error (p < .001) following Palmer and Strobeck (1986). Absolute (i.e., unsigned) FA of hind tibiae and wings of females (r = .038, p > .1, N = 1562; controlling for body size) and males (r = .079, p = .003, N = 1397) were overall merely weakly positively correlated within in- dividuals, suggesting low general developmental in- stability in response to diverse environmental stressors 3.3 \| Survival costs of spermathecal expression context (r = −0.374, p < .01; total N = 192 families with variable spermatheca numbers; cf. Figure 4a). At the same time in a separate experiment, pupal mortality at stressfully high temperatures increased with temperature (24–27°C), as expected (χ2 = 13.67, p < .001; GLM with binomial errors and sex and temperature as fixed effects; total N = 136 pupae). The smaller females generally survived better than the larger males (χ2 = 3.84, p = .051), an effect that may be (solely?) mediated by their size difference (dimorphism), as pupal size statis- tically replaced the sex effect in an equivalent model (χ2 = 4.03, p = .045). In contrast to the overall juvenile egg‐to‐adult mortality results reported above, however, female 4S pupae survived extremely hot temperatures better than 3S pupae except at the highest viable tem- perature(s) (analogous GLM using only female pupae; spermatheca main effect: χ2 = 9.54, p = .002; temperature Across the entire range of rearing conditions and temperatures (12–27°C), family‐wise egg‐to‐adult sur- vival rates (of both sexes) correlated negatively with the mean frequency of female 4S expression in those families (when ignoring temperature treatment), sug- gesting life‐history costs of this trait and a trade‐off (simple linear correlation: r = −0.210, p < .05; N = 507 families). This negative relationship remained when including family as random effect (effective N = 408 families, because in some data subsets broods were split among temperature environments; see Section 2.3). This result was even clearer when a ma- jority of N = 316 families with only 3S females were excluded because they are uninformative in this 9 WALTERS ET AL. effect: χ2 = 23.13, p < .001; interaction: χ2 = 9.46, p = .002; Figure 4b; N = 73 female pupae). 3.4 \| Spermathecal expression and fluctuating asymmetry as indicators of developmental instability Based on a subset of ca 800 flies of various treatments (hot temperatures, ivermectin, low food; all flies pooled regardless of treatment). Nevertheless, both tibia and wing FA were positively correlated with growth rate in males (partial r = .124 and .111, respectively, both p < .001) and, more weakly so, in females (partial r = .031, p > .1 and r = .109, p < .001, respectively). 3.5 \| Spermatheca plasticity of a closely related species Finally, the increase in the number of spermathecae in S. stercoraria associated with faster growth is not a neces- sary outcome of warmer temperatures, as spermathecal 10 \| 10 WALTERS ET AL. 10 FIGURE 5 Square‐root‐transformed absolute proportional fluctuating asymmetry (FA) in wing shape of 3S (N = 638) versus 4S (N = 235) females (F1,871 = 4.18; p = .041) ± SE. The wing landmarks used are displayed FIGURE 6 Spermathecal expression increases with temperature in Scathophaga stercoraria (green; N = 609 females) but decreases with temperature in the closely related Scathophaga suilla (gray; N = 73 females). Simple line graphs with individual SE FIGURE 5 Square‐root‐transformed absolute proportional fluctuating asymmetry (FA) in wing shape of 3S (N = 638) versus 4S (N = 235) females (F1,871 = 4.18; p = .041) ± SE. The wing landmarks used are displayed FIGURE 6 Spermathecal expression increases with temperature in Scathophaga stercoraria (green; N = 609 females) but decreases with temperature in the closely related Scathophaga suilla (gray; N = 73 females). Simple line graphs with individual SE are depicted FIGURE 5 Square‐root‐transformed absolute proportional fluctuating asymmetry (FA) in wing shape of 3S (N = 638) versus 4S (N = 235) females (F1,871 = 4.18; p = .041) ± SE. The wing landmarks used are displayed FIGURE 6 Spermathecal expression increases with temperature in Scathophaga stercoraria (green; N = 609 females) but decreases with temperature in the closely related Scathophaga suilla (gray; N = 73 females). Simple line graphs with individual SE are depicted FIGURE 6 Spermathecal expression increases with p p temperature in Scathophaga stercoraria (green; N = 609 females) but decreases with temperature in the closely related Scathophaga suilla (gray; N = 73 females). Simple line graphs with individual SE are depicted number was observed to decrease with temperature in the closely related S. suilla (Figure 6). This striking contrast across species demonstrates that developmental instability does not have a simple directional effect across related taxa, but that these plastic responses, presumably mediated by developmental instability, have evolved in opposing directions within this clade. may at least initially be triggered stochastically by likely nonadaptive developmental errors (see also Berger et al., 2011; Schäfer et al., 2013). 3.5 \| Spermatheca plasticity of a closely related species At the same time, how- ever, we found overall mortality costs, as well as benefits (Figure 4), of 4S pheno‐ and genotypes (also affecting the male siblings), which by contrast suggest a potentially adaptive heritable life‐history trade‐off moderating 4S ex- pression in yellow dung flies (once present). We integrate these contrasting results in our discussion below. 4 \| DISCUSSION stercoraria, spermathecal and growth rate variation has a (strong) direct heritable component (Figure 2), and may additionally show heritable genotype‐ by‐environment (GxE) interactions that can respond to selection in cases of erratic or systematic environmental change (e.g., climate warming; Berger et al., 2011; Blanckenhorn, 1998, 2015; González‐Tokman et al., 2021; Schäfer et al., 2013). Thus, should there be a general ad- vantage in having more (rather than fewer) sperm storing organs (Demont et al., 2012, 2021; Ward, 2000, 2007), then the warmer climes of southern Europe (as shown by Berger et al. [2011]), or other regions now facing climate warming and/or more extensive heat periods in these flies' subarctic ranges (Blanckenhorn et al., 2018; Schäfer et al., 2018), could augment the opportunity for selection on sperm competition and cryptic female choice me- chanisms in yellow dung flies (Simmons et al., 2020; Ward, 1998). Whatever the impact of developmental error in produ- cing the 4S phenotype of S. stercoraria, direct evidence that female 4S expression is adaptive remains equivocal even after this study. Fitness differences between the two alter- native spermathecal phenotypes are difficult to show by di- rect experimental comparison in a sexual selection context (Schäfer et al., 2013), as are unambiguous demonstrations of sperm selection by females, regardless of their spermathecal number (Demont et al., 2012, 2021). Somewhat reduced fe- cundity of 4S females has been reported in previous studies (Ward et al., 2008), whereas our selection experiment here resulted in larger clutches laid by 4S line females, but also in a negative relationship, that is, a potential trade‐off, between a female's spermathecal volume, which was greater in 3S lines, and her clutch size. Moderate egg‐to‐adult survival costs of 4S expression could be demonstrated here (Figure 4a) and before (Schäfer et al., 2013). However, at stressfully high temperatures, at which mortality quickly approaches 100%, 4S female pupae apparently survived bet- ter than 3S pupae (Figure 4b), the opposite mortality pattern. These conflicting fitness effects possibly suggest that weak antagonistic pleiotropy across developmental stages and/or environmental contexts may further contribute to the di- versification of female sperm storage organs. Female reproductive structures, including the number of spermathecae, repeatedly have been found to vary con- siderably within and across even closely related insect taxa (Minder et al., 2005; Pitnick et al., 1999; Presgraves et al., 1999; Puniamoorthy et al., 2010). 4 \| DISCUSSION 11 flies, FA is not heritable (Blanckenhorn & Hosken, 2003) and a response to hot temperature stress (Hosken et al., 2000), but not responsive to food stress, inbreeding (Hosken et al., 2000), or parasiticide exposure (ivermectin: Floate & Coughlin, 2010). Regardless, 4S females do show greater FA than 3S females, strengthening the hypothesis of a relationship between spermathecal expression and developmental instability (Figure 5). Moreover, the over- expression of female spermathecae at high temperatures is specific, hence perhaps unique to S. stercoraria, as in the closely related and ecologically similar S. suilla heat ex- posure reduced rather than increased the number of ex- pressed spermathecae (Figure 6). in these flies. Diminished growth would also be observed in cases of high larval competition due to food (dung) limitation or high fly population densities, even in warmer climes, thus possibly more than offsetting any growth acceleration by warmer temperatures, and instead leading to lower effective expression of the 4S phenotype in nature (Berger et al., 2011; Jann et al., 2000; Schäfer et al., 2013, 2018). In this way, lasting environmental changes of diverse kinds (e.g., when pesticides are in- troduced: Blanckenhorn, Puniamoorthy, Schäfer, et al., 2013; Blanckenhorn, Puniamoorthy, Scheffczyk, et al., 2013; Puniamoorthy et al., 2014) may contribute to trait diversification, potentially by genetic assimilation (Flatt, 2005; Waddington, 1953; West‐Eberhard, 2003), which ul- timately can lead to speciation. The contrasting sper- mathecal responses to temperature of the two closely related, diversified S. stercoraria and S. suilla (Figure 6) may be such an example. As an alternative hypothesis, the observed sper- mathecal variation might reveal some ancestral form of plasticity that might have been adaptive in the past—for instance, reflecting the optimal solution to a trade‐off be- tween costs of expressing a fourth spermatheca versus the benefits of improved female choice—but which in modern day populations may have become mostly hidden devel- opmental noise exposed only under conditions allowing fast growth. Crucially, however, environmentally medi- ated plasticity in spermathecal expression, and by exten- sion plasticity in any other morphological, physiological, behavioral, or life‐history character, in most cases changes the stage for natural selection by spontaneously exposing previously unexpressed (genetic) variation (McGuigan & Sgrò, 2009). In S. 4 \| DISCUSSION The spermathecal polymorphism of yellow dung fly females, which has a demonstrable effect on post‐copulatory sexual and fecundity selection (Berger et al., 2011; Demont et al., 2012, 2021; Schäfer et al., 2013; Simmons et al., 2020; Ward, 2000, 2007), shows a strong heritable component but also exhibits substantial phenotypic plasticity in re- sponse to various environmental factors (Figure 2). This equally applies to development and growth rate (Blanckenhorn, 1998, 2009). Our study suggests that any factor that retards or accelerates growth (such as food limitation, warm temperatures, human‐introduced pesti- cides, etc.) strongly modifies the production during juve- nile development of a fourth spermatheca in this species (as opposed to the standard three; Figures 1 and 3). The spontaneous increase in 4S expression from 2% in the field to roughly 25% under overabundant food conditions in the laboratory (Figure 2), the tight relationship of 4S expres- sion with growth rate (Figure 3), and an admittedly weak but positive correlation of 4S expression with both fluc- tuating asymmetry of wings and legs and growth rate (Figures 3, 5 and 6) suggest that the production of a fourth spermatheca by yellow dung fly females is plastically triggered by any environment conducive to fast growth. We therefore conclude that this natural polymorphism A tight plastic relationship with growth rate (Figure 3) could indicate that the formation of spermathecae is error‐ prone during development, potentially induced by pleio- tropic relationships with other growth and developmental traits (Berger et al., 2011; Thüler et al., 2011). In this sense, spermatheca expression may be a nonadaptive con- sequence of developmental instability in contemporary yellow dung fly populations, as has been found and argued occasionally for D. melanogaster in the past (Hadorn & Garber, 1944; Mather & Harrison, 1949; Singh et al., 2006; Wexelson, 1928). In D. melanogaster, a supernumerary third spermatheca can also occur by loss of function of a particular gene (Gef26; Singh et al., 2006), which is un- likely to be the cause here given our evidence for con- tinuous, environmentally induced evolutionary responses. Our direct evidence for the aberration hypothesis remains limited, however, given merely weak intra‐individual correlations with another common and well‐established indicator of developmental instability, fluctuating asym- metry of paired morphological structures in bilaterally symmetric organisms (FA, here assessed for hind tibiae and wings: Palmer & Strobeck, 1986; Parsons, 1992; Polak, 2003; Van Valen, 1962; Figure 5). In yellow dung WALTERS ET AL. diversification in the end resulted from adaptation by nat- ural selection, or from nonadaptive (random, pleiotropic) side‐effects of standard (temperature‐dependent) metabolic processes, cannot be easily distinguished or reconstructed. Regardless of whether we consider the spermathecal plas- ticity documented here and elsewhere (Berger et al., 2011; Schäfer et al., 2013) adaptive, its evolution will be mediated by the usual mixture of selective and neutral processes in- teracting in spatio‐temporally variable environments (i.e., fluctuating‐stabilizing selection; sensu Istock, 1981). This is because any short‐term environmental change at an evo- lutionary, perhaps even ecological time scale may be sufficient to significantly and sustainably change the micro‐ evolutionary trajectories of certain traits (e.g., here sper- mathecal number) in certain altered environments (e.g., by warming climate or human‐mediated pesticides). AUTHOR CONTRIBUTIONS All authors contributed to conceptualizing this study. The MS was written by Wolf U. Blanckenhorn with input from all others. Karin Thüler performed the spermatheca selection experiment under direct supervision of Luc F. Bussière & Wolf U. Blanckenhorn. Richard J. Walters, David Berger, Luc F. Bussière, Wolf U. Blanckenhorn, and Martin A. Schäfer conducted the rearing experi- ments that generated the survival and fluctuating asym- metry data. Patrick T. Rohner reanalyzed these data using up‐to‐date statistical methods. Ralf Jochmann conducted the comparative survey presented in Table S1. Blanckenhorn, W. U. (2015). Investigating yellow dung fly body size evolution in the field: Response to climate change? Evolution, 69, 2227–2234. https://doi.org/10.1111/evo.12726 Blanckenhorn, W. U., & Hosken, D. J. (2003). The heritability of three condition measures in the yellow dung fly. Behavioral Ecology, 14, 612–618. Blanckenhorn, W. U., Pemberton, A. J., Bussière, L. 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https://openalex.org/W2054138009	https://europepmc.org/articles/pmc3988934?pdf=render	English	null	The Combination Therapy with Zoledronic Acid and Propranolol Improves the Trabecular Microarchitecture and Mechanical Property in an Rat Model of Postmenopausal Osteoporosis	Journal of osteoporosis	2,014	cc-by	7,413	Hindawi Publishing Corporation Journal of Osteoporosis Volume 2014, Article ID 586431, 10 pages http://dx.doi.org/10.1155/2014/586431 Hindawi Publishing Corporation Journal of Osteoporosis Volume 2014, Article ID 586431, 10 pages http://dx.doi.org/10.1155/2014/586431 Hindawi Publishing Corporation Journal of Osteoporosis Volume 2014, Article ID 586431, 10 pages http://dx.doi.org/10.1155/2014/586431 Deepak Kumar Khajuria,1,2 Rema Razdan,1 and D. Roy Mahapatra2 1 Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore 560027, India 2 Department of Aerospace Engineering, Indian Institute of Science, Bangalore 560012, India Deepak Kumar Khajuria,1,2 Rema Razdan,1 and D. Roy Mahapatra 1 Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore 560027, India 2 Department of Aerospace Engineering, Indian Institute of Science, Bangalore 560012, India 1 Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore 560027, India 2 Department of Aerospace Engineering, Indian Institute of Science, Bangalore 560012, India Correspondence should be addressed to Deepak Kumar Khajuria; deepak kumarkhajuria@yahoo.co.in Received 30 November 2013; Revised 19 January 2014; Accepted 27 February 2014; Published 30 March 2014 Academic Editor: Harri Siev¨anen Copyright © 2014 Deepak Kumar Khajuria et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We conducted the present study to investigate the therapeutic effects of propranolol (PRO), alone and in combination with the antiresorptive agent ZOL, in a rat model of postmenopausal osteoporosis. Female Wistar rats were OVX or sham-operated at 3 months of age. Twelve weeks after surgery, rats were randomized into six groups: (1) sham + vehicle, (2) OVX + vehicle, (3) OVX + ZOL (100 𝜇g/kg, i.v. single dose), (4) OVX + ZOL (50 𝜇g/kg, i.v. single dose), (5) OVX + PRO (0.1 mg/kg, s.c. 5 days per week), and (6) OVX + ZOL (50 𝜇g/kg, i.v. single dose) + PRO (0.1 mg/kg, s.c. 5 days per week) for 12 weeks. At the end of treatment study, various bone parameters were evaluated. With respect to improvement in the mechanical strength of the lumbar spine and the femoral mid-shaft, the combination treatment of ZOL and PRO was more effective than each drug administered as a monotherapy. Moreover, combination therapy using ZOL and PRO preserved the trabecular microarchitecture better than single-drug therapy using ZOL or PRO in OVX rats. These data suggest that combination therapy with ZOL plus PRO represents a potentially useful therapeutic option for patients with osteoporosis. 1. Introduction Femurs and LV4 bones were soaked in saline solution gauze and frozen at −20∘C for the microarchitectural and biomechanical tests (Figure 1). The procedure for killing of the animals was in accordance with the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines. Combination drug therapy seems to be promising be- cause, in some cases, it can increase the effectiveness of treatment and is now the subject of extensive investigation [22, 23]. Rodrigues et al. demonstrated that low doses of PRO suppress bone resorption by inhibiting receptor acti- vator of nuclear factor kappa-B ligand (RANKL) mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters [24]. This result is sup- ported by a previous finding, which showed that propranolol stimulates osteoprotegerin (OPG) on its own in osteoblast cells [25]. PRO, which could directly prevent bone loss and biomechanical alteration by increasing bone formation and decreasing bone resorption, may be the next osteogenic agent for osteoporosis treatment [8, 16, 24]. To the best of our knowledge, no study has assessed whether the combination of ZOL and PRO enhances bone properties in rat model of experimental osteoporosis. Owing to the different mechanisms of action of ZOL and PRO, our hypothesis was that the combination of ZOL and PRO would facilitate greater improvements in bone properties than either intervention alone. 1. Introduction Zoledronic acid (ZOL) is a third generation nitrogen- containing bisphosphonate that has been shown to signifi- cantly reduce the risk of fractures in patients who receive the once-yearly dosing regimen for the treatment of post- menopausal osteoporosis [3]. ZOL interferes with osteoclas- tic activity by inhibiting osteoclast formation and osteoclast bone resorptive activities and by inducing osteoclast apop- totic cell death. On osteoclast stimulation of bone resorp- tion, the bisphosphonate is released and internalized by the osteoclasts, interfering with osteoclast formation, function, and survival [4–6]. Moreover, the results obtained from several histologic and microcomputed tomography studies have shown that ZOL can be highly effective in prevention of bone loss in ovariectomized (OVX) rats [7–10]. The use of ZOL therapy has been associated with adverse events, such Osteoporosis is a degenerative disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and increased fracture risk. Osteoporosis is considered an important public health issue increasing rapidly in the elderly population [1, 2]. The fractures caused by osteoporosis have clinical and public health impacts, as they are often associated with increased morbidity, mortality, and enormous healthcare expenditure [1]. For those already affected by osteoporosis, timely diagnosis of bone loss, assessment of fracture risk, and selection of optimal treatment at appropriate stages of the disease are very important for effective management of osteoporosis. 2 2 Journal of Osteoporosis as osteonecrosis of jaw, renal dysfunction, hypocalcemia, and atrial fibrillation [1, 7–11]. obtaining ethical clearance from the Institutional Animal Ethical Committee. obtaining ethical clearance from the Institutional Animal Ethical Committee. obtaining ethical clearance from the Institutional Animal Ethical Committee. i 𝛽-Blockers are now considered to be the potential can- didates of therapeutic drugs under investigation for fracture healing and more specifically for osteoporosis therapy [8– 10]. Many genetic and pharmacological studies have shown that activation of 𝛽-adrenergic receptors in osteoblasts and stromal cells leads to inhibition of bone formation and intensification of bone resorption [12–15]. In an animal study, a lower dose of propranolol (PRO), a nonselective 𝛽- blocker, has been shown to increase bone mass in different experimental models of bone disorders [8–10, 16–18]. Results of some prior epidemiological studies confirm the hypothesis that 𝛽-blockers use is associated with a decrease in fracture risk [19–21]. 2.3. Preclinical Study Design. 1. Introduction At three months of age, ovariec- tomy was performed under ketamine and xylazine anaesthe- sia (80 mg/kg and 10 mg/kg; i.p.) according to the method described by Khajuria et al. [26]. The SHAM-operation rats were subject to SHAM surgery exposure without removing the ovaries. At 3 months after ovariectomy (age: 6 months), animals were divided into 6 groups of 6 animals each. The SHAM group and one OVX group served as negative and positive controls, respectively. A group of OVX rats were left untreated (untreated OVX). Both the untreated OVX and the sham OVX groups were used as controls. Two OVX groups were treated with single intravenous dose of ZOL at 50 𝜇g/kg (ZOL 50 group) and 100 𝜇g/kg (ZOL 100 group), administered into tail vein as a slow intravenous injection over 30 s under light inhalation anesthesia. One OVX group was treated with PRO at a dose of 0.1 mg/kg, injected subcutaneously five days per week, for 12 weeks. Treatment on the remaining OVX group was initiated with a 50 𝜇g/kg ZOL and 0.1 mg/kg PRO combination-treated (ZOL 50 + PRO) group. Subcutaneous injections five days per week in case of OVX groups treated with PRO and ZOL 50 + PRO require some animal handling and create some stress to the animals. Therefore, apart from positive and negative control groups, OVX groups treated with ZOL (100 𝜇g/kg and 50 𝜇g/kg, intravenous single dose) were also subcutaneously administered vehicle (normal saline, 5 days per week) for 12 weeks. The medication dosages used in this experiment were selected from previous studies on rat osteoporosis model. In our previous study, we used 100 𝜇g/kg single intravenous injection of ZOL in OVX rats and showed that this dose of ZOL restores microstructural and biomechanical features of bone. For this reason, 100 𝜇g/kg single intravenous injection of ZOL was used in this experiment [8, 9]. Similarly, we showed that subcutaneous administration of 0.1 mg/kg PRO given 5 days per week for 10 weeks prevents OVX induced bone loss by increasing bone formation and decreasing bone resorption, and thus 0.1 mg/kg given for 5 days per week of PRO was selected in this study [8, 9]. Body weight (expressed in grams) was monitored at the end of the experiments. All groups were euthanized by an overdose of anesthesia. In all rats, femurs and lumbar vertebrae (LV4) were excised and cleared of fat and connective tissues. 2. Materials and Methods Figure 1: A flow chart showing the preclinical study protocol. isotropic voxel size resolution of 16 𝜇m. In total, 120 high- resolution slices (=2 mm length, same length as chosen for the Micro-CT) with a pixel matrix of 2048 × 2048 were measured using an effective energy of 70 keV and a current intensity of 114 mA. After preprocessing the images with the same Gaussian filter (∑= 0.7, support = 1), a threshold was selected at 28% of the maximal grey-scale value, which corresponds to the peak for bone tissue in the histogram of the grey value dis- tribution in the image. Bone volume (BV/TV, fractional bone volume), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were evaluated. In addition to the computation of metric parameters, values of nonmetric parameters were calculated to describe the 3D nature of the trabecular bone sample. The structure model index (SMI) quantifies the plate—rod characteristic of 3D trabecular structure. 2.6. Statistical Analysis. All data were expressed as the mean ± standard deviation (SD). For all the data, com- parisons between different treatments were analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. In all cases, a probability error of less than 0.05 was selected as the criterion for statistical significance. Graphs were drawn using Graph Pad Prism (version 5.0 for Win- dows). 2.5. Biomechanical Bone Strength Testing. The structural properties of left femurs and LV4 were determined by three- point bending or compression, respectively, using a universal testing machine (BISS Makron, Bangalore, India). 2.5.1. 3-Point Bending Test. Femur strength was assessed by 3-point bending as previously described [8]. Briefly, femurs were removed from the −20∘C freezer and rehydrated in a saline solution for 4 h at room temperature. Hydrated weight of the bones was determined using a four-decimal place digital scale. Length of the bones was measured with calipers. Specimens were placed on two supports that were separated by a distance of 12 mm and bent until fracture by lowering the crosshead positioned at the mid-shaft at a constant speed of 0.033 mm/s. From the load-displacement curve, the peak load (N), the ultimate stiffness (N/mm), and the toughness (mJ) were obtained. Ultimate stress (strength) and Young’s modulus were derived from load- deformation curves obtained by using equations described by Khajuria et al. [8]. 2. Materials and Methods 2.1. Drugs, Chemicals, and Other Materials. ZOL was ob- tained from Naprod Life Sciences, Maharashtra, India. PRO, ketamine, xylazine, and xylene were obtained from Aurobindo Pharma (Hyderabad, India), GlaxoSmithKline Pharmaceuticals (Mumbai, India), Neon Pharma (Mum- bai, India), Indian Immunologicals (Hyderabad, India), and S.D. Fine Chemicals (Mumbai, India), respectively. Ethicon chromic sutures-3/0 and Ethicon mersilk sutures-3/0 were obtained from Johnson & Johnson Ltd., Baddi, Himachal Pradesh, India. 2.4. Microcomputed Tomography (Micro-CT) Analysis of Bone. The distal region of the right femora was used for three- dimensional Micro-CT analysis. The Micro-CT and software used for this experiment were from Scano Medical (micro- CT40, Scanco Medical AG, Bassersdorf, Switzerland). Anal- ogous to the Micro-CT measurements, the distal femoral metaphysis was scanned, whereby in this case with a higher 2.2. Experimental Animals. In-house laboratory bred healthy female Wistar rats of 3 months of age were included for the study. Animals were maintained under controlled tempera- ture at 25∘C ± 2∘C with 12 h light/dark cycle with food and water provided ad libitum. The experiments were conducted as per the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines after 3 Journal of Osteoporosis Female rats Ovariectomy OVX rats OVX rats Drug administration OVX rats End of the experiment Evaluation of various bone parameters Development of osteoporosis 3months 3months 3months Age: 3months Age: 6months Age: 9months Figure 1: A flow chart showing the preclinical study protocol. Table 1: Effects of zoledronic acid, propranolol, or zoledronic acid plus propranolol on body weight. Table 1: Effects of zoledronic acid, propranolol, or zoledronic acid plus propranolol on body weight. 3months Group Body weight (g) SHAM 304.0 ± 10.65∗∗∗ OVX 396.6 ± 8.92 ZOL 100 355.0 ± 8.51 ZOL 50 352.0 ± 9.82 PRO 367.0 ± 8.45 ZOL 50 + PRO 359.6 ± 13.14 Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗∗∗𝑃< 0.001, compared to OVX group. All groups except SHAM group undergo ovariectomy. Group Body weight (g) SHAM 304.0 ± 10.65∗∗∗ OVX 396.6 ± 8.92 ZOL 100 355.0 ± 8.51 ZOL 50 352.0 ± 9.82 PRO 367.0 ± 8.45 ZOL 50 + PRO 359.6 ± 13.14 Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗∗∗𝑃< 0.001, compared to OVX group. All groups except SHAM group undergo ovariectomy. 2. Materials and Methods Development of osteoporosis 3months Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗∗∗𝑃< 0.001, compared to OVX group. All groups except SHAM group undergo ovariectomy. 3months 2.5.2. Compression Test. To assess the biomechanical prop- erties of an individual lumbar vertebra, briefly, the LV4 body was separated from the epiphyseal ends, the posterior pedicle, and the spinous process using a low speed diamond band saw. By removing the cranial and caudal ends of the specimen, the planoparallel surfaces were obtained for compression testing. An electronic caliper was used to determine the dimensions of the LV4 body. From the vertebral body, a central cylinder with planoparallel ends and a height of approximately 6 mm was obtained. A vertical compressive force was applied to the specimen in the craniocaudal direction using steel compression plates at a deformation rate of 0.033 mm/sec. The ultimate compressive load (N), the stiffness (N/mm), and the toughness (mJ) were calculated as the mechanical properties from the load-displacement curve. The modulus of elasticity for a material is the slope of its stress-strain plot within the elastic range. The load-deflection curve was converted to stress-strain curve by dividing force by the initial area of the vertebral body and deflection by the original length of the same specimen [9]. 2.5.2. Compression Test. To assess the biomechanical prop- erties of an individual lumbar vertebra, briefly, the LV4 body was separated from the epiphyseal ends, the posterior pedicle, and the spinous process using a low speed diamond band saw. By removing the cranial and caudal ends of the specimen, the planoparallel surfaces were obtained for compression testing. An electronic caliper was used to determine the dimensions of the LV4 body. From the vertebral body, a central cylinder with planoparallel ends and a height of approximately 6 mm was obtained. A vertical compressive force was applied to the specimen in the craniocaudal direction using steel compression plates at a deformation rate of 0.033 mm/sec. The ultimate compressive load (N), the stiffness (N/mm), and the toughness (mJ) were calculated as the mechanical properties from the load-displacement curve. The modulus of elasticity for a material is the slope of its stress-strain plot within the elastic range. The load-deflection curve was converted to stress-strain curve by dividing force by the initial area of the vertebral body and deflection by the original length of the same specimen [9]. 3. Results Correspondingly, a significantly higher Tb.Sp and SMI were observed for the OVX group rats compared to that of the SHAM rats, suggesting that ovariectomy caused the trabecular bone to become thin and sparse and disrupted the bone structure. Significant improvements, however, were observed in BV/TV, Tb.Sp, Tb.Th, Tb.N, and SMI values in all single treatments compared with the OVX group. In the ZOL 50 + PRO group, there were significant improvements in these microstructural indices compared with the OVX group. Moreover, the BV/TV, Tb.Th, and Tb.N in the ZOL 50 + PRO group were significantly higher than those in the ZOL 100, ZOL 50, and PRO groups. Correspondingly, a significantly higher Tb.Sp was observed for all single treatment groups compared to that of ZOL 50 + PRO group. Therefore, with respect to improvement of the microstructural parameters of trabecular bone, ZOL and PRO were considered to be independent and additive. trabecular bone of the distal femur was readily observed in OVX group (Figure 2(b)) compared with numerous and well- developed trabeculae in SHAM group (Figure 2(a)). ZOL 100 (Figure 2(c)), ZOL 50 (Figure 2(d)), PRO (Figure 2(e)), and ZOL 50 + PRO (Figure 2(f)) groups prevented bone loss from OVX induced osteopenia. The quantification of trabecular bone changes in the distal femoral metaphysis is shown in Figure 3. As compared to SHAM group, the OVX group showed significant decrease in BV/TV, Tb.Th, and Tb.N. Correspondingly, a significantly higher Tb.Sp and SMI were observed for the OVX group rats compared to that of the SHAM rats, suggesting that ovariectomy caused the trabecular bone to become thin and sparse and disrupted the bone structure. Significant improvements, however, were observed in BV/TV, Tb.Sp, Tb.Th, Tb.N, and SMI values in all single treatments compared with the OVX group. In the ZOL 50 + PRO group, there were significant improvements in these microstructural indices compared with the OVX group. Moreover, the BV/TV, Tb.Th, and Tb.N in the ZOL 50 + PRO group were significantly higher than those in the ZOL 100, 3.3. Effects on the Mechanical Properties in the Femoral Mid- Shaft. Figures 4(a)–4(e) show the peak load, ultimate stiff- ness, toughness, ultimate strength, and Young’s modulus in the femoral midshaft, respectively. Three-point bending tests of the right femur indicated that ovariectomy caused significant reductions in the peak load, ultimate stiffness, toughness, ultimate strength, and Young’s modulus compared with those in SHAM group. 3. Results 3.1. Effects on the Body Weight. The final body weights were significantly greater for animals in the OVX treatment and untreated groups compared with the SHAM animals. There were no statistically significant differences in weights ob- served between any of the active treatment groups and that of the untreated OVX control group (Table 1). 3.2. Trabecular Structure Parameters Using Micro-CT. 3D re- construction of the trabecular bone structures in the distal femoral metaphysis by Micro-CT is shown in Figure 2. Dete- rioration of the trabecular architecture due to destruction of 4 Journal of Osteoporosis 0.000 0.228 (mm) 1.0 mm (a) 0.000 0.228 (mm) 1.0 mm (b) 0.000 0.312 (mm) 1.0 mm (c) 0.000 0.240 (mm) 1.0 mm (d) 0.000 0.216 (mm) 1.0 mm (e) 0.000 0.252 (mm) 1.0 mm (f) Figure 2: Three-dimensional architecture of trabecular bone within the distal metaphyseal femur region. (a) SHAM group. (b) OVX group. (c) OVX + ZOL 100 group. (d) OVX + ZOL 50 group. (e) OVX + PRO group. (f) OVX + ZOL 50 + PRO group. All groups except SHAM group undergo ovariectomy. 0.000 0.312 (mm) 1.0 mm (c) 0.000 0.228 (mm) 1.0 mm (b) 0.000 0.228 (mm) 1.0 mm (a) 0.000 0.228 (mm) 1.0 mm (b) (b) (c) (a) (c) 0.000 0.252 (mm) 1.0 mm (f) 0.000 0.216 (mm) 1.0 mm (e) 0.000 0.240 (mm) 1.0 mm (d) (e) (d) (f) Figure 2: Three-dimensional architecture of trabecular bone within the distal metaphyseal femur region. (a) SHAM group. (b) OVX group. (c) OVX + ZOL 100 group. (d) OVX + ZOL 50 group. (e) OVX + PRO group. (f) OVX + ZOL 50 + PRO group. All groups except SHAM group undergo ovariectomy. trabecular bone of the distal femur was readily observed in OVX group (Figure 2(b)) compared with numerous and well- developed trabeculae in SHAM group (Figure 2(a)). ZOL 100 (Figure 2(c)), ZOL 50 (Figure 2(d)), PRO (Figure 2(e)), and ZOL 50 + PRO (Figure 2(f)) groups prevented bone loss from OVX induced osteopenia. The quantification of trabecular bone changes in the distal femoral metaphysis is shown in Figure 3. As compared to SHAM group, the OVX group showed significant decrease in BV/TV, Tb.Th, and Tb.N. Moreover, in ZOL 100, ZOL 50, PRO, and ZOL + PRO groups, the ultimate strength of the femur was significantly higher than in the OVX group. In all single treatments, the ultimate strength was significantly lower than that in the ZOL 50 + PRO group (𝑃< 0.001). Furthermore, Young’s modulus of the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups was 3. Results In the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups, the peak load of the femur was significantly higher than in the OVX group. Similarly, in the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups, the ultimate stiffness of the femur was significantly higher than in the ournal of Osteoporosis 5 SHAM OVX ZOL 100 ZOL 50 PRO 0.0 0.1 0.2 0.3 0.4 0.5 BV/TV (%) ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (a) 0.00 0.02 0.04 0.06 0.08 0.10 Tb.Th (mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗ ∗∗ ∗a ∗a ∗a ZOL 50 + PRO (b) 0 2 4 6 8 10 Tb.N (1/mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (c) 0.0 0.2 0.4 0.6 Tb.Sp (mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (d) 0.0 0.5 1.0 1.5 SMI SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗ ∗ ∗ ZOL 50 + PRO (e) igure 3: Effects of zoledronic acid and propranolol, alone or in combination, on bone parameters measured by Micro-CT at the distal emur region of ovariectomized rats. Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗𝑃< 0.05; ∗𝑃< 0.01; ∗∗∗𝑃< 0.001, compared to OVX group; a𝑃< 0.05; b𝑃< 0.01, compared to ZOL + PRO group. All groups except SHAM group ndergo ovariectomy. OVX group. In contrast, the ultimate stiffness in all single treatments was significantly lower than that in the ZOL 50 + PRO group. The toughness of the femur in the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups was significantly higher than in the OVX group. In all single treatments, the toughness was significantly lower than that in the ZOL 50 + PRO groups. 3. Results 5 Journal of Osteoporosis Journal of Osteoporosis SHAM OVX ZOL 100 ZOL 50 PRO 0.0 0.1 0.2 0.3 0.4 0.5 BV/TV (%) ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (a) 0.00 0.02 0.04 0.06 0.08 0.10 Tb.Th (mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗ ∗∗ ∗a ∗a ∗a ZOL 50 + PRO (b) SHAM OVX ZOL 100 ZOL 50 PRO 0.0 0.1 0.2 0.3 0.4 0.5 BV/TV (%) ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (a) 0.00 0.02 0.04 0.06 0.08 0.10 Tb.Th (mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗ ∗∗ ∗a ∗a ∗a ZOL 50 + PRO (b) 0 2 4 6 8 10 Tb.N (1/mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (c) 0.0 0.2 0.4 0.6 Tb.Sp (mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (d) SHAM OVX ZOL 100 ZOL 50 PRO ZOL 50 + PRO (a) SHAM OVX ZOL 100 ZOL 50 PRO ZOL 50 + PRO (b) 0 2 4 6 8 10 Tb.N (1/mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (c) 0.0 0.2 0.4 0.6 Tb.Sp (mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (d) 1 5 (e) Figure 3: Effects of zoledronic acid and propranolol, alone or in combination, on bone parameters measured by Micro-CT at the distal femur region of ovariectomized rats. Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗𝑃< 0.05; ∗∗𝑃< 0.01; ∗∗∗𝑃< 0.001, compared to OVX group; a𝑃< 0.05; b𝑃< 0.01, compared to ZOL + PRO group. All groups except SHAM group undergo ovariectomy. OVX group. In contrast, the ultimate stiffness in all single treatments was significantly lower than that in the ZOL 50 + PRO group. The toughness of the femur in the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups was significantly higher than in the OVX group. In all single treatments, the toughness was significantly lower than that in the ZOL 50 + PRO groups. 3. Results 6 6 Journal of Osteoporosis 0 50 100 150 200 Peak load (N) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗ ∗ ∗ ZOL 50 + PRO (a) 0 100 200 300 Ultimate stiffness (N/mm) ∗∗∗ ∗∗∗ SHAM OVX ZOL 100 ZOL 50 PRO ∗a ∗∗a ∗∗a ZOL 50 + PRO (b) 0 10 20 30 40 50 Toughness (mJ) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗a ∗a ∗a ZOL 50 + PRO (c) 0 100 200 300 Ultimate strength (MPa) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗c ∗∗c ∗c ZOL 50 + PRO (d) 0 2 4 6 Young’s modulus (GPa) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗∗a ZOL 50 + PRO (e) ure 4: Effects of zoledronic acid, propranolol, or zoledronic acid plus propranolol on the mechanical strength of the femoral diaphys e diaphysis was subjected to three-point bending to failure, which provided data on peak load (a), ultimate stiffness (b), toughness ( mate strength (d), and Young’s modulus (e). Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison te < 0.05; ∗∗𝑃< 0.01; ∗∗∗𝑃< 0.001, compared to OVX group; a𝑃< 0.05; b𝑃< 0.01; c𝑃< 0.001 compared to ZOL + PRO group. All grou ept SHAM group undergo ovariectomy. (b) (d) (e) Figure 4: Effects of zoledronic acid, propranolol, or zoledronic acid plus propranolol on the mechanical strength of the femoral diaphysis. The diaphysis was subjected to three-point bending to failure, which provided data on peak load (a), ultimate stiffness (b), toughness (c), ultimate strength (d), and Young’s modulus (e). Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗𝑃< 0.05; ∗∗𝑃< 0.01; ∗∗∗𝑃< 0.001, compared to OVX group; a𝑃< 0.05; b𝑃< 0.01; c𝑃< 0.001 compared to ZOL + PRO group. All groups except SHAM group undergo ovariectomy. Figure 4: Effects of zoledronic acid, propranolol, or zoledronic acid plus propranolol on the mechanical strength of the femoral diaphysis. The diaphysis was subjected to three-point bending to failure, which provided data on peak load (a), ultimate stiffness (b), toughness (c), ultimate strength (d), and Young’s modulus (e). Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. 3. Results ∗𝑃< 0.05; ∗∗𝑃< 0.01; ∗∗∗𝑃< 0.001, compared to OVX group; a𝑃< 0.05; b𝑃< 0.01; c𝑃< 0.001 compared to ZOL + PRO group. All groups except SHAM group undergo ovariectomy. 7 Journal of Osteoporosis significantly increased when compared with the OVX group. In all single treatments, Young’s modulus was significantly lower as compared to ZOL 50 + PRO group. and beneficial effects in treating osteoporosis when compared with the therapeutic dose of ZOL (100 𝜇g/kg, i.v.) reported by Cheng et al. [27]. As compared to ZOL 100 group, ZOL 50 group has comparable beneficial effects on conserving BV/TV, Tb.Th, Tb.N, and Tb.Sp, which was confirmed by the measurements using Micro-CT in this study. Furthermore, results of the bending and compression test in ZOL 50 group indicate similar ultimate peak load, stiffness, energy, bending stress, and Young’s modulus compared to ZOL 100 treated rats. Based on our collective structural and biomechanical analysis, we showed that ZOL 50 may have similar potency in treating osteoporosis in estrogen deficient rats. 3.4. Effects on the Mechanical Properties in the Fourth Lumbar Vertebra. Figures 5(a)–5(e) show the peak load, ultimate stiffness, toughness, ultimate strength, and Young’s modulus in the LV4, respectively. Mechanical compression testing of the LV4 indicated that ovariectomy caused significant reductions in the peak load, ultimate stiffness, toughness, ultimate strength, and Young’s modulus compared with those in sham group. In the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups, the peak load of the vertebral body was significantly higher than in the OVX group. Likewise, in all the therapeutic interventions, the ultimate stiffness of the vertebral body was significantly higher than in the OVX group. In contrast, the ultimate stiffness in ZOL 100, ZOL 50, and PRO groups was significantly lower than that in the ZOL 50 + PRO group. Moreover, the toughness and ultimate strength of the vertebral body in the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups were significantly higher than that in the OVX group. In all single treatment groups, the toughness and ultimate strength were significantly lower than that in the ZOL 50 + PRO group. Furthermore, Young’s modulus of the ZOL 100, ZOL 50, PRO, and ZOL + PRO groups was significantly increased when compared with the OVX group. In all single treatment groups, Young’s modulus was significantly lower than that in the ZOL 50 + PRO group. 4. Discussion The different and complementary modes of action of antios- teoporotic drugs allow a considerable range of combination therapies for treating osteoporosis. In the present preclinical study, we analyzed the skeletal effects of a nonselective 𝛽- blocker, PRO, in combination with antiresorptive ZOL ther- apy in OVX rats. We asked whether single intravenous dose of ZOL would (1) inhibit bone loss and microarchitectural changes associated with estrogen deficiency in adult rats and (2) enhance anabolic effects of PRO. Firstly, we clearly demonstrated that combination therapy using ZOL and PRO preserves the trabecular microarchitecture better than single- drug therapy using ZOL or PRO in OVX rats. Secondly and most interestingly, the combination treatment of ZOL and PRO administered improved the mechanical properties of the lumbar spine and femoral midshaft in an OVX rat model of osteoporosis better than single-drug therapy using ZOL or PRO. These findings suggest that the combination treatment of ZOL and PRO had a therapeutic advantage over either the ZOL or PRO monotherapy for treating osteoporosis. g y Currently, drugs used in the treatment of osteoporosis are primarily antiresorptive agents, and therefore, PRO, a strong inducer of bone formation [16, 17, 24], is expected to be a use- ful and effective drug for patients with osteoporosis [19–21]. Studies have demonstrated that low doses of PRO suppress bone resorption by inhibiting RANKL mediated osteoclas- togenesis as well as inflammatory markers without affecting haemodynamic parameters [24]. This result is supported by a previous finding, which showed that PRO stimulates OPG on its own in osteoblast cells [25]. Antiosteoporotic activity of PRO (0.1 mg/kg) is associated with no adverse effect on cardiac functions in the rats [16]. This selective action of PRO for bone tissues may be useful in terms of reduced risk of untoward effects observed with ZOL. One of the important factors which may increase renal toxicity of ZOL is higher dose [7, 8, 11, 29]. Serious atrial fibrillation has been reported more frequently in the ZOL-treated patients [1, 8, 30]. Significantly, PRO is among the safest antiarrhythmic drugs currently available which are effective in conversion of atrial fibrillation to normal sinus rhythm [31, 32]. An appealing hypothesis is that PRO when combined with an antiresorptive drug ZOL may result in superior effects on the skeleton and may prevent or reduce the risk of atrial Body weight in the OVX group was greater than in the SHAM group. 3. Results i Biomechanical testing provides a direct method to study mechanical traits of bones among various experimental animals with different testing protocols [28]. The bone strength is determined by the bone mass and the intrinsic properties of the bone material [8–10]. ZOL plus PRO was statistically superior to all other treatments at increasing stiffness, toughness, ultimate strength, and Young’s modulus of the femoral midshaft as well as lumbar vertebrae. This meant that the femora and lumbar vertebrae of rats receiving the combination of ZOL and PRO could resist deformation with the force applied, much better than single-drug therapy using ZOL or PRO. g In the analysis of the architecture of distal femoral tra- becular bone using Micro-CT, it was found that combination therapy with ZOL plus PRO was statistically superior to ZOL or PRO monotherapy in suppressing the decrease in BV/TV and the increase in Tb.Sp; both changes are due to ovariectomy. Moreover, combination therapy with ZOL plus PRO was statistically superior to ZOL or PRO monotherapy in increasing Tb.Th and suppressing the decrease in Tb.N, both of which are indices for the trabecular network. These parameters indicate that combination therapy with ZOL plus PRO promotes osteogenesis, thickens trabecular bone, and strengthens trabecular connectivity. 4. Discussion This indicates that the increase in body weight was caused by ovary extraction. Previous studies from our laboratory and others have shown a similar increase in body weight after OVX, mainly because of the increase in estrogen deficiency [8].h i The results of this current study also showed that lower dose of ZOL (50 𝜇g/kg, i.v.) had showed similar promising 8 Journal of Osteoporosis Journal of Osteopo 0 200 400 600 Peak load (N) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗ ∗∗ ∗∗ ZOL 50 + PRO (a) 0 200 400 600 800 Ultimate stiffness (N/mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗∗a ∗∗∗a ∗∗∗b ZOL 50 + PRO (b) 0 50 100 150 Toughness (mJ) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗∗a ZOL 50 + PRO (c) 0 20 40 60 Ultimate strength (MPa) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗∗a ZOL 50 + PRO (d) 0.0 0.5 1.0 1.5 Young’s modulus (GPa) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗a ZOL 50 + PRO (e) Effects of zoledronic acid, propranolol, or zoledronic acid plus propranolol on the mechanical strength of the fourth lumbar ve 4 was compressed to failure to provide data on peak load (a), ultimate stiffness (b), toughness (c), ultimate strength (d), and Y e). Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗𝑃< 0.05; ∗∗𝑃< 0.01; ∗∗∗𝑃< to OVX group; a𝑃< 0.05; b𝑃< 0.0, compared to ZOL + PRO group. All groups except SHAM group undergo ovariectomy. 4. Discussion 0 200 400 600 Peak load (N) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗ ∗∗ ∗∗ ZOL 50 + PRO ( ) 0 200 400 600 800 Ultimate stiffness (N/mm) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗∗a ∗∗∗a ∗∗∗b ZOL 50 + PRO (b) 0 200 400 Peak load (N) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗ ∗∗ ∗∗ ZOL 50 + PRO (a) 0 200 400 600 Ultimate stiffness (N/mm SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗a ∗∗∗a ∗∗∗b ZOL 50 + PRO (b) 0 50 100 150 Toughness (mJ) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗∗a ZOL 50 + PRO (c) 0 20 40 60 Ultimate strength (MPa) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗∗a ZOL 50 + PRO (d) (b) (a) (b) 0 50 100 150 Toughness (mJ) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗∗a ZOL 50 + PRO (c) 0 20 40 60 Ultimate strength (MPa) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗∗a ZOL 50 + PRO (d) (c) (c) (d) 0.0 0.5 1.0 1.5 Young’s modulus (GPa) SHAM OVX ZOL 100 ZOL 50 PRO ∗∗∗ ∗∗∗ ∗∗a ∗∗a ∗a ZOL 50 + PRO (e) (e) Figure 5: Effects of zoledronic acid, propranolol, or zoledronic acid plus propranolol on the mechanical strength of the fourth lumbar vertebra (LV4). LV4 was compressed to failure to provide data on peak load (a), ultimate stiffness (b), toughness (c), ultimate strength (d), and Young’s modulus (e). Data are shown as the mean ± SD (𝑛= 6), evaluated by Tukey’s multiple comparison test. ∗𝑃< 0.05; ∗∗𝑃< 0.01; ∗∗∗𝑃< 0.001, compared to OVX group; a𝑃< 0.05; b𝑃< 0.0, compared to ZOL + PRO group. All groups except SHAM group undergo ovariectomy. Journal of Osteoporosis 9 fibrillation. These investigations suggest that the strategy of combined treatment should be a logical and effective therapeutic approach for osteoporosis. In postmenopausal osteoporosis, the tight coupling of bone formation and bone resorption may be compromised by estrogen deficiency which might contribute to cause bone loss. 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W4205454442.txt	https://www.mdpi.com/1422-0067/23/1/420/pdf?version=1640929298	en		Effects of a Novel Cold Atmospheric Plasma Treatment of Titanium on the Proliferation and Adhesion Behavior of Fibroblasts	International journal of molecular sciences	2,021	cc-by	5,773	International Journal of Molecular Sciences Article Effects of a Novel Cold Atmospheric Plasma Treatment of Titanium on the Proliferation and Adhesion Behavior of Fibroblasts Ming Yan 1, * , Philip Hartjen 1 , Martin Gosau 1 , Tobias Vollkommer 1 , Audrey Laure Céline Grust 1 , Sandra Fuest 2 , Lan Kluwe 1 , Simon Burg 1 , Ralf Smeets 1,2 and Anders Henningsen 1 1 2 * Citation: Yan, M.; Hartjen, P.; Gosau, M.; Vollkommer, T.; Grust, A.L.C.; Fuest, S.; Kluwe, L.; Burg, S.; Smeets, R.; Henningsen, A. Effects of a Novel Cold Atmospheric Plasma Treatment of Titanium on the Proliferation and Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; p.hartjen@uke.de (P.H.); m.gosau@uke.de (M.G.); t.vollkommer@uke.de (T.V.); a.grust@uke.de (A.L.C.G.); lkluwe@uke.de (L.K.); s.burg@uke.de (S.B.); r.smeets@uke.de (R.S.); a.henningsen@uke.de (A.H.) Department of Oral and Maxillofacial Surgery, Division of Regenerative Orofacial Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; s.fuest@uke.de Correspondence: cnming.yan@hotmail.com; Tel.: +49-152-2690-8655; Fax: +49-407-4105-9665 Abstract: Cold plasma treatment increases the hydrophilicity of the surfaces of implants and may enhance their integration with the surrounding tissues. The implaPrep prototype device from Relyon Plasma generates cold atmospheric plasma via dielectric barrier discharge (DBD). In this study, titanium surfaces were treated with the implaPrep device for 20 s and assessed as a cell culture surface for fibroblasts. One day after seeding, significantly more cells were counted on the surfaces treated with cold plasma than on the untreated control titanium surface. Additionally, the viability assay revealed significantly higher viability on the treated surfaces. Morphological observation of the cells showed certain differences between the treated and untreated titanium surfaces. While conventional plasma devices require compressed gas, such as oxygen or argon, the implaPrep device uses atmospheric air as the gas source. It is, therefore, compact in size and simple to handle, and may provide a safe and convenient tool for treating the surfaces of dental implants, which may further improve the implantation outcome. Adhesion Behavior of Fibroblasts. Int. J. Mol. Sci. 2022, 23, 420. https:// Keywords: cold atmospheric plasma; osteoblast-like cells; titanium; proliferation doi.org/10.3390/ijms23010420 Academic Editors: Gianrico Spagnuolo and Marco Tatullo Received: 18 November 2021 Accepted: 28 December 2021 Published: 31 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction The failure of implants to achieve good osseointegration within the surrounding bone tissue after implantation is often an important reason for the failure of implant surgery [1]. It has always been the research direction for the majority of scholars to improve the success rate of implantation by changing the surface properties of the implant, increasing the biocompatibility between the implant and the surrounding tissue, and reducing the risk of infection after implantation [2–6]. However, the success of implants requires not only good osseointegration but also a good soft tissue seal [7]. A good soft tissue seal can reduce the influence of microorganisms on the implant and protect the osseointegration [8]. The soft tissue around the implant is composed of epithelial tissue consisting of epithelial cells and connective tissue consisting of fibroblasts. It was found that epithelial cells have migrate along the surfaces of the implant towards the root, while healthy connective tissue can prevent the migration of epithelial cells towards the root. A good bond between connective tissue and the implant means that fibroblast can adhere and proliferate well on the implant surface. The factors that influence the cell adhesion and proliferation are the surface roughness, surface morphology, surface chemistry, and hydrophilicity [9]. Plasma is a term used to describe partially ionized gases, which can be classified into thermal plasma and low-temperature plasma. Thermal plasma can be used as a surface coating technology to spray metals and ceramics onto other materials. Metallic particles Int. J. Mol. Sci. 2022, 23, 420. https://doi.org/10.3390/ijms23010420 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 420 morphology, surface chemistry, and hydrophilicity [9]. Plasma is a term used to describe partially ionized gases, which can be classified into thermal plasma and low-temperature plasma. Thermal plasma can be used as a surface coating technology to spray metals and ceramics onto other materials. Metallic particles such as titanium, silver, and 2 of 10 hydroxyapatite are treated using thermal plasma spraying technology to enhance the biocompatibility of dental or orthopedic implants [10]. In contrast, low-temperature plasma has a lower temperature and can be used to directly treat living tissues to achieve such as titanium, silver, and hydroxyapatite are treated using thermal plasma spraying sterilization, coagulation, wound healing, andor tissue regeneration [11]. It can technology toblood enhance the biocompatibility of dental orthopedic implants [10]. In also be used to indirectly treat living tissues by means of implanting plasma-treated materials contrast, low-temperature plasma has a lower temperature and can be used to directly [12,13]. In our previous work, the surfaces of coagulation, titanium specimens were treated with an treat living tissues to achieve sterilization, blood wound healing, and tissue argon–oxygen plasma s [14,15]. It was found plasma treatment can increase regeneration [11]. It can jet alsofor be60 used to indirectly treat livingthat tissues by means of implanting plasma-treated our titanium previous work, theand surfaces titanium specimens the wettabilitymaterials of the [12,13]. surfacesIn of sheets can ofalso promote the early were treatedofwith an argon–oxygen for 60 s [14,15]. It wasspecimens, found that plasma attachment gingival fibroblastsplasma on thejetsurfaces of titanium which may treatment can increase the between wettability ofimplant the surfaces sheets soft and tissue, can also enhance the sealing effect the and of thetitanium surrounding reduce promote the early attachment of gingival fibroblasts on the surfaces of titanium specimens, the risks of infection by pathogenic bacteria, and enhance the ability of the implant to which may enhance the sealing effect between the implant and the surrounding soft integrate within the surrounding bone tissues. The traditional plasma treatment systems tissue, reduce the risks of infection by pathogenic bacteria, and enhance the ability of (including vacuum devices and gas storage devices) are large in size, use high external the implant to integrate within the surrounding bone tissues. The traditional plasma voltages, require external working gas, and which extremely treatmentand systems (including vacuum devices gasmakes storagethem devices) are largeinconvenient in size, for clinical applications. With the development of industrial technology, the atmospheric use high external voltages, and require external working gas, which makes them extremely plasma devices theapplications. advantagesWith of being small in size and lighttechnology, in weight the and can inconvenient forhave clinical the development of industrial directly use air as the working which is convenient forinclinical atmospheric plasma devices have gas, the advantages of being small size andapplications. light in weightIn this and can use air as the working which is convenient for clinical applications. study, thedirectly implaPrep prototype devicegas, from Relyon Plasma (Figure 1) was used to activate In this study, the implaPrep prototype device from Relyon Plasma (Figure 1) used and the surfaces of the materials, and the effects of the activation on the was adhesion to activate the surfaces of the materials, and the effects of the activation on the adhesion proliferation of fibroblasts were observed to explore whether atmospheric plasma can and proliferation of fibroblasts wereofobserved to explore whetherproviding atmospheric plasma for improve the biological properties bone implant materials, a reference can improve the biological properties of bone implant materials, providing a reference for clinical applications. clinical applications. Figure 1. (A) The implaPrep prototype device from Relyon Plasma is based on a cylindrical dielectric barrier1.discharge withprototype an inner diameter measuring approximately 1.5 cm, by an Figure (A) The system implaPrep device from Relyon Plasma is based on powered a cylindrical alternating current. The equipment parameters were adjusted to 24 V and 1.5 A. (B) Traditional dielectric barrier discharge system with an inner diameter measuring approximately 1.5 cm, device reactor frequency input power 24 W, system pressure 1tombar, powered by an(generator alternating current. 100 ThekHz, equipment parameters were adjusted 24 Vgas andflow 1.5 A. rate 1.25 sccm, and gas reactor purity >99.5%). (B) Traditional device (generator frequency 100 kHz, input power 24 W, system pressure 1 mbar, gas flow rate 1.25 sccm, and gas purity >99.5%). 2. Results 2.1. Water Immersion Angle 2. Results Implants that were plasma-treated for 20 s showed increased wettability with regard 2.1. Water Immersion Angle to the immersion angle measurement compared to the untreated reference (immersion angle 120◦ vs. that 63◦ ) were (Figure 2). Implants plasma-treated for 20 s showed increased wettability with regard to the immersion angle measurement compared to the untreated reference (immersion angle 120° vs. 63°) (Figure 2). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 22, 23, x FOR PEER REVIEW 3 of 10 3 of 11 Int. J. Mol. Sci. 2022, 23, 420 3 of 10 Figure 2. Dental implants with acid-etched and sandblasted titanium surfaces were analyzed Figure 2. Dental implants with acid-etched and sandblasted titanium surfaces were analyzed regardregarding the water immersion angle before and after plasma treatment for 20 s. The columns ing the water immersion angle before and after plasma treatment for 20 s. The columns represent represent mean values, while the error bars represent the standard deviation. mean with values, while the error represent titanium the standard deviation. Figure 2. Dental implants acid-etched and bars sandblasted surfaces were analyzed regarding the water immersion angle before and after plasma treatment for 20 s. The columns 2.2. Early Cell Adhesion 2.2. Early Cell Adhesion represent mean values, while the error bars represent the standard deviation. Figure 33shows showsthe the early adhesion of L929 onsurfaces the surfaces both materials. Figure early adhesion of L929 cellscells on the of bothofmaterials. Most Most of the cells adhered to theofsurfaces of the a spherical shape and of the cells adhered to the surfaces the materials in materials a sphericalinshape and pseudopodia 2.2. Early Cell Adhesion pseudopodia butobvious there was no obvious in theHowever, control groups. form, but thereform, was no spreading in thespreading control groups. most of However, the cells Figure 3 shows theinofearly adhesion of cellsstate, on the surfaces of both materials. most cells were in aL929 spread polygonal, with thickextending and longaround pseudopodia were a the spread state, polygonal, with thick and long pseudopodia them extending around them in the experimental groups (Figure 3). Most of the cells in adhered to the surfaces of the3).materials in a spherical shape and the experimental groups (Figure pseudopodia form, but there was no obvious spreading in the control groups. However, most of the cells were in a spread state, polygonal, with thick and long pseudopodia extending around them in the experimental groups (Figure 3). Figure 3. Cont. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW Int. J. Mol. Sci. 2022, 23, 420 4 of 10 4 of 10 Figure 3. (A) Acid-etched and sandblasted titanium surface. (B) Untreated titanium surface. C attachment and morphology results for different surface-treated titanium specimens after 2, 8 48, and 72 h. Cells were cytoskeleton-stained with phalloidin. The cells increased in volume a Figure3.3.(A) (A)the Acid-etched and sandblasted titanium surface. (B)Untreated Untreated titaniumand surface. Cell were la Figure Acid-etched sandblasted titanium surface. titanium surface. adhered to surfaces.and The morphology of the cells (B) was more extended theCell cells attachment attachmentand andmorphology morphologyresults resultsfor fordifferent differentsurface-treated surface-treated titanium titanium specimens specimens after after 2, 2, 8, 8, 24, 24, especially on plasma- and UV-light-treated surfaces. In the plasma- and UV-light-treated gro 48, and 72 h. Cells were cytoskeleton-stained with phalloidin. The cells increased in volume and 48, and 72 h. Cells were cytoskeleton-stained with phalloidin. The cells increased in volume and the cells were most extended and showed more filopodia-like structures. adhered to the surfaces. The morphology of the cells was more extended and the cells were larger, adhered to the surfaces. The morphology of the cells was more extended and the cells were larger, especially and UV-light-treated UV-light-treatedsurfaces. surfaces.InInthe theplasmaplasmaand UV-light-treated groups, especially on on plasmaplasma- and and UV-light-treated groups, the the cells were most extended and showed more filopodia-like structures. 2.3. Cell Proliferation cells were most extended and showed more filopodia-like structures. The proliferation of L929 cells was determined using an XTT assay. As show 2.3.Cell CellProliferation Proliferation 2.3. Figure 4, the numbers of cells in the two groups of materials gradually increased The proliferation proliferation of L929 cells was determined The determined using using an an XTT XTTassay. assay. As Asshown showninin time. The numbers of cells in the experimental groups were higher than in the co Figure 4, the numbers of cells in the two groups of materials gradually increased over time. Figure the numbers of cells in the two groups of materials gradually increased over groups 24,of 48, and the differences were statistically significant (p < The numbers cellsof incells the72 experimental groups were higher than in thethan control groups at time. Theatnumbers inh,theand experimental groups were higher in the control 24, 48, and 72 h, the72differences were statistically significant (p < 0.05) (Figure 4). (Figure 4).24, There were no statistical differences among the experimental groups. groups at 48,and and h, and the differences were statistically significant (p <There 0.05) were no4). statistical differences amongdifferences the experimental (Figure There were no statistical amonggroups. the experimental groups. Figure 4. (A) Acid-etched and sandblasted titanium surface. (B) Untreated titanium surface. The Figure 4. (A) Acid-etched and sandblasted titanium surface. (B) Untreated titanium surface. The cell cell counts (proliferation) were analyzed using XTT at 24, 48,surface. and 72 h.(B) Untreated titanium surface. T Figure 4. (A) Acid-etched and sandblasted titanium counts (proliferation) were analyzed using XTT at 24, 48, and 72 h. cell counts (proliferation) were analyzed using XTT at 24, 48, and 72 h. 2.4. 2.4.Cell CellCount Count The cell on the the surfaces surfacesof ofthe thetitanium titaniumspecimens specimens TheCount cellcount count results results for for the the two two treatments treatments on 2.4. Cell at presented as as means means±± standard deviations deviations in in Figure Figure5.5.Here, Here, atdifferent different time time points are presented The cell count results fortrend the two treatments on the surfaces of the (x s)s)depicts cell growth trend based number of on cells the titanium titanium (x−−±± depicts the cell growth based on on the the number of cells theon titanium speci- speci at different time points arethe presented as means standard inofFigure 5. specimens. The results showed that thewere cells were able±to todeviations the of surfaces the mens. The results showed that cells able to adhere toadhere the surfaces the titanium − specimens in each experimental group within 24based h ofwithin cellon culture slowly, while thethe tita titanium specimens in cell each growth experimental 24 hbut of grew cell culture but on grew (x ± s) depicts the trendgroup the number of cells number of cells in each group did not change much, which was considered the incubation slowly, while the number of cells in each group did not change much, which specimens. The results showed that the cells were able to adhere to the was surfaces o consideredspecimens the incubation period of the cells. The numbers cellsculture in the but titanium in and eachadaptation experimental group within 24 h ofofcell slowly, while the number of cells in each group did not change much, which considered the incubation and adaptation period of the cells. The numbers of cells i Int.J.J.Mol. Mol.Sci. Sci.2022, 2022,23, 23,x420 Int. FOR PEER REVIEW of 10 55 of 10 and adaptation period of the cells. The numbers of cells in the experimental groups were experimental groups were higher than in the control groups at 24, 48, and 72 h, and the higher than in the control groups at 24, 48, and 72 h, and the differences were statistically differences were statistically significant (p < 0.05) (Figure 5). significant (p < 0.05) (Figure 5). Figure 48,48, Figure5. 5.(A,B) (A,B)The Thecell cellcounts counts(proliferation) (proliferation)were wereanalyzed analyzedusing usingImageJ ImageJsoftware softwareatat2,2,8,8,24,24, and 72 h. (C,D) The morphologies of the fibroblasts were determined at 2, 8, 24, 48, and 72 h by and 72 h. (C,D) The morphologies of the fibroblasts were determined at 2, 8, 24, 48, and 72 h by direct direct observation light microscope after live–dead staining. Scale100 bar: 100(A,C) μm. (A,C) Acidobservation with awith lightamicroscope after live–dead staining. Scale bar: µm. Acid-etched etched and sandblasted titanium surface. (B,D) Untreated titanium surface. and sandblasted titanium surface. (B,D) Untreated titanium surface. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW Int. J. Mol. Sci. 2022, 23, 420 6 of 10 6 of 10 2.5. Assessment of Cytotoxicity 2.5. Assessment of Cytotoxicity As expected, cytotoxicity was not detected on any of the surfaces, regardless of the As expected, was not detected on any of the surfaces, regardless of the treatment (Figurecytotoxicity 6). treatment (Figure 6). Figure of different surface-treated titanium specimens after 24, 48, and24, 7248, h, measured Figure6.6.Cytotoxicity Cytotoxicity of different surface-treated titanium specimens after and 72 h, using an LDH assay. (A) Acid-etched and sandblasted titanium surface. (B) Untreated measured using an LDH assay. (A) Acid-etched and sandblasted titanium surface.titanium (B) Untreated surface. significant detected.were detected. titaniumNo surface. No differences significantwere differences 3. Discussion 3. Discussion The advantageous mechanical and biological properties of titanium have gained The advantageous mechanical and biological properties of titanium have gained increasing attention from dental professionals. With continuous improvements in dental implant technology, titanium has good application prospects as a dental material [16]. increasing attention from dental professionals. With continuous improvements in denta However, titanium andtitanium its alloyshas are good biologically inert materials, which onlymaterial form implant technology, application prospects as a can dental [16] mechanical bonds with growing bone, not chemical bonds [17]. Therefore, it is necessary However, titanium and its alloys are biologically inert materials, which can only form to perform a surface treatment on titanium and its alloys to promote biological activities, mechanical bonds with growing bone, not chemical bonds [17]. Therefore, it is necessary such as enhanced adhesion, proliferation, protein synthesis, contact angles, and matrix to perform a surface treatment on titanium and its alloys to promote biological activities mineralization of osteoblasts on its surfaces, which could accelerate osseointegration. The such as enhanced adhesion, proliferation, proteinsuccess. synthesis, contact angles, and matrix establishment of osseointegration is the key to implant mineralization of osteoblasts on its surfaces,angle which osseointegration. The The determination of the water immersion is acould simpleaccelerate method used to measure establishment of osseointegration is the key to implant success. the hydrophilicity of surfaces [18]. As can be seen from Figure 2, the plasma treatment resultedThe in increased wettability of the titanium surface, angle as evidenced by a reduced determination of the water immersion is a simple methodimmersion used to measure angle. This increasedof wettability cellular the hydrophilicity surfaceslikely [18]. promotes As can be seen attachment. from Figure 2, the plasma treatmen During process ofwettability osseointegration, adhesionsurface, and proliferation on the surface resulted in the increased of thecell titanium as evidenced by a reduced of the implant are prerequisites [19–21]. Therefore, when evaluating the biological activity immersion angle. This increased wettability likely promotes cellular attachment. of implant materials, the material’s ability to promote the early adhesion of osteoblasts on During the process of osseointegration, cell adhesion and proliferation on the surface the surface and the formation of good cell morphology should be observed as important of the implant are prerequisites [19–21]. evaluating the biological indicators. Moreover, the adhesion of cells toTherefore, materials iswhen the foundation of tissue engineer- activity of implant materials, the material’s ability to promote the early adhesion of on ing research. The adhesion and aggregation of cells on the surface of the implantosteoblasts plays surface theosteogenesis formation of good morphology should observed as important athe key role in and contact [22]. Thecell experiments showed that be surface-coarsening treatment the implantsthe could promoteof thecells adhesion and proliferation of osteoblasts. indicators.of Moreover, adhesion to materials is the foundation of tissue Within 24 h in culture, mouse fibroblasts had attached well to the surfaces of titanium engineering research. The adhesion and aggregation of cells on the surface of the implant sheets group, although there [22]. was no significant changeshowed in the number plays in a each key treatment role in contact osteogenesis The experiments that surfaceof fibroblasts. However, after 24 h in culture, the number of fibroblasts began to increase coarsening treatment of the implants could promote the adhesion and proliferation of linearly, and the number of fibroblasts in the experimental group was greater than that in osteoblasts. Within 24 h in culture, mouse fibroblasts had attached well to the surfaces of the control group at each time point. In addition, the fibroblasts on the surfaces of titanium titanium sheets each treatment group, there was no significant change in the specimens in eachintreatment group were wellalthough stretched and showed a prolonged-spindle number of fibroblasts. However, after 24 h in culture, the number ofgroup fibroblasts shape, while the antennae and pseudopodia were not obvious in the control and thebegan to increaseoflinearly, the number of fibroblasts in the experimental group was greater number adhered and cells was also smaller. The cold plasma (traditional and atmospheric) and treatment groupsgroup were superior to thepoint. untreated group in the thisfibroblasts regard. thanUV that in the control at each time In addition, on the surfaces Prior to the clinical application of any biomaterial, a biological evaluation should of titanium specimens in each treatment group were well stretched and be showed a conducted in accordance with the relevant biosafety inspection criteria, including three in the prolonged-spindle shape, while the antennae and pseudopodia were not obvious stages of primary toxicity screening, animal experiments, and clinical trials [23,24]. The control group and the number of adhered cells was also smaller. The cold plasma primary rapid toxicity screening program is divided into two systems: in vitro and in vivo. (traditional and atmospheric) and UV treatment groups were superior to the untreated group in this regard. Prior to the clinical application of any biomaterial, a biological evaluation should be conducted in accordance with the relevant biosafety inspection criteria, including three Int. J. Mol. Sci. 2022, 23, 420 7 of 10 In vitro cytotoxicity testing is one of the important aspects. At present, the lactate dehydrogenase release assay is used to test the cytotoxicity of plant materials. This method has the advantage of sensitivity, simplicity, and objective quantification. In recent years, it has been gradually adopted to evaluate the cytotoxicity of biological and dental materials [25]. Lactate dehydrogenase (LDH) in culture medium was used as an indicator in this study [26]. Therefore, the cytotoxicity test was used in this experiment to study the titanium sheets treated by different methods. The experimental results showed that mouse fibroblasts could adhere to the surfaces of titanium sheets in each treatment group. There were no statistically significant differences among the groups (p > 0.05). In vivo experiments should become an indispensable part of the soft tissue exploration process, especially because in vitro experiments cannot simulate the peri-implant fiber pathway. Further studies with in vivo experiments will be summarized in our next paper. Currently, there is no relevant standard for plasma devices in clinical applications. Factors such as the gas type, treatment time, generated power, and exposure methods should be considered during application, because different conditions may have different biological effects. In this experiment, the implaPrep device developed by Relyon Plasma was applied to change the use of compressed gas to pure air, which can provide a more uniform, safe, and convenient medical treatment method. We believe that the application of atmospheric plasma in the field of stomatology has bright prospects. 4. Materials and Methods 4.1. Measurement of the Water Immersion Angle before and after Plasma Treatment Dental implants with acid-etched and sandblasted titanium surfaces were analyzed regarding the water immersion angles before and after plasma treatment for 20 s. The immersion angle measurements were performed 30 min after plasma treatment. 4.2. Experimental Groups and Material Surface Activation Treatment Two kinds of titanium material (acid-etched and sandblasted titanium and untreated titanium) were used in this experiment. Samples were randomly divided into one group of non-treated samples (controls) and three experimental groups, with three samples in each group. The materials were immersed in isopropanol and dried for disinfection. The disks in the novel plasma group were treated using the implaPrep prototype device for 20 s (relyon plasma is based on a cylindrical dielectric barrier discharge with an inner diameter of approximately 1.5 cm, powered by alternating current were adjusted to 24 V and 1.5 A) (Relyon Plasma GmbH, Regensburg, Germany). Disks in the traditional plasma group were treated with argon plasma for 60 s, using an NTP reactor (pressure 1 mbar, gas flow rate 1.25 sccm, and gas purity > 99.5%) (Diener Electronic GmbH, Ebhausen, Germany). Disks in the UV light experimental group were treated using a UV light oven for 12 min, which generated UV light with an intensity of 0.15 mW/cm2 (λ = 253.7 nm). Both of the low-temperature plasma systems are shown in Figure 1. 4.3. Cell Culture L929 murine fibroblast cells (Sigma–Aldrich, Munich, Germany) were cultured in endothelial cell medium (ECM) containing 5% fetal bovine serum, 1% endothelial growth factor, and 1% penicillin/streptomycin at 37 ◦ C and 5% CO2 . The cell growth was observed and the medium was changed once every two days. 4.4. Cell Adhesion Fluorescence-labeled phalloidin (biotinylated phalloidin, Alexa Fluor 488 green, 1:1000; Thermo Fisher Scientific, Waltham, MA, USA) and DAPl were used to label cytoskeleton filaments and nuclei, respectively. The following specific steps of staining were performed. The culture medium in the Petri dish was aspirated and then cells were washed with sterile PBS 3 times for 5 min and fixed with paraformaldehyde (40 g/L) for 30 min. The cells were washed again with sterile PBS three times, then 0.3% tritonX-100 solution (Gibco, Int. J. Mol. Sci. 2022, 23, 420 8 of 10 Invitrogen, Paisley, UK) was added and the cells were incubated for 5 min to increase their permeability. The TritonX-100 solution was removed and DAPI was added to stain the cells for 15 min, then they washed with PBS three times. Phalloidin was added to stain the cells for 30 min in the dark, then they were washed with sterile PBS three times. Finally, the stained cells were visualized under a confocal laser scanning microscope. 4.5. Cell Proliferation Assay Here, 1 mL of cell suspension with a density of 8 × 104 cells per mL was seeded on the surfaces of the materials in experimental groups and control groups in 12-well plates. The cell growth on the surface of the material was observed under a microscope at 8, 24, 48, and 72 h, respectively. Next, 500 µL of XTT labeling solution was added to each well at 24, 48, and 72 h, which was further cultured at 37 ◦ C for 4 h. Then, the 12-well plates were shaken for 1 min. Next, 100 µL of the supernatant from each well was transferred into a 96-well plate and three parallels were set for every sample. The OD values of the supernatants were measured at 450 nm and a reference wavelength of 650 nm. 4.6. Lactate Dehydrogenase Release Assay Here, 10 ul of supernatant from each well was collected at 24, 48, and 72 h in culture. Then, LDH (LDH Cytotoxicity Assay Kit II, BioVision, Milpitas, CA, USA) reaction mix solution was added to the corresponding well of the 96-well plate and incubated in the dark for 30 min at room temperature. The OD values were measured at 450 and 650 nm (reference). 4.7. Observation of Cell Growth with Live–Dead Staining The cells were washed twice with sterile PBS and then live–dead cell fluorescent dye (containing 100 µg/mL FDA and 60 µg/mL PI) was added to cells, which were then incubated in the dark for 3 min. The cells were washed twice again with sterile PBS and then cell viability was observed under a microscope. Five regions of interest were randomly selected and photographed and the numbers of cells were determined using Image J software. 4.8. Statistical Analysis All experiments were repeated three times independently in duplicate wells. Statistical analysis was performed using SPSS 21 (IBM, Armonk, NY, USA). The significance of differences in viability and toxicity was assessed using t-tests. For all results, significant differences were defined as p < 0.05. 5. Conclusions In this study, titanium was activated by atmospheric low-temperature plasma and the results showed that the activated titanium has promoting effects on cell adhesion and proliferation, and at the same time has no adverse effects on cytocompatibility. In the next step, our group will further study the role of titanium activated by atmospheric low-temperature plasma on enhancing osteogenesis. Author Contributions: A.H.: study conception and design, data analysis and interpretation, critical editing of the manuscript. M.Y.: study conception and design, experimental operation, data collection, analysis and interpretation, critical editing of the manuscript. L.K.: study conception and design, experimental operation, data collection and analysis. P.H., T.V. and S.F.: experimental operation, data collection and analysis. S.B., A.L.C.G., R.S. and M.G.: study conception, discussion and critical editing. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by Relyon Plasma GmbH (Regensburg, Germany). M.Y. was supported by the Merit Scholarship for International Students (No.7238065). Institutional Review Board Statement: Not applicable. Int. J. Mol. Sci. 2022, 23, 420 9 of 10 Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Clark, D.; Levin, L. 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https://openalex.org/W4392617907	https://dergipark.org.tr/tr/download/article-file/3427293	Breton	null	Seasonal Changes in Body Composition in Elite Male Handball Players	International journal of disabilities sports & health sciences :	2,024	cc-by-sa	6,322	Abstract bst act Having knowledge of an athlete's body composition values enables individuals to assess their physical condition and performance. It has become increasingly important in recent years to demonstrate the significant relationship between body composition and athletes' physical performance. The aim of this study is to examine the changes in body composition of elite male handball players during preparation and competition periods (42 weeks). Twenty-three elite male handball players volunteered for this study The Tanita brand body analysis device was used to evaluate body composition and eight measurements were taken. Athletes were divided according to their playing positions (playmakers n=8 and goalkeepers n=4, wings n=7 and pivots n=4). 7 measurements were evaluated, which were weight (W), body fat percentage (BF%), fat mass (FM), muscle mass (MM), total body water (TBW), body mass index (BMI), and basal metabolic rate (BMR). Wing players had lower body fat percentage and body fat mass than all other playing positions and regardless of position, all athletes' body fat percentage and body fat mass significantly decreased at T2 (p<0.05). As a result, increase in body fat percentage and fat mass was observed during the competition period, and this increase continued (p<0.05). The increase in fat mass during the competition period may be due to different training content and higher training intensity during the preparation period, changes in nutrition habits due to camps, long trips, and tournaments, as well as the ef-fects of fatigue and stress. Further studies are needed in these areas. Keywords Body Composition, Fat Mass, Handball, Male Body Composition, Fat Mass, Handball, Male International Journal of Disabilities Sports and Health Sciences e-ISSN: 2645-9094 RESEARCH ARTICLE Nebahat ELER1 1Department of Coaching , Physical Education and School, Zonguldak Bulent Ecevit University, Zonguldak/Türkiye Corresponding author: nebahateler@beun.edu.tr Received: 22 September 2023 ; Revised ; 19 November 2023 ; Accepted: 07 March 2024; Published: 25 March 2024 How to cite this article: Eler, N. (2024). Seasonal Changes in Body Composition in Elite Male Handball Players. Int J Disabil Sports Health Sci;7(2):274- 281. https://doi.org/10.33438/ijdshs.1364866 Seasonal Changes in Body Composition in Elite Male Handball Players Nebahat ELER1 Eler, N., Int J Disabil Sports Health Sci; 2024;7(2):274-281 DOI:10.33438/ijdshs.1364866 Eler, N., Int J Disabil Sports Health Sci; 2024;7(2):274-281 DOI:10.33438/ijdshs.1364866 N. (2024). Seasonal Changes in Body Composition in Elite Male Handball Players. Int J Disabil Sports Health Sci;7(2):274- shs.1364866 INTRODUCTION to show that there is a significant relationship between body composition and athletes' physical performance (Rybakova et. al., 2020). Human physique varies in many ways, and this diversity in physical characteristics in certain sports turns into specific advantages for athletes during the game. Each sport requires a specific body type. A body type unsuitable for a sport can be a significant obstacle to a player's progress. Also, knowing and understanding the effect of training and competition on body composition can help athletes control their weight and safely change their body composition. Additionally, tracking body composition trends in specific sports enables coaches to prepare their athletes correctly for specific competi-tions or positions (Singh et. al., 2011). Handball is an Olympic team sport Body composition is only one of the many factors (physical, physiological, genetic, and psychological) that determine athletic performance. In addition to body size, the structure of body composition components is also important. Body weight can be divided into various components to achieve a more balanced and ap- propriate distribution of fat, muscle, and bone mass (Sundgot-Borgen et.al., 2013). Determining athletes' body composition (low fat and muscle ratios) is an important factor in increasing performance. Having knowledge of an individual's body composition values allows for interpretation of their physical condition and performance. In recent years, it has become increasingly important Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 .Page 274 / 281. Seasonal Changes in Body Composition characterized by defensive actions and fast-paced offensive actions to score goals (Martínez- Rodríguez et.al., 2021). It is difficult to determine the factors that affect performance in handball because it is complex and multifactorial, characterized by high-intensity explosive movements. Handball players must coordinate running, jumping, pushing, changing directions, passing, catching, throwing, and blocking movements well. The intensities during the game always vary between standing, walking, sprinting, running at a moderate pace, moving sideways and backward, so a high level of endurance is essential to maintain a high game tempo throughout the game (Póvoas et. al., 2012; Michalsik et. al., 2015). Due to the high physical demands of handball, players need highly devel-oped anthropometric and physical attributes (linear speed, change of direction speed, aerobic capacity, muscle strength, and power) to succeed (Bilge, 2012) al., 2017). INTRODUCTION Through the use of profiles, coaches and coaches can gain valuable information about each player's unique physical and physiological characteristics, which can be used to improve their overall performance and contribute to team success. Evaluation of body composition, with a focus on fat and skeletal muscle content, is particularly important in sports science and practice (Cavedon et. al., 2018). Previous research suggests that certain physical characteristics, including high body mass and stature, are associated with high-level handball performance (Lidor et. al., 2005). In addition, higher values of fatfree mass were associated with better performance, particularly due to increased muscular power and strength (Granados et. al., 2013) and physical characteristics can vary considerably across different player positions in the team (Karcher & Buchheit, 2014; Lidor et. al., 2005). In talent selection, assessing players' body composition is important in determining their playing positions and developing training programs (Schwesig et al., 2017; Fieseler et. al., 2017). Studies on anthropometry and body composition in handball are available (Milanese et. al., 2011; Owen et. al., 2017). However, no study has been found that examines changes in body composition values in elite male handball players during a season. The aim of this study is to examine changes in body composition between different playing positions in elite male handball players during the preparation and competition periods (42 weeks). We have two hypotheses in this study. Especially in fat and muscle mass: 1- We expect a decrease in fat mass, an increase in muscle mass, at the end of the preparation periods and during the competition period, 2- A decrease in fat mass and an increase in muscle mass between positions. In handball, there are generally four playing positions: 1) goalkeeper: stops the ball in the goal and prevents the opposing team from scoring; 2) playmakers (right-left-center): they are the ones who indicate where the attacks should start, serving as an extension of the coach on the field, and command the team in both offense and defense. The right-left playmakers are effective in both offense and defense; 3) wings: they are the ones who stop closed defenses in the goal area and often assist in scoring; 4) pivot: responsible for creating space by entering the defense wall at possible openings (Karcher & Buchheit, 2014). INTRODUCTION Handball players need to possess well-developed physical characteristics, including linear and change of direction speed, aerobic capacity, muscular strength, and power, to be successful in the sport. These anthropometric and physical qualities are strongly associated with handball performance and play a crucial role in determining a player's success on the court. Evaluating the match between physical structure and composition, weight, and height is considered important factors in performance, even depending on the sport and player positions (Martínez-Rodríguez et.al., 2020). Study Design Twenty-three elite male handball players participated in this study voluntarily (age: 25.41 ± 5.44 yr, height: 190.53 ± 5.79 cm, body weight: 93.71 ± 10.9 kg). After explaining the objectives of the study, written informed consent was obtained from each subject. This study followed ethical standards and received approval from the Zonguldak Bülen Ecevit University Non-invasive In the sport of handball, player profiling can be an effective method of identifying talent, assessing strengths and weaknesses, determining optimal playing positions, and designing personalized strength and conditioning training programs (Karcher & Buchheit, 2014; Schwesig et Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 .Page 275 / 281. Seasonal Changes in Body Composition Clinical Research Ethics Committee, reference number (no: 2023/01, date 11.01.2023). Participant provided informed consent, with the volunteer form covering research details, risks, benefits, confidentiality, and participant rights. The research strictly adhered to the ethical principles of the Declaration of Helsinki, prioritizing participant's rights and well-being in design, procedures, and confidentiality measures.This study was designed to examine the Clinical Research Ethics Committee, reference number (no: 2023/01, date 11.01.2023). Participant provided informed consent, with the volunteer form covering research details, risks, benefits, confidentiality, and participant rights. The research strictly adhered to the ethical principles of the Declaration of Helsinki, prioritizing participant's rights and well-being in design, procedures, and confidentiality measures.This study was designed to examine the changes in body composition during the preparation and competition period (42 weeks) of a handball team competing in the super league in the 2021-2022 season in Turkey. Measurements were taken at 8 different times: preseason (T1), end of preparation period (T2), end of tournament and preparation match period (T3), end of first half (T4), end of second preparation period (T5), second half (T6), after Turkish cup at the end of second half (T7), and end of season (T8) (Table 1). Table 1. Study Design Body composition testing timeline Month August September October November December Week 1 2 3 4 1 2 3 4 5 1 1 2 3 4 1 2 3 4 1 2 3 4 5 Monday T1 TR TR TR TR TO TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR Tuesday C TR TR TR TO R TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR Wednesday C TR TR TR TO TO TR TR TR TR TR TR TR M TR TR TR TR TR TR TR TR TR Thursday C TR TR TR TO TO TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR Friday C TR TR TR TO TO TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR Saturday C TR TR T2 TO TO TR M M M M M EC TR T4 M M M M M M TR TR Sunday R R R R TO R R T3 TR R R R EC R TR R R R R R T4 R R Month January February March April May June Week 1 2 3 4 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2 3 4 1 2 Monday TR TR TR TR TR TR R R TR R R R T7 TR TR R R R R R R T8 R Tuesday TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR R R Wednesday TR TR TR TO M TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR TR R R Thursday TR TR TO TR TR TR TR TR TR TR TR TC TC TR TR TR TR TR TR TR TR R R Friday TR TR TO TO TR TR TR TR TR TR TR TC TR TR TR TR TR TR TR TR TR R R Saturday TR TR TO TR TR TR TR M TR TR TR TC TR M TR TR TR TR TR TR TR R R Sunday R T5 R T6 R M M R M M M TC R TR M M M M M M M R R T- Measurement, C-Camp, TR- Training, TO-Tournament, M-Match, EC-European cup, TC- Turkey cup Table 1. Statistical Analysis y The mean and standard deviation values of the weight, body fat percentage, fat mass, muscle mass, total body water, body mass index, and basal metabolic rate.variables were given according to the positions and repetition numbers of all players. p The Wilcoxon test was used to determine which measurement caused the difference between positions. Statistical significance was set at p < 0.05 in all cases. All analyses were performed using IBM-SPSS 21 software. Table 2. Body composition in handball players according to four playing positions at eight different points across the season (mean ± SD, n = 23) The Wilcoxon test was used to determine which measurement caused the difference between positions. Statistical significance was set at p < 0.05 in all cases. All analyses were performed using IBM-SPSS 21 software. The mean and standard deviation values of the weight, body fat percentage, fat mass, muscle mass, total body water, body mass index, and basal metabolic rate.variables were given according to the positions and repetition numbers of all players. Table 2. Study Design Body composition testing timeline The bioelectrical impedance analysis (BIA) is a frequently used method for the evaluation of body composi-tions due to its safety, speed, non- invasiveness, and relatively low cost (Özçetin et. Al., 2017). The assessment of body composition by BIA is based on the passage of a lowintensity (800 μA) and fixedfrequency (50kHz) electrical current through the body. When the electrical current is applied to the body, there is a resistance that opposes the current, called impedance. The drop in voltage between the electrodes provides a measurement of impedance. This is a vector sum of the resistance and conductance properties of body tissues, as well as a reactance (response) that arises from the resistance of cell membranes. Body water is a good conductor of electricity, and muscle clusters, like other tissues, are filled with electrolytes and water. Water offers little re- sistance to the passage of electrical current. Since body fat has a low hydration index, it has a high bioimpedance. Therefore, muscle conducts electricity more easily than fat, and the fat content can be calculated based on the difference between total weight, fatfree body mass, and fat mass (Sampei & Sigulem, 2009). p g The Tanita (BC418) body composition analyzer was used to evaluate body composition. The measurement was conducted by an expert, and all measurements were supervised and performed by the sports science and medical team of the club. Athletes were instructed to avoid alcohol and caffeine for 24 hours before the test and to avoid food and fluids for at least four to five hours before the test. They were also instructed not to engage in any physical activity 12 hours before the test. Each measurement was taken at 10:00 in the morning. Athletes were divided into groups according to their playing positions (8 setters and 4 .Page 276 / 281. Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 Seasonal Changes in Body Composition Seasonal Changes in Body Composition The normality assumptions of the variables were examined using the Kolmogorov-Smirnov test. As the variables did not come from a normal distribution, the nonparametric Two-Way Friedman Test for repeated measures was performed. goalkeepers, 7 wings and 4 pivots). Seven measurements were evaluated, including weight, body fat percentage, fat mass, muscle mass, total body water, body mass index, and basal metabolic rate. Statistical Analysis Body composition in handball players accordin the season (mean ± SD, n = 23) he season (mean ± SD, n = 23) T1: pre-season; T2: end of preparation period; T3: end of tournament and preparation match period; T4: end of first half; T5: end of second preparation period; T6: second half; T7: after Turkish cup at the end of second half; T8: end of season 7 T1 T2 T3 T4 T5 T6 T7 T8 Chi- Sq p value Weight (kg) All players 93.70 ± 10.92 93.71 ± 10.82 94.62 ± 10.66 94.68 ± 10.95 94.33 ± 10.74 94.92 ± 10.93 95.04 ± 10.45 95.04 ± 10.02 10.191 0.178 Goalkeeper 97.50 ± 11.88 97.05 ± 9.40 99.75 ± 5.30 97.95 ± 8.13 98.70 ± 8.20 101.15 ± 7.28 100.60 ± 7.50 100.20 ± 6.65 7.509 0.378 Playmaker 98.15 ± 7.55 97.58 ± 8.05 98.00 ± 8i31 99.10 ± 8.50 98.63 ± 8.90 98.60 ± 9.09 98.75 ± 9.23 98.53 ± 8.25 7.043 0.424 Wings 84.22 ± 7.81 84.58 ± 7.43 85.42 ± 8.82 85.24 ± 8.47 84.98 ± 8.13 85.06 ± 7.52 85.96 ± 7.87 86.48 ± 8.35 10.531 0.16 Pivot 104.70 ± 8.63 105.45 ± 11.24 105.70 ± 7.07 106.15 ± 7.71 104.75 ± 6.86 106.00 ± 6.65 104.75 ± 5.73 104.30 ± 6.08 4.939 0.667 Body fat Percentage (%) All players 14.95 ± 4.28 14.01 ± 4.16 14.78 ± 4.36 15.59 ± 3.8 15.38 ± 4.07 16.10 ± 4.14 16.01 ± 4.06 16.72 ± 3.75 27.53 0.000 Goalkeeper 19.20 ± 1.13 17.30 ± 0.14 19.60 ± 3.54 19.45 ± 1.06 19.80 ± 0.99 21.10 ± 1.41 21.50 ± 2.69 22.10 ± 3.25 11.275 0.127 Playmaker 14.53 ± 1.75 14.20 ± 1.91 14.08 ± 1.42 15.93 ± 1.07 15.58 ± 1.45 15.98 ± 1.20 15.43 ± 1.05 16.05 ± 1.00 8.413 0.298 Wings 11.98 ± 4.85 11.04 ± 5.07 11.70 ± 4.32 12.66 ± 4.33 12.12 ± 4.30 12.88 ± 4.26 12.86 ± 3.54 14.28 ± 3.74 13.95 0.050* Pivot 19.00 ± 1.13 17.75 ± 0.35 19.10 ± 1.27 18.40 ± 2.26 18.75 ± 1.48 19.40 ± 1.70 19.55 ± 0.64 18.80 ± 1.27 5.282 0.626 Body Fat Mass (kg) All players 14.28 ± 4.91 13.39 ± 4.65 14.24 ± 4.92 14.98 ± 4.33 14.79 ± 4.75 15.6 ± 4.96 15.47 ± 4.83 16.07 ± 4.36 30.04 0.000* Goalkeeper 18.65 ± 1.20 16.75 ± 1.48 19.45 ± 2.47 19.00 ± 0.57 19.50 ± 0.71 21.30 ± 0.14 21.55 ± 1.06 22.05 ± 1.77 11.048 0.137 Playmaker 14.30 ± 2.48 13.88 ± 2.39 13.80 ± 1.70 15.70 ± 0.86 15.45 ± 2.75 15.83 ± 2.41 15.30 ± 2.19 15.75 ± 0.73 9.839 0.198 Wings 10.26 ± 4.42 9.54 ± 4.57 10.14 ± 4.07 11.02 ± 4.16 10.46 ± 3.90 11.16 ± 4.02 11.18 ± 3.50 12.50 ± 3.75 15.737 0.028* Pivot 19.95 ± 2.76 18.70 ± 1.56 20.15 ± 0.07 19.45 ± 0.92 19.60 ± 0.28 20.55 ± 0.49 20.45 ± 0.49 19.65 ± 7.36 5.626 0.584 Muscle Mass (kg) All players 79.43 ± 7.53 80.33 ± 7.51 80.39 ± 7.75 79.71 ± 8 79.55 ± 7.3 79.34 ± 7.14 79.58 ± 7.22 78.98 ± 7.36 10.757 0.15 Goalkeeper 78.85 ± 10.68 80.30 ± 7.92 80.30 ± 7.78 78.95 ± 7.57 79.20 ± 7.50 79.85 ± 7.14 79.05 ± 8.56 78.15 ± 8.41 7.503 0.378 Playmaker 83.85 ± 5.67 83.73 ± 6.90 84.20 ± 7.53 83.40 ± 8.01 83.20 ± 6.18 82.78 ± 6.82 83.45 ± 7.26 82.78 ± 7.77 4.896 0.673 Wings 73.98 ± 6.14 75.04 ± 5.22 75.30 ± 7.03 74.24 ± 5.88 74.52 ± 6.60 73.92 ± 4.99 74.80 ± 6.07 74.00 ± 5.89 3.759 0.807 Pivot 84.80 ± 5.80 86.80 ± 9.62 85.60 ± 7.07 86.75 ± 8.70 85.15 ± 7.14 85.50 ± 7.21 84.30 ± 5.23 84.65 ± 3.61 2.19 0.949 Total Body Water All players 58.15 ± 5.52 58.81 ± 5.49 58.85 ± 5.67 58.35 ± 5.84 58.23 ± 5.35 58.08 ± 5.22 58.26 ± 5.28 57.82 ± 5.39 10.673 0.154 Goalkeeper 57.70 ± 7.78 58.80 ± 5.80 58.80 ± 5.66 57.80 ± 5.52 58.00 ± 5.52 58.50 ± 5.23 57.85 ± 6.29 57.25 ± 6.15 7.503 0.378 Playmaker 61.40 ± 4.17 61.28 ± 5.04 61.65 ± 5.49 61.05 ± 5.86 60.90 ± 4.54 60.58 ± 4.98 61.10 ± 5.31 60.60 ± 5.69 4.803 0.684 Wings 54.14 ± 4.49 54.94 ± 3.83 55.12 ± 5.16 54.36 ± 4.28 54.56 ± 4.86 54.12 ± 3.67 54.78 ± 4.45 54.16 ± 4.30 3.693 0.814 Pivot 62.10 ± 4.24 63.55 ± 7.00 62.65 ± 5.16 63.50 ± 6.36 62.30 ± 5.23 62.60 ± 5.23 61.70 ± 3.82 61.95 ± 2.62 2.506 0.927 Body Mass Index (kg/m2) All players 26.04 ± 2.04 25.98 ± 2.08 26.18 ± 1.99 26.14 ± 1.98 26.09 ± 1.89 26.27 ± 2.06 26.28 ± 1.87 26.28 ± 1.81 7.276 0.4 Goalkeeper 27.20 ± 1.70 26.95 ± 1.20 27.75 ± 0.07 27.10 ± 0.99 27.50 ± 0.85 28.10 ± 0.57 27.95 ± 0.64 28.05 ± 0.21 8.383 0.3 Playmaker 26.40 ± 1.05 26.13 ± 1.14 26.28 ± 1.22 26.50 ± 1.15 26.38 ± 1.33 26.43 ± 1.39 26.40 ± 1.37 26.28 ± 1.07 7.27 0.401 Wings 24.52 ± 1.99 24.62 ± 1.98 24.78 ± 1.99 24.74 ± 2.08 24.66 ± 1.81 24.70 ± 1.85 25.02 ± 1.98 25.16 ± 2.01 13.041 0.071 Pivot 27.95 ± 2.47 28.15 ± 3.18 27.95 ± 2.47 27.95 ± 2.47 27.70 ± 1.98 28.05 ± 2.33 27.55 ± 1.91 27.35 ± 2.19 7.286 0.4 Basal Metabolic Rate All players 2364.4±241.9 2389.1±245.77 2394.6±249.11 2376.3±259.68 2368.4±237.51 2363.6 ± 235.3 2370.6±233.73 2353.6±236.68 11.001 0.139 Goalkeeper 2342.0±39.41 2377.0±257.39 2387.0±239.00 2343.0±241.83 2352.0±240.42 2379.0±227.69 2356.0±267.29 2328.0±260.22 7.503 0.378 Playmaker 2477.2±191.13 2472.0±227.46 2487.7±246.34 2470.2±260.03 2460.2±210.85 2449.0±229.29 2469.0±243.39 2450.2±251.85 4.902 0.672 Wings 2197.2±197.42 2226.6±170.44 2238.4±227.98 2208.0±194.49 2212.8±212.66 2192.4±165.90 2221.2±197.74 2200.2±194.32 4.394 0.733 Pivot 2579.5±193.04 2642.0±315.37 2607.0±224.86 2642.5±275.06 2590.5±225.57 2605.5±226.98 2562.0± 61.22 2569.5±116.67 3.564 0.828 RESULTS Changes in body mass percentage at eight different points across the season by playing position a Significantly different from T1, b Significantly different from T2, c Significantly different from T3, d Significantly different from T4, e Significantly different from T5, f Significantly different from T6,g Significantly different from T7, h Significantly different from T8. Figure 2. Changes in body fat mass at eight different points across the season by playing position a Significantly different from T1, b Significantly different from T2, c Significantly different from T3, d Significantly different from T4, e Significantly different from T5, f Significantly different from T6,g Significantly different from T7, h Significantly different from T8. Figure 1. Changes in body mass percentage at eight different points across the season by playing position a Significantly different from T1, b Significantly different from T2, c Significantly different from T3, d Significantly different from T4, e Significantly different from T5, f Significantly different from T6,g Significantly different from T7, h Significantly different from T8. Figure 2. Changes in body fat mass at eight different points across the season by playing position a Significantly different from T1, b Significantly different from T2, c Significantly different from T3, d Significantly different from T4, e Significantly different from T5, f Significantly different from T6,g Significantly different from T7, h Significantly different from T8. Figure 2. Changes in body fat mass at eight different points across the season by playing position a Significantly different from T1, b Significantly different from T2, c Significantly different from T3, d Significantly different from T4, e Significantly different from T5, f Significantly different from T6,g Significantly different from T7, h Significantly different from T8. Figure 2. Changes in body fat mass at eight different points across the season by playing position a Significantly different from T1, b Significantly different from T2, c Significantly different from T3, d Significantly different from T4, e Significantly different from T5, f Significantly different from T6,g Significantly different from T7, h Significantly different from T8. RESULTS between all athletes and wing players based on the eight measurements (p<0.05) (Figure 1, 2). Regardless of the positions, the body fat percentage and body fat mass of all athletes significantly decreased in T2 compared to T1. Compared to T2, an increase was observed in T4, T5, T6, T7, and T8. A decrease was observed in T5 compared to T4. In T6, an increase continued compared to T1, T2, T3, and T5, and in T7, it continued compared to T1 and T2. In T8, an The average values ± SD of the body composition (weight, body fat percentage, fat mass, muscle mass, total body water, body mass index, and basal metabolic rate) arranged according to all players and game positions for the eight applied measurements are shown in Table 2. In the analysis, only differences were found in body fat percentage and body fat mass values Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 .Page 277 / 281. Seasonal Changes in Body Composition increase was observed again except for T6 and T7. Among the wing players, an increase was observed in T4 compared to T3, and a significant increase was observed in T2, T3, T5, and T7 compared to T8. Wing players had lower body fat percentage and body fat mass than all other game positions. Although it showed a decrease on average in T2 compared to T3, it was not significant. T4 showed an increase compared to T3. Similarly, in T8, it was significantly higher than T2, T3, T5, and T7. When evaluated both in all athletes and in positions, body fat percentage and body fat mass decreased significantly in the second measurement and increased significantly in the last measurement in wingers. However, in players playing in all positions, a decrease in body fat percentage, fat mass and body mass index was expected, and an increase in muscle mass, especially in the 2nd measurement and afterwards. Additionally, no significant changes were observed in weight, total body water, and basal metabolic rate measurements. Figure 1. Changes in body mass percentage at eight different points across the season by playing position a Significantly different from T1, b Significantly different from T2, c Significantly different from T3, d Significantly different from T4, e Significantly different from T5, f Significantly different from T6,g Significantly different from T7, h Significantly different from T8. Figure 1. DISCUSSION several points, including T4, T5, T6, T7 y T8. It was also observed that wing players had lower body fat percentage and body fat mass than other playing positions. Finally, no significant changes were observed in weight, muscle mass, total body water, body mass index, and basal metabolic rate measurements. The aim of this study was to examine changes in body composition of elite male handball players during preparation and competition periods (42 weeks). The main findings of these results are that during the preparation period before the season, all athletes experienced a significant decrease in body fat percentage and body fat mass. However, over the course of the season, a significant increase in body fat percentage and body fat mass was observed at As in the studies conducted by Owen et al. (2018), Lago Peñas et al. (2013) and Madic et al. (2018), it was found that there was a significant decrease in fat mass compared to preseason training start (T1) at T2 ( Owen et.al., 2018; Lago- Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 .Page 278 / 281. Seasonal Changes in Body Composition wing players had significantly lower body fat percentage and body fat mass compared to other positions, which is consistent with previous research on handball players (Martínez-Rodríguez et. al., 2020). This may be due to the specific physical demands of the wing position, which requires lighter and faster bodies to adcuire higher level of speed and capacity to change of movement rapidly (Bojić-Ćaćić et. al., 2018). Furthermore, no significant changes were observed in weight, muscle mass, total body water, body mass index, and basal metabolic rate measurements in all measurements. These findings are consistent with previous research on handball players after preseason training (Cichy et. al., 2020) and may indicate that changes in body composition during the season are primarily due to changes in body fat mass. However, there is limited distribution of body fat mass, there is not a sig-nificant change in most body composition parameters during the competition period (Milanese et. al., 2011). Penas et. al., 2013; Madic et. al., 2018). The decrease in body fat percentage and fat mass at the end of the general preparation period indicates the result of the high level of aerobic, anaerobic and strength training applied during this period Reilly (1996). DISCUSSION Although there was no significant difference in body fat percentage and fat mass at the end of the special preparation period for all players except for wing players playing in other positions, a decrease was observed in the averages. However, unlike the studies by Madic et al. (2018) and Ostojic (2003), in our study, the decrease in body mass percentage and fat mass did not continue after T2 with the competition period (Madic et. al., 2018; Ostojic, 2003). It was observed that there was an increase in body fat percentage and fat mass after T2, and this increase continued. This trend has been observed in other studies on team sport athletes Ostojic, (2003) and may be due to factors such as decreased training intensity and volume during the competitive season, increased travel, and changes in dietary habits. It is important to note that the study was conducted on a single team and may not be representative of other teams or sports. Additionally, the study only measured body composition and did not take into account other factors that may have contributed to changes in performance, such as changes in strength, power, and sprint. Further research is needed to better understand the relationship between changes in body composition and changes in performance in handball. In team sports, each game position has its own physiological characteristics due to different technical and tactical demands on the field. In handball, wing players have been measured to spend an average of 15 minutes more on the field than other players during the effective game time of 60 minutes. It has also been shown that wing players cover 1,200 meters more total distance than playmakers (2,882 m) or pivots (2,702 m), have two to three times more total sprint distance throughout the match, and have less body fat weight compared to players in other positions (Póvoas et. al., 2012; Büchel et. al., 2019; Mohoric et. al., 2022). Sibila et al. noted that goalkeepers and pivots generally have a higher height than wings, and pivots have more athleticism (more muscle mass) and a strong shot. Numerous studies have shown that wing players are fast, agile, and have a high jumping capacity with a low body mass index, and therefore generally have less weight and lower body fat percentage with the highest aerobic capacity (Schwesig et. al., 2017; Ghobadi et. al., 2013; Sporis et.al., 2010; Massuca et. REFERENCES Martínez-Rodríguez A, Martínez-Olcina M, Hernández- García M, Rubio-Arias JÁ, Sánchez-Sánchez, J., Sánchez-Sáez, JA. (2020). Body composition characteristics of handball players: Systematic review. Arch Med del Deport; 37:52–61. Bilge, M. (2012). Game Analysis of Olympic, World and European Championships in Men’s Handball. J Hum Kinet; 35:109–18. [PubMed] Bilsborough, JC., Greenway, K., Livingston, S., Cordy, J., Coutts, AJ. (2016). Changes in Anthropometry, Upper-Body Strength, and Nutrient Intake in Professional Australian Football Players During a Season. Int J Sports Physiol Perform; 11:290–300. [PubMed] Martínez-Rodríguez, A., Martínez-Olcina, M., Hernández- García, M., Rubio-Arias, JÁ., Sánchez-Sánchez, J., Lara-Cobos, D., et al. (2021). Mediterranean Diet Adherence, Body Composition and Performance in Beach Handball Players: A Cross Sectional Study. Int J Environ Res Public Health; 18. [PubMed] Bojić-Ćaćić, L., Vuleta, D., Milanović, D. (2018). Position- related differences in morphological characteristics of U14 female handball players, Kinesiology; 50:235– 42. [CrossRef] Massuca, L., Branco, B., Miarka, B., Fragoso, I. (2015). Physical Fitness Attributes of Team-Handball Players are Related to Playing Position and Performance Level. Asian J Sports Med; e24712. [PubMed] Büchel, D., Jakobsmeyer, R., Döring, M., Adams, M., Rückert, U., Baumeister, J. (2019). Effect of playing position and time on-court on activity profiles in german elite team handball. Int J Perform Anal Sport; 19:832–44. [CrossRef] Michalsik, LB., Madsen, K., Aagaard, P. (2015). Technical match characteristics and influence of body anthropometry on playing performance in male elite team handball. J strength Cond Res; 2015;29:416–28. [PubMed] Cavedon, V., Zancanaro, C., Milanese, C. (2018). Anthropometric prediction of DXA-measured body composition in female team handball players. PeerJ; 6:e5913. [PubMed] Milanese, C., Piscitelli, F., Lampis, C., Zancanaro, C. (2011). Anthropometry and body composition of female handball players according to competitive level or the playing position. J Sports Sci; 29:1301–9. [PubMed] Cichy, I., Dudkowski, A., Kociuba, M., Ignasiak, Z., Sebastjan, A., Kochan, K., et al. (2020). Sex Differences in Body Composition Changes after Preseason Training in Elite Handball Players. Int J Environ Res Public Health; 17:3880. [PubMed] Mohoric, U., Abazovic, E., Paravlic, AH. (2022). Morphological and Physical Performance-Related Characteristics of Elite Male Handball Players: The Influence of Age and Playing Position. Appl Sci; 12:11894. [CrossRef] Fieseler, G., Hermassi, S., Hoffmeyer, B., Schulze, S., Irlenbusch, L., Bartels, T., et al. (2017). Differences in anthropometric characteristics in relation to throwing velocity and competitive level in professional male team handball: a tool for talent profiling. J Sports Med Phys Fitness; 57:985–92. [P bM d] Ostojic, SM. (2003). DISCUSSION al., 2015), while pivots are among the longest, heaviest, and slowest players Mohoric et. al., (2022), consisting of strong players constantly in contact and fighting with opponents (Sibila & Pori, 2009). These findings are in line with the results of our study, As a result, unexpected results were obtained in our study, except for T2. An increase in body fat percentage and fat mass was observed during the competition period, and this increase continued. The increase in fat mass during the competition period could be attributed to the different training content and higher training intensity during the preparation period, changes in nutrition habits due to camps, long trips, and tournaments, as well as the effects of fatigue and stress. Further research is needed on these issues. Examining body composition is important in sports such as handball, where body weight needs to be moved against gravity. In this study, norms were established for the body composition status of elite male handball players during the preparation and competition periods, and changes in specific periods were monitored. It was observed that handball players improved their body composition during the preparation period. .Page 279 / 281. Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 Seasonal Changes in Body Composition Ghobadi, H., Rajabi, H., Farzad, B., Bayati, M., Jeffreys, I. (2013). Anthropometry of World-Class Elite Handball Players According to the Playing Position: Reports From Men’s Handball World Championship 2013. J Hum Kinet; 39:213–20. [PubMed] The increase in body fat percentage and fat mass during the competition process and at the end of the season showed that the transition period after the season should be well evaluated. Conflict of interest Granados, C., Izquierdo, M., Ibáñez, J., Ruesta, M., Gorostiaga, EM. (2013). Are there any differences in physical fitness and throwing velocity between national and international elite female handball players? J strength Cond Res; 27:723–32. [PubMed] No conflict of interest is declared by the authors. In addition, no financial support wasreceived. Ethics Statements Karcher, C., Buchheit, M. (2014). On-court demands of elite handball, with special reference to playing positions. Sports Med; 44:797–814. [PubMed] This study followed ethical standards and received approval from the Zonguldak Bülen Ecevit University Non-invasive Clinical Research Ethics Committee, reference number (no: 2023/01, date 11.01.2023). Lago-Penas, C., Rey, E., Lago-Ballesteros, J., Dominguez E, Casais L. (2013). Seasonal variations in body composition and fitness parameters according to individual percentage of training completion in professional soccer players. Int Sport J; 14:205–15. Author Contributions Lidor, R., Falk, B., Arnon, M, Cohen, Y., Segal, G., Lander, Y. (2005). Measurement of talent in team handball: the questionable use of motor and physical tests. J strength Cond Res; 19:318–25. [PubMed] Study Design, Data Collection, Statistical Analysis, Data Interpretation, Article Preparation, Literature Review processes were carried out by the Author. The author has read and accepted the published version of the article. Study Design, Data Collection, Statistical Analysis, Data Interpretation, Article Preparation, Literature Review processes were carried out by the Author. The author has read and accepted the published version of the article. Madic, DM., Andrasic, S., Gusic, M., Molnar, S., Radanovic, D., Trajkovic, N. (2018). Seasonal body composition variations in adolescent soccer players. Int J Morphol; 36:877–80. [CrossRef] REFERENCES Seasonal Alterations in Body Composition and Sprint Performance of Elitesoccer Players. J Exerc Physiol Online; 6:24–7. Owen, AL., Lago-Peñas, C., Dunlop, G., Mehdi, R., Chtara, M., Dellal, A. (2018). Seasonal Body Composition Variation Amongst Elite European Professional p g [PubMed] Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 .Page 280 / 281. Seasonal Changes in Body Composition Soccer Players: An Approach of Talent Identification. J Hum Kinet; 62:177–84. [PubMed] Özçetin, M., Khalilova, F., Kılıç, A. (2017). An unusual method for assessing nutritional status: BIA. Journal of Children; 17:61–6. [CrossRef] Póvoas, SCA., Seabra, AFT., Ascensão, AAMR., Magalhães, J., Soares, JMC., Rebelo, ANC. (2012). Physical and physiological demands of elite team handball. J strength Cond Res; 26:3365–75. [PubMed] Reilly, T. (1996). Science and Soccer. London, U.K.: E&FN. Rousanoglou, EN., Noutsos, KS., Bayios, IA. (2014). Playing level and playing position differences of anthropometric and physical fitness characteristics in elite junior handball players. J Sports Med Phys Fitness; 54:611–21. [PubMed] Rybakova, E., Shutova, T., Vysotskaya, T. (2020). Sports training of ski jumpers from a springboard based on body composition control and physical fitness. J Phys Educ Sport; 20:752–8. Sampei, MA., Sigulem, DM. (2009). Field methods in the evaluation of obesity in children and adolescents. Rev Bras Saúde Matern Infant; 9:21–9. [CrossRef] Schwesig, R., Hermassi, S., Fieseler, G., Irlenbusch, L., Noack, F., Delank, KS, et al. (2017). Anthropometric and physical performance characteristics of professional handball players: influence of playing position. J Sports Med Phys Fitness; 57:1471–8. [PubMed] Sibila, M., Pori, P. (2009). Position-related differences in selectedmorphological body characteristics of top-level handball players. Coll Antropol; 33:1079–86. [PubMed] Singh, NN., Singh, RR., Singh, SK. (2010). Study of trunk flexibility and body composition between football and badminton players. J Phys Educ Spor; 2011;11:18. Sporis, G., Vuleta, D., Vuleta, D., Milanović, D. (2010). Fitness profiling in handball: physical and physiological characteristics of elite players. Coll Antropol; 34:1009–14. [PubMed] Sundgot-Borgen, J., Meyer, NL., Lohman, TG., Ackland, TR., Maughan, RJ., Stewart, AD., et al. (2013). How to minimise the health risks to athletes who compete in weight-sensitive sports review and position statement on behalf of the Ad Hoc Research Working Group on Body Composition, Health and Performance, under the auspices of the IOC Medical Commission. Br J Sports Med; 47:1012–22. [PubMed] This work is distributed under https://creativecommons.org/licenses/by-sa/4.0/ .Page 281 / 281. Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281 Eler, N., Int J Disabil Sports Health Sci, 2024;7(2);274-281
https://openalex.org/W2076395508	https://europepmc.org/articles/pmc3032782?pdf=render	English	null	Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence	PloS one	2,011	cc-by	7,861	Abstract Most viruses express during infection products that prevent or neutralize the effect of the host dsRNA activated protein kinase (PKR). Translation of Sindbis virus (SINV) mRNA escapes to PKR activation and eIF2 phosphorylation in infected cells by a mechanism that requires a stem loop structure in viral 26S mRNA termed DLP to initiate translation in the absence of functional eIF2. Unlike the rest of viruses tested, we found that Alphavirus infection allowed a strong PKR activation and eIF2a phosphorylation in vitro and in infected animals so that the presence of DLP structure in mRNA was critical for translation and replication of SINV. Interestingly, infection of MEFs with some viruses that express PKR inhibitors prevented eIF2a phosphorylation after superinfection with SINV, suggesting that viral anti-PKR mechanisms could be exchangeable. Thus, translation of SINV mutant lacking the DLP structure (DDLP) in 26S mRNA was partially rescued in cells expressing vaccinia virus (VV) E3 protein, a known inhibitor of PKR. This case of heterotypic complementation among evolutionary distant viruses confirmed experimentally a remarkable case of convergent evolution in viral anti-PKR mechanisms. Our data reinforce the critical role of PKR in regulating virus-host interaction and reveal the versatility of viruses to find different solutions to solve the same conflict. Citation: Domingo-Gil E, Toribio R, Na´jera JL, Esteban M, Ventoso I (2011) Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence. PLoS ONE 6(2): e16711. doi:10.1371/journal.pone.0016711 Editor: Jean-Pierre Vartanian, Institut Pasteur, France Citation: Domingo-Gil E, Toribio R, Na´jera JL, Esteban M, Ventoso I (2011) Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence. PLoS ONE 6(2): e16711. doi:10.1371/journal.pone.0016711 di Pi i i P Editor: Jean-Pierre Vartanian, Institut Pasteur, France Received August 12, 2010; Accepted January 11, 2011; Published February 2, 2011 Received August 12, 2010; Accepted January 11, 2011 mingo-Gil et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Domingo-Gil et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Diversity in Viral Anti-PKR Mechanisms: A Remarkable Case of Evolutionary Convergence Elena Domingo-Gil1., Rene´ Toribio1., Jose´ Luis Na´jera2, Mariano Esteban2, Iva´n Ventoso1* 1 Departamento de Biologı´a Molecular and Centro de Biologı´a Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Universidad Auto´noma de Madrid, Madrid, Spain, 2 Centro Nacional de Biotecnologı´a (CSIC), Campus Universidad Auto´noma de Madrid, Madrid, Spain Abstract Funding: This work was supported in part from the VIRUS-HOST interaction programme (Comunidad de Madrid) and by grants from the Ministerio de Ciencia e Innovacio´n (SAF2006-09810; SAF2008-02036) and the Fundacio´n Mutua Madrilen˜a (FMM 2008). The institutional support from Fundacio´n Ramo´n Areces is also acknowledged. E.D was a recipient of VIRUS-HOST programme postdoctoral contract. R.T. was a recipient of the SAF2006-09810 contract and I.V. was a researcher of Ramo´n y Cajal Programme. The elaboration of this work took about 2 years and the estimated cost was 3000 euros excluding salaries. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: iventoso@cbm.uam.es . These authors contributed equally to this work. . These authors contributed equally to this work. precedes the eventual secretion of IFN. PKR is present at basal levels in most of mammalian tissues, but its amount increases after priming cells with IFNa/b [15,16]. PKR binds dsRNA molecules generated during the replication of RNA viruses as well as in some transcripts from DNA viruses, leading to the activation of the kinase by a sequential wave of autophosphorylation events [17,18,19,20]. Activated PKR phosphorylates and inactivates eukaryotic initiation factor 2 (eIF2), the only well-described substrate of the kinase that is also phosphorylated by other members of eIF2-kinase family in vertebrates [17,21]. As result of this, the general translation is rapidly inhibited in an attempt of the infected cells to block viral translation and abort virus spreading [16,22]. However, as in other pathways of innate response, viruses have evolved a variety of strategies to prevent or overcome the activation of PKR in infected cells (reviewed in [12]). Among these mechanisms, the most frequent found are viral products that prevent the activation of PKR by sequestering its activator (dsRNA), by direct binding to the regulatory element of the kinase or by expressing a pseudosubstrate that competes with eIF2 for binding to the kinase (see figure 1). Other viruses such as poliovirus and Rift Valley virus induce the degradation of PKR by a mechanism that has not been well characterized yet [23,24]. In some cases such as Herpes virus-infected cells, eIF2 phosphory- lation is rapidly reversed by the action of viral phosphatases that PLoS ONE \| www.plosone.org Introduction During virus-host coevolution, the acquisition of an antiviral pathway by the host was generally followed by the appearance of a viral countermeasure so that the current host-parasite interactions are keeping on a dynamic equilibrium (the Red Queen principle) [1,2,3]. A paradigmatic example of these complex interactions are the different mechanisms that viruses have evolved to evade or subvert the antiviral effect of interferons (IFNs) and other pro- inflammatory cytokines that are secreted by vertebrate cells in response to virus and other pathogens [4,5,6,7,8,9]. Thus, viruses express products that impair the detection of viral proteins or nucleic acids by host pattern-recognition receptors (PRRs; Toll- like receptors and RIG-I-like receptors), block the signaling pathways that lead to the synthesis of IFNa/b and other cytokines, or prevent the activation of some IFN-stimulated genes such as dsRNA-activated kinase (PKR) [6,9,10,11,12,13,14]. Moreover, in some cases such as poxvirus or HCV, one or more viral product can interfere with IFN secretion or signalling at multiple points, ensuring a more efficient viral escape to innate immunity of the host [6,9]. Among innate antiviral mechanisms of vertebrates, PKR activation constitutes one of the first line of antiviral defense acting at the immediate-early phase of virus replication that PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16711 1 are expressed along the infection [25,26,27]. For Alphavirus, the strategy is markedly different; PKR is strongly activated upon infection with SINV and Semliki Forest (SFV) viruses so that eIF2 The existence of these different mechanism stress the critical role of PKR in regulating host-virus interaction and show a remarkable example of convergent evolution to generate different Figure 1. Current known viral strategies to prevent or counteract PKR activation can be grouped into three categories. A) First, viruses can prevent the activation of PKR by expressing proteins or RNAs that bind the kinase in an inhibitory manner, by sequestering dsRNA activator molecules or by inducing the degradation of PKR. Examples of this group are vaccinia E3 and K3, influenza NS1 and Adenovirus VAI RNAs and Rift Valley virus NS1 protein. In the second category are viruses that promote eIF2a dephosphorylation such as Herpes virus c34.5 protein which acts as a regulatory subunit of cellular protein phosphatase 1a (PP1). Herpesvirus also express products from the first group (Us11 gene, a PKR inhibitor). Introduction In the third category, some viruses do not prevent PKR-mediated eIF2a phosphorylation, but viral mRNA can initiate translation in an eIF2-independent manner by means of a RNA structure (DLP) that stalls the scanning 40S ribosome on the initiation codon. To date, this strategy has been found only in Alphavirus. B) Secondary structure prediction of the 59extreme of 26S mRNAs of SINV and SFV. The stability of DLP structure (DGu) is expressed in kcal. mol21. Arrows show the initiation codon. Circled nucleotides in SINV were mutated to adenines in DDLP mutant virus as described previously [29]. doi:10.1371/journal.pone.0016711.g001 Diversity in Viral Anti-PKR Mechanisms Diversity in Viral Anti-PKR Mechanisms Figure 1. Current known viral strategies to prevent or counteract PKR activation can be grouped into three categories. A) First, viruses can prevent the activation of PKR by expressing proteins or RNAs that bind the kinase in an inhibitory manner, by sequestering dsRNA activator molecules or by inducing the degradation of PKR. Examples of this group are vaccinia E3 and K3, influenza NS1 and Adenovirus VAI RNAs and Rift Valley virus NS1 protein. In the second category are viruses that promote eIF2a dephosphorylation such as Herpes virus c34.5 protein which acts as a regulatory subunit of cellular protein phosphatase 1a (PP1). Herpesvirus also express products from the first group (Us11 gene, a PKR inhibitor). In the third category, some viruses do not prevent PKR-mediated eIF2a phosphorylation, but viral mRNA can initiate translation in an eIF2-independent manner by means of a RNA structure (DLP) that stalls the scanning 40S ribosome on the initiation codon. To date, this strategy has been found only in Alphavirus. B) Secondary structure prediction of the 59extreme of 26S mRNAs of SINV and SFV. The stability of DLP structure (DGu) is expressed in kcal. mol21. Arrows show the initiation codon. Circled nucleotides in SINV were mutated to adenines in DDLP mutant virus as described previously [29]. doi:10.1371/journal.pone.0016711.g001 Figure 1. Current known viral strategies to prevent or counteract PKR activation can be grouped into three categories. A) First, viruses can prevent the activation of PKR by expressing proteins or RNAs that bind the kinase in an inhibitory manner, by sequestering dsRNA activator molecules or by inducing the degradation of PKR. Examples of this group are vaccinia E3 and K3, influenza NS1 and Adenovirus VAI RNAs and Rift Valley virus NS1 protein. Introduction In the second category are viruses that promote eIF2a dephosphorylation such as Herpes virus c34.5 protein which acts as a regulatory subunit of cellular protein phosphatase 1a (PP1). Herpesvirus also express products from the first group (Us11 gene, a PKR inhibitor). In the third category, some viruses do not prevent PKR-mediated eIF2a phosphorylation, but viral mRNA can initiate translation in an eIF2-independent manner by means of a RNA structure (DLP) that stalls the scanning 40S ribosome on the initiation codon. To date, this strategy has been found only in Alphavirus. B) Secondary structure prediction of the 59extreme of 26S mRNAs of SINV and SFV. The stability of DLP structure (DGu) is expressed in kcal. mol21. Arrows show the initiation codon. Circled nucleotides in SINV were mutated to adenines in DDLP mutant virus as described previously [29]. doi:10.1371/journal.pone.0016711.g001 The existence of these different mechanism stress the critical role of PKR in regulating host-virus interaction and show a remarkable example of convergent evolution to generate different patterns of molecular mimicry [8]. For example, VV K3L mimics eIF2a acting as a competitive inhibitor of PKR, whereas Adenovirus VAI RNAs mimics dsRNA activators of PKR by binding the kinase in an inhibitory manner [8,12]. Fingerprints of this antagonist coevolution has been found recently in the are expressed along the infection [25,26,27]. For Alphavirus, the strategy is markedly different; PKR is strongly activated upon infection with SINV and Semliki Forest (SFV) viruses, so that eIF2 factor is completely phosphorylated [28,29]. Translation of viral subgenomic mRNAs (26S), however, resists due to the presence of a prominent hairpin loop structure in mRNA located downstream of the initiation codon (DLP) that allows the 40S ribosome to initiate in the absence of eIF2 [29]. February 2011 \| Volume 6 \| Issue 2 \| e16711 February 2011 \| Volume 6 \| Issue 2 \| e16711 PLoS ONE \| www.plosone.org 2 Diversity in Viral Anti-PKR Mechanisms accelerated rates of positive evolution found in PKR gene for defeating viral anti-PKR products such as poxvirus E3 and K3 [30,31]. However, an important issue that has not been addressed to date is whether these different anti-PKR mechanisms might be exchanged between non related viruses to get a functional complementation. In this work we show that a cis-acting RNA structure of Alphaviruses responsible for counteracting PKR activation can be replaced by a viral protein such as vaccinia E3. [25,36,37,38]. Alphavirus are unique in allowing PKR activation in cultured cells and infected animals We and others have reported before that infection of 3T3 fibroblasts with SINV virus triggered the complete phosphoryla- tion of eIF2 due to a strong PKR activation [28,29] (Figure 2). We wanted to test whether such phosphorylation of host eIF2a was also detected in cells infected with other Alphaviruses and with members of other unrelated families of RNA viruses. For this, we infected cells with SFV (Alphavirus), VSV (Rhabdovirus), Influenza A (Orthomyxovirus) and EMCV (Picornavirus) (Figure 2A). Equivalent or even stronger PKR activation was observed in cells infected with SFV as judged by the mobility shift of PKR band in SDS-PAGE that was indicative of autopho- sphorylation associated to kinase activation (Figure 2B). As for SINV, activation of PKR in SFV-infected cells induced a strong phosphorylation of eIF2a that was not observed in the rest of viruses analyzed with the exception of VSV, where a slight increase in eIF2 phosphorylation was observed as reported before [16]. The use of PKR gene knock-out cells (PKRo/o) showed that eIF2 phosphorylation in Alphavirus-infected cells was almost completely attributable to PKR activation as described before (Figure 2B and [29]. With the exception of Alphavirus, the rest of RNA viruses tested here did not trigger a substantial activation of eIF2 kinases in infected cells. Rescue of SINV DDLP translation by poxvirus E3 protein In a first attempt to test whether anti-PKR mechanisms could be exchangeable among viruses, we analyzed the phosphorylation state of eIF2 in SINV-infected cells that had been first infected with low doses of some RNA (Influenza A, VSV and EMVC) and DNA (VV) viruses (Figure 4A). Time-lag between infections was set to allow the simultaneous expression of products from both viruses. Thus, the previous infection with the indicated viruses did not prevent the replication and accumulation of SINV proteins in doubly infected cells (Figure 4A). Interestingly, pre-infection with VV and EMCV viruses completely prevented eIF2 phosphoryla- tion triggered by SINV superinfection. However, other viruses such as VSV were unable to prevent PKR activation after superinfection with SINV, whereas Influenza A infection exerted only a moderate preventive effect (,50% reduction). In this experiment we also included a VV mutant lacking the E3L gene, the main PKR inhibitor in VV-infected cells [39,40,41]. Notably, mutant VV-DE3L was unable to prevent eIF2a phosphorylation after SINV superinfection, showing that the inhibitory potential of VV on PKR is largely attributable to the action of E3 protein. Discussion Adaptation of non-related parasites to the same host or ecological niche is generally associated to events of convergent evolution (homoplasy), where analogous genetic elements arise during the acquisition of the same biological trait [8,43]. A paradigmatic example of this is the plethora of viral mechanisms to counteract the innate response of vertebrates (reviewed in [6]). Collectively, viruses have acquired mechanisms to subvert or counteract virtually all the biochemical routes of the host that lead into the production of IFN and other antiviral cytokines (reviewed in [6,8]). Among them, the appearance of mechanisms to prevent or counteract PKR activation likely increased the replication capabilities of viruses during their adaptation to vertebrate hosts. Alphavirus are unique in allowing PKR activation in cultured cells and infected animals This trans-inhibitory activity of E3 opened the possibility to rescue translation of SINV DDLP mutant by expressing VV E3L gene in MEF cells. For this, we used a previous established MEF line that expressed the VV E3 protein in an inducible manner after tetracycline withdrawal [42]. As is shown is Figure 4B, induction of E3L gene partially prevented eIF2 phosphorylation after SINV infection. Moreover, mobility shift of PKR in SDS-PAGE was prevented by expression of E3 protein, showing that this viral product targeted PKR to block its activation. Notably, expression of E3 protein partially restored translation of DDLP virus mRNA that was strongly inhibited due to eIF2 phosphorylation in non induced cells. This allowed a ,50 fold increase of DDLP virus yield in induced cells, showing that a deficiency in a cis-acting RNA structure involved in translation can be complemented by an unrelated product but that participates in the same biological trait. Next, we analyzed whether eIF2a phosphorylation was also detected in animals infected with Alphavirus. For this, mice were infected with SINV by the intranasal route, and 4 days later brains were subjected to immunofluorescence (IF) analysis. As described previously, SINV showed a marked tropism for cortical and spinal cord neurons [32,33,34,35]. Groups of neurons expressing viral antigens were easily detected in pyriform, motor and somatosen- sorial areas of brain cortex as well in hippocampus. Notably, the vast majority of these infected cells showed a strong staining of phospho eIF2 (Figure 3A), with no signal detected outside of areas of viral replication. Clearly, no eIF2 phosphorylation was detected in infected neurons of PKRo/o mice, showing that as occurred in vitro PKR was responsible for eIF2 phosphorylation in response to infection of animals (Figure 3A). We extended this in vivo analysis to other viruses endowed with anti-PKR mechanisms such as VV. Clearly, no eIF2 phosphorylation was detected in spleens of mice infected with VV despite the efficient replication detected in this organ (Figure 3B). The absence of eIF2 phosphorylation in mice infected with VV was not attributable to a low expression of the kinase in spleen, since a comparable expression of PKR was detected in all murine organs analyzed (Figure 3C). Taken together, our results show that PKR-induced eIF2 phosphoryla- tion seems to be a specific feature of animals infected with Alphavirus. Introduction To test whether translational resistance of SINV virus to PKR activation was important for replication in vivo, we compared the replication of wild type virus and a SINV mutant lacking the DLP structure (DDLP) in 26S mRNA. This structure in RNA has been reported before to be essential for translation and virus replication in 3T3 cells [29]. Clearly, replication of DDLP virus was greatly hampered in wild type, but not in PKRo/o mice (Figure 3D). About 3–4 log reduction in viral yield of DDLP virus were found in PKR+/+ mice (wild type) when compared to PKRo/o counterparts, whereas wild type viruses replicated at similar levels in both animal types. This result shows that as predicted from other viruses, PKR resistance in Alphavirus is essential for efficient replication in vivo. Resistance to PKR activation is critical for replication of Alphavirus in animals Adjacent sections were incubated with anti-SINV and anti-phosphoeIF2a antibodies as described in Materials and Methods. B) IF analysis of spleens from mice infected with VV-Luc at 1dpi. Sections were incubated with anti- VVp14 (reactive against the envelope protein A27) and anti-phosphoeIF2a antibodies. Note that spleen cells expressing viral antigens did not react with anti- phosphoeIF2a antibodies. C) Expression of PKR in different mouse organs from uninfected animals. Equivalent amounts of protein extracts were analyzed by immunoblot against PKR, total eIF2a and b-actin. D) Attenuation of DDLP mutant virus in wild type, but not in PKRo/o mice. Animals were inoculated with 107 of WT and 26107 of DDLP mutant viruses. Viral yields in mouse brains at 4 dpi were titrated by plaque assay. Results are the mean from 10 animals inoculated for each group in three independent experiments. SD from each group is also showed. doi:10.1371/journal.pone.0016711.g003 Figure 3. eIF2a phosphorylation and translational resistance of SINV virus also operates in infected animals. A) IF analysis of brains from wild type and PKR knock-out mice infected with SINV at 4 dpi. Adjacent sections were incubated with anti-SINV and anti-phosphoeIF2a antibodies as described in Materials and Methods. B) IF analysis of spleens from mice infected with VV-Luc at 1dpi. Sections were incubated with anti- VVp14 (reactive against the envelope protein A27) and anti-phosphoeIF2a antibodies. Note that spleen cells expressing viral antigens did not react with anti- phosphoeIF2a antibodies. C) Expression of PKR in different mouse organs from uninfected animals. Equivalent amounts of protein extracts were analyzed by immunoblot against PKR, total eIF2a and b-actin. D) Attenuation of DDLP mutant virus in wild type, but not in PKRo/o mice. Animals were inoculated with 107 of WT and 26107 of DDLP mutant viruses. Viral yields in mouse brains at 4 dpi were titrated by plaque assay. Results are the mean from 10 animals inoculated for each group in three independent experiments. SD from each group is also showed. doi:10.1371/journal.pone.0016711.g003 Thus, the type of anti-PKR mechanism found in a given virus seems to be greatly influenced by the genomic organization and the natural history of the virus during the adaptation to vertebrate hosts. Thus, the singular mechanism of PKR resistance found in Alphavirus probably reflects a peculiar evolutionary adaptation to vertebrates from arthropods (mainly mosquitoes), the vectors that Alphavirus and other Arbovirus use for transmission [35,44,45]. Resistance to PKR activation is critical for replication of Alphavirus in animals Viral anti-PKR products commonly act as virulence factors necesary for efficient viral replication and pathogenesis in animals PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16711 February 2011 \| Volume 6 \| Issue 2 \| e16711 3 Diversity in Viral Anti-PKR Mechan Diversity in Viral Anti-PKR Mechanisms Diversity in Viral An Figure 2. Alphaviruses are unique in allowing PKR activation and eIF2a phosphorylation in infected cells. A) Susceptibility of MEFs to viruses used in this study. Cells were infected with the indicated viruses to a moi of 25 pfu/cell and 6 h later pulsed with [35S]-Met for 30 min. Labeled proteins were resolved by SDS-PAGE followed of autoradiography. B) PKR activation and eIF2a phosphorylation in wild type or PKRo/o MEFs infected with the indicated viruses at 4 and 6 hpi. Note the mobility shift of PKR band upon activation in SINV and SFV-infected cells (upper panel). eIF2a phosphorylation in wild type (+/+) and PKR knock-out cells (o/o) infected with the indicated viruses. Only Alphavirus-infected cultures showed a strong eIF2a phosphorylation. For VSV-infected cells, a slight increase in eIF2a phosphorylation was also observed. doi:10.1371/journal.pone.0016711.g002 PLoS ONE \| www.plosone.org 4 February 2011 \| Volume 6 \| Issue 2 \| e16711 Figure 2. Alphaviruses are unique in allowing PKR activation and eIF2a phosphorylation in infected cells. A) Susceptibility of MEFs to viruses used in this study. Cells were infected with the indicated viruses to a moi of 25 pfu/cell and 6 h later pulsed with [35S]-Met for 30 min. Labeled proteins were resolved by SDS-PAGE followed of autoradiography. B) PKR activation and eIF2a phosphorylation in wild type or PKRo/o MEFs infected with the indicated viruses at 4 and 6 hpi. Note the mobility shift of PKR band upon activation in SINV and SFV-infected cells (upper panel). eIF2a phosphorylation in wild type (+/+) and PKR knock-out cells (o/o) infected with the indicated viruses. Only Alphavirus-infected cultures showed a strong eIF2a phosphorylation. For VSV-infected cells, a slight increase in eIF2a phosphorylation was also observed. doi:10.1371/journal.pone.0016711.g002 February 2011 \| Volume 6 \| Issue 2 \| e16711 PLoS ONE \| www.plosone.org 4 Diversity in Viral Anti-PKR Mechanisms Figure 3. eIF2a phosphorylation and translational resistance of SINV virus also operates in infected animals. A) IF analysis of brains from wild type and PKR knock-out mice infected with SINV at 4 dpi. Resistance to PKR activation is critical for replication of Alphavirus in animals Three hours later, cells were superinfected with SINV at moi of 25 pfu/cell and 5 h later lysed in sample buffer for immunoblot analysis against SINV capsid (SINV C, upper panel) and anti-phosphoeIF2a (bottom panel). B) Expression of VV E3L gene rescued translation of DDLP SINV mutant. MEF-E3L cells were induced for the expression of VV E3L by tetracycline withdrawal and infected with SINV-WT or SINV-DDLP mutant. Five hours later, cells were labeled with [35S]-Met+Cys for 30 min and analyzed by SDS-PAGE followed of autoradiography and by immunoblot against anti-E3, anti-PKR and anti-phospho eIF2a. Parallel cultures were infected at moi of 5 pfu/cell and viral yields were determined by plaque assay 2 days later. Data are the mean 6SD from three and two independent experiments in PKR+/+ and PKRo/o cells, respectively. doi:10.1371/journal.pone.0016711.g004 Figure 4. Anti-PKR mechanisms can be exchanged between viruses. A) Previous infection with some viruses prevented eIF2a Figure 4. Anti-PKR mechanisms can be exchanged between viruses. A) Previous infection with some viruses prevented eIF2a phosphorylation after SINV virus superinfection. The protocol followed for mixed infections is outlined. Wild type MEFs were infected with the indicated viruses at a moi of 2 pfu/cell. Three hours later, cells were superinfected with SINV at moi of 25 pfu/cell and 5 h later lysed in sample buffer for immunoblot analysis against SINV capsid (SINV C, upper panel) and anti-phosphoeIF2a (bottom panel). B) Expression of VV E3L gene rescued translation of DDLP SINV mutant. MEF-E3L cells were induced for the expression of VV E3L by tetracycline withdrawal and infected with SINV-WT or SINV-DDLP mutant. Five hours later, cells were labeled with [35S]-Met+Cys for 30 min and analyzed by SDS-PAGE followed of autoradiography and by immunoblot against anti-E3, anti-PKR and anti-phospho eIF2a. Parallel cultures were infected at moi of 5 pfu/cell and viral yields were determined by plaque assay 2 days later. Data are the mean 6SD from three and two independent experiments in PKR+/+ and PKRo/o cells, respectively. doi:10.1371/journal.pone.0016711.g004 levels in PKRo/o mice where no eIF2a phosphorylation in infected neurons was detected. This result showed that cis-acting DLP structure in SINV 26S mRNA is only required to overcome the effect of eIF2a phosphorylation on translation of viral mRNA, in contrast with other scenarios such as Influenza NS1 or VV E3 proteins whose important functions extend beyond to PKR inhibition [36,38,42,50]. Resistance to PKR activation is critical for replication of Alphavirus in animals Moreover, the appearance of cis-acting RNA structures such as DLP to counteract PKR-mediated eIF2a phosphorylation was probably limited to simple monocistronic RNA virus such as Alphavirus, whereas in polycistronic RNA viruses (e.g. Influenza A) and in complex DNA viruses (e.g. Poxvirus) the acquisition of trans-acting PKR inhibitors allowed viral mRNAs to be collectively translated in infected cells. Although being functional equivalent, viral anti-PKR mechanisms that prevent or allow PKR activation could have different biological consequences. The strong eIF2a phosphorylation found in neurons of SINV-infected mice is enough to completely block translation of non-viral mRNAs throughout the infection [46], that probably leads to a rapid and irreversible death of infected cells. This early shut-off of host translation by eIF2a phosphorylation in SINV-infected cells could also suppress the synthesis of IFN and ISG as described recently for HCV infection [47]. This possibility agrees well with previous findings showing that infection with many Alphaviruses prevented the secretion of IFNa/b in infected cultured cells and animals, despite SINV showed an exquisite sensitivity to IFNa/b when cells were primed with this cytokine before infection [32,48,49]. For other viruses such as poxviruses, however, preventing PKR activation during in vivo replication could maintain protein synthesis in infected cells for more time, allowing the virus to express the late genes required for the culmination of the cycle [7,36]. In this case, viruses had to further acquire a more specific mechanism to prevent the synthesis and secretion of interferons by blocking activation of NFkB or IRF7 factors [6,9]. Our results show that, in addition to VV, EMCV expresses a trans-acting inhibitor that prevented the activation of PKR in response to SINV superinfection. However, unlike to that found in poliovirus-infected human cells, anti-PKR activity of EMCV Our results show that, in addition to VV, EMCV expresses a trans-acting inhibitor that prevented the activation of PKR in response to SINV superinfection. However, unlike to that found in poliovirus-infected human cells, anti-PKR activity of EMCV February 2011 \| Volume 6 \| Issue 2 \| e16711 PLoS ONE \| www.plosone.org 5 Diversity in Viral Anti-PKR Mechanisms Figure 4. Anti-PKR mechanisms can be exchanged between viruses. A) Previous infection with some viruses prevented eIF2a phosphorylation after SINV virus superinfection. The protocol followed for mixed infections is outlined. Wild type MEFs were infected with the indicated viruses at a moi of 2 pfu/cell. Resistance to PKR activation is critical for replication of Alphavirus in animals At least for SINV virus, our results demonstrate that resistance to PKR is exerted exclusively at translational level and that probably only involves overcoming eIF2 phosphorylation. In our opinion this is a remarkable point because the existence of other substrates of PKR apart of eIF2a has been suggested before [51,52] despite the exquisite catalytic specificity that PKR shows for eIF2 [17,21]. does not seem to involve degradation of the kinase since no alteration in the steady state of PKR was found in EMCV- infected MEFs (Figure 2). For VSV, the absence of effect on SINV-triggered eIF2a phosphorylation was expected in part given the exquisite sensitivity that VSV showed to PKR activation and eIF2a phosphorylation. Thus, VSV seems to lack of an autonomous anti-PKR activity as suggested from previous results [16], suggesting that this virus could hide the replicative forms of dsRNA in a way that go unnoticed for host PKR [20]. For VV, our results show that E3 protein accounted for almost all the anti-PKR activity in doubly infected cells, stressing the main role of this protein over the K3 product that probably has only a marginal contribution to the anti-PKR activity of the virus as suggested before [39]. The heterotypic complementation ob- tained for SINV DDLP mutant in cells expressing E3 revealed a notable case of functional analogy that reinforced the importance of anti-PKR mechanisms for virus replication in vertebrates. Thus, as showed for other viruses such as VV, Herpes and Influenza A, elimination of anti-PKR mechanism drastically reduced the replication of SINV virus in wild type animals (Figure 4). However, SINV DDLP mutant replicated to wild type does not seem to involve degradation of the kinase since no alteration in the steady state of PKR was found in EMCV- infected MEFs (Figure 2). For VSV, the absence of effect on SINV-triggered eIF2a phosphorylation was expected in part given the exquisite sensitivity that VSV showed to PKR activation and eIF2a phosphorylation. Thus, VSV seems to lack of an autonomous anti-PKR activity as suggested from previous results [16], suggesting that this virus could hide the replicative forms of dsRNA in a way that go unnoticed for host PKR [20]. February 2011 \| Volume 6 \| Issue 2 \| e16711 Acknowledgments We are in debt to J. Ma Almendral for having allowed us to work at his laboratory since 2005 and J.J. Berlanga (Centro de Biologı´a Molecular Severo Ochoa) for providing us with mice. Thanks go to J. C. Bell (University of Ottawa, Canada) and B.R. Williams (Cleveland Clinic, USA) for PKR knock-out mice and MEFs, respectively. Infection of animals 4–6 week old mice of 129sv strain were infected with 107 pfu of SINV by intranasal inoculation. PKR knock-out mice in the same genetic background were kindly provided by J.C. Bell (University of Ottawa, Canada) [55]. At 4–5 days postinfection, animals were sacrificed by cervical dislocation, decapitated and brain extracted. For VV inoculation, 5 weeks old mice of BALB/c strain were inoculated by the intraperitoneal route with 26107 pfu of a recombinant virus that expresses luciferase (VV-Luc) [54] and organs were extracted at 1dpi. Brains and spleens were fixed O/N with 4% of PFA, cryoprotected with 20% sucrose in PBS and frozen for cryosectioning. Brains and spleens of some animals were taken to titrate viral yields at 5 dpi. In these cases, brains were homogenated in 1 mL of PBS, centrifuged 10 min at 8,0006g and Ethics Statement This study was carried out in strict accordance with the recommendations in the Directive 86/609/EEC of the European Union on the protection of animals used for experimental and other scientific purposes, and that was implemented by the Spanish Government under approval Nu 1201/2005. The protocol was approved by the committee on the ethics of animal experiments of Universidad Auto´noma de madrid (Permit Number: CEI 20-419). All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering. Resistance to PKR activation is critical for replication of Alphavirus in animals For VV, our results show that E3 protein accounted for almost all the anti-PKR activity in doubly infected cells, stressing the main role of this protein over the K3 product that probably has only a marginal contribution to the anti-PKR activity of the virus as suggested before [39]. The heterotypic complementation ob- tained for SINV DDLP mutant in cells expressing E3 revealed a notable case of functional analogy that reinforced the importance of anti-PKR mechanisms for virus replication in vertebrates. Thus, as showed for other viruses such as VV, Herpes and Influenza A, elimination of anti-PKR mechanism drastically reduced the replication of SINV virus in wild type animals (Figure 4). However, SINV DDLP mutant replicated to wild type Finally, the concept of molecular mimicry where pathogens have taken on proteins or protein domains from their hosts to gain evolutionary advantages could be also applicable to RNA structures such as DLP involved in translational control of some cellular mRNA. Thus, it is possible that Alphavirus had copied a mechanism of translation initiation that already operated in the cell. In our opinion, this is a very exciting possibility that deserves further investigation. February 2011 \| Volume 6 \| Issue 2 \| e16711 PLoS ONE \| www.plosone.org 6 Diversity in Viral Anti-PKR Mechanisms viral titres in the supernatants were quantified by plaque assay in BHK21 cells. Author Contributions Conceived and designed the experiments: IV. Performed the experiments: EDG RT IV JLN. Analyzed the data: IV EDG. Contributed reagents/ materials/analysis tools: JLN ME. Wrote the paper: IV. Conceived and designed the experiments: IV. Performed the experiments: EDG RT IV JLN. Analyzed the data: IV EDG. Contributed reagents/ materials/analysis tools: JLN ME. Wrote the paper: IV. Immunofluorescence of brain sections Brains were cryosectioned at ,15 mm and the resulting slides were post-fixed with 4% PFA for 209 at RT, treated with 50 mM of NH4Cl, permeabilized with 0.2% Triton X-100, blocked in 5% of BSA and incubated O/N at 4uC with the indicated antibodies in a wet chamber. Primary antibodies used were: anti-phospho eIF2a (Cell Signalling, 1:250 dilution), anti-SINV capsid (1:500) and anti-VVp14 (1:500). Slides were then incubated with secondary antibodies coupled to Alexa 488 or 595 for 2 h at RT, washed with PBS-0.1% Triton X-100, stained with DAPI and mounted. Slides were examined and photographed in a Leika confocal microscope. Metabolic labeling and Immunoblot analysis Cells growing in 24 well plates were infected with the indicated virus at a moi of 25 pfu/cell. At the indicated times, cultures were labeled with 25 mCi/ml of Met+Cys (NEB) for 309, washed with DMEN lacking serum and lysed in sample buffer as described previously. Samples were analyzed in a 12% SDS-PAGE, soaked in 1 M sodium salicylate, dried and exposed to X-rays films (AGFA). For immunoblot, proteins were transferred to nitrocel- lulose membrane and probed with the indicated antibodies: anti- PKR (1:250, Santa Cruz Biotech.), anti-phosphoeIF2a (1:500, Invitrogen), anti-eIF2 (1:500 Santa Cruz) and anti-b-actin (1:2000, Sigma). For PKR detection, protein transfer was carried out O/N at 4uC in a wet transfer apparatus (BIORAD), whereas a semi-dry transfer (BIORAD) was used for detection of eIF2 and b-actin proteins. To detect phosphoeIF2a, blots were first probed with anti-phosphoeIF2a antibody, incubated with mild stripping buffer (0.1 M NaOH, 159) and then probed with anti-total eIF2a antibody. Blots were revealed by ECL as described previously [29]. 14. 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Cell Host Microbe 6: 513–522. 26. He B, Gross M, Roizman B (1997) The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 94: 843–848. 48. Burke CW, Gardner CL, Steffan JJ, Ryman KD, Klimstra WB (2009) Characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses. Virology 395: 121–132. yp p gy 49. Frolova EI, Fayzulin RZ, Cook SH, Griffin DE, Rice CM, et al. References 1. Van Valen L (1974) Two modes of evolution. Nature 252: 298–300. 10. Kang DC, Gopalkrishnan RV, Wu Q, Jankowsky E, Pyle AM, et al. (2002) mda- 5: An interferon-inducible putative RNA helicase with double-stranded RNA- dependent ATPase activity and melanoma growth-suppressive properties. Proc Natl Acad Sci U S A 99: 637–642. 1. Van Valen L (1974) Two modes of evolution. Nature 252: 298–300. 2. Webber C, Ponting CP (2004) Genes and homology. Curr Biol 14: R 2. Webber C, Ponting CP (2004) Genes and homology. Curr Biol 14: R332–333. 3. Paterson S, Vogwill T, Buckling A, Benmayor R, Spiers AJ, et al. (2010) Antagonistic coevolution accelerates molecular evolution. Nature 464: 275–278. 11. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, et al. (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA- induced innate antiviral responses. Nat Immunol 5: 730–737. 4. Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124: 783–801. 5. Pichlmair A, Reis e Sousa C (2007) Innate recognition of viruses. Immunity 27: 370–383. p 12. Garcia MA, Gil J, Ventoso I, Guerra S, Domingo E, et al. (2006) Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev 70: 1032–1060. 6. Bowie AG, Unterholzner L (2008) Viral evasion and subversion of pattern- recognition receptor signalling. Nat Rev Immunol 8: 911–922. 13. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, et al. (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101–105. 7. Sadler AJ, Williams BR (2008) Interferon-inducible antiviral effectors. Nat Rev Immunol 8: 559–568. 8. Elde NC, Malik HS (2009) The evolutionary conundrum of pathogen mimicry. Nat Rev Microbiol 7: 787–797. 9. Suthar MS, Gale M, Jr., Owen DM (2009) Evasion and disruption of innate immune signalling by hepatitis C and West Nile viruses. Cell Microbiol 11: 880–888. PLoS ONE \| www.plosone.org 7 February 2011 \| Volume 6 \| Issue 2 \| e16711 Diversity in Viral Anti-PKR Mechanisms Diversity in Viral Anti-PKR Mechanisms Abraham N, Stojdl DF, Duncan PI, Methot N, Ishii T, et al. 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https://openalex.org/W4297979925	https://pure.mpg.de/pubman/item/item_2434406_4/component/file_2462936/tp2016292a.pdf	English	null	Genetic effects influencing risk for major depressive disorder in China and Europe	Carolina Digital Repository (University of North Carolina at Chapel Hill)	2,017	cc-by	9,526	OPEN OPEN Citation: Transl Psychiatry (2017) 7, e1074; doi:10.1038/tp.2016.292 www.nature.com/tp 1Department of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA; 2Department of Psychiatry, Charite Universitatsmedizin Berlin Campus Benjamin Franklin, Berlin, Germany; 3Medical and Population Genetics, Broad Institute, Cambridge, MA, USA; 4Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; 5Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia; 6Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia; 7Department of Biological Psychology, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 8Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich, Germany; 9Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; 10Department of Psychiatry, VU University Medical Center and GGZ inGeest, Amsterdam, The Netherlands; 11Institute of Epidemiology and Social Medicine, University of Muenster, Münster, Germany; 12Division of Psychiatry, University of Edinburgh, Edinburgh, UK; 13King's College London, NIHR BRC for Mental Health, London, UK; 14King's College London, MRC Social Genetic and Developmental Psychiatry Centre, London, UK; 15Department of Clinical Medicine, Translational Neuropsychiatry Unit, Aarhus University, Aarhus, Denmark; 16Department of Biomedicine, University of Basel, Basel, Switzerland; 17Division of Medical Genetics, University of Basel, Basel, Switzerland; 18Institute of Neuroscience and Medicine (INM-1), Research Center Juelich, Jülich, Germany; 19Institute of Human Genetics, University of Bonn, Bonn, Germany; 20Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia; 21Centre for Advanced Imaging, University of Queensland, Brisbane, QLD, Australia; 22Department of Psychological Medicine, Cardiff University, Cardiff, UK; 23EMGO+ Institute, VU University Medical Center, Amsterdam, The Netherlands; 24Department of Genomics, Life and Brain Center, University of Bonn, Bonn, Germany; 25Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; 26Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA; 27Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; 28Department of Psychiatry and Behavioral Sciences, State University of New York Downstate Medical Center, Brooklyn, NY, USA; 29Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany; 30Department of Psychiatry, Trinity College Dublin, Dublin, Ireland; 31Department of Psychiatry and Psychotherapy, University Medicine Greifswald, Greifswald, Germany; 32Department of Psychiatry, Kaiser-Permanente Northern California, San Fransisco, CA, USA; 33Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland; 34Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 35Department of Psychiatry, Washington University in Saint Louis School of Medicine, St Louis, MO, USA; 36Division of Medical Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland; 37Brain and Mind Research Institute, University of Sydney, Sydney, NSW, Australia; 38Human Genomics Research Group, Department of Biomedicine, University of Basel, Basel, Switzerland; 39Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine and Ernst Moritz Arndt University Greifswald, Greifswald, Germany; 40Max Planck Institute of Psychiatry, Munich, Germany; 41Department of Psychiatry and The Behavioral Sciences, University of Southern California, Los Angeles, CA, USA; 42Neuroscience Therapeutic Area, Janssen Research and Development, LLC, Titusville, NJ, USA; 43Department of Psychiatry, Washington University in Saint Louis School of Medicine, St Louis, MO, USA; 44School of Psychology, University of Queensland, Brisbane, QLD, Australia; 45Department of Psychiatry, New York State Psychiatric Institute, Columbia University College of Physicians and Surgeons, New York, NY, USA; 46Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, UK; 47Institute for Molecular Biology, University of Queensland, Brisbane, QLD, Australia; 48Psychosis Research Unit, Aarhus University Hospital, Risskov, Denmark; 49Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK; 50Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Greifswald, Germany; 51Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia; 52MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University School of Medicine, Cardiff, UK; 53Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, USA; 54Department of Psychiatry, Harvard Medical School, Boston, MA, USA; 55Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA; 56Medical Genetics Section, CGEM, IGMM, University of Edinburgh, Edinburgh, UK; 57Department of Psychiatry, University of Iowa, Iowa, IA, USA; 58Department of Psychiatry, Washington University in Saint Louis, St Louis, MO, USA; 59Department of Human and Molecular Genetics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA; 60Department of Biochemistry and Molecular Biology II, Institute of Neurosciences, Center for Biomedical Research, University of Granada, Granada, Spain; 61King’s College London, MRC Social Genetic and Developmental Psychiatry Centre, London, UK; 62Department of Psychiatry, University of Groningen, University of Medical Center Groningen, Groningen, The Netherlands; 63Institute of Psychiatric Phenomics and Genomics, Medical Center of the University of Munich, Campus Innenstadt, Munich, Germany; 64Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Göttingen, The Netherlands; 65Department of Psychiatry and Behavioral Sciences, Johns Hopkins University, Baltimore, MD, USA; 66Human Genetics Branch, NIMH Division of Intramural Research Programs, Bethesda, MD, USA; 67Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA; 68Division of Psychiatry, Group Health, Seattle, WA, USA; 69Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany; 70Department of Psychiatry, Leiden University Medical Center, Leiden, The Netherlands; 71Division of Epidemiology, New York State Psychiatric Institute, New York, NY, USA; 72Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, NY, USA; 73Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA; 74King's College London, Department of Medical and Molecular Genetics, London, UK; 75Merton College, University of Oxford, Oxford, UK; 76Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK; 77Medical Epidemiology and Biostatistics, Karolinska Institutet, Solna, Sweden; 78Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA and 79Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Correspondence: Dr TB Bigdeli, Department of Psychiatry, Virginia Commonwealth University School of Medicine, Virginia Institute for Psychiatric and Behavioral Genetics, 800 E. Leigh Street, Biotech One, Suite 101, Richmond, VA 23219-1534, USA. E-mail: tim.bigdeli@gmail.com ORIGINAL ARTICLE Genetic effects inﬂuencing risk for major depressive disorder in Chi d E TB Bigdeli1, S Ripke2,3,4, RE Peterson1, M Trzaskowski5,6, S-A Bacanu1, A Abdellaoui7, TFM Andlauer8,9, ATF Beekman10, K Berger11, DHR Blackwood12, DI Boomsma7, G Breen13,14, HN Buttenschøn15, EM Byrne6, S Cichon16,17,18,19, T-K Clarke12, B Couvy-Duchesne6,20,21, N Craddock22, EJC de Geus7,23, F Degenhardt19,24, EC Dunn25,26,27, AC Edwards1, AH Fanous28, AJ Forstner19,24, J Frank29, M Gill30, SD Gordon20, HJ Grabe31, SP Hamilton32, O Hardiman33, C Hayward34, AC Heath35, AK Henders6, S Herms19,24,36, IB Hickie37, P Hoffmann19,24,38, G Homuth39, J-J Hottenga7, M Ising40, R Jansen10, S Kloiber40, JA Knowles41, M Lang29, QS Li42, S Lucae40, DJ MacIntyre12, PAF Madden43, NG Martin20,44, PJ McGrath45, P McGufﬁn14, AM McIntosh12,46, SE Medland20, D Mehta6, CM Middeldorp7, Y Milaneschi10, GW Montgomery47, O Mors48, B Müller-Myhsok8,9,49, M Nauck50, DR Nyholt51, MM Nöthen19,24, MJ Owen52, BWJH Penninx10, ML Pergadia53, RH Perlis54,55, WJ Peyrot10, DJ Porteous56, JB Potash57, JP Rice58, M Rietschel29, BP Riley1,59, M Rivera60,61, R Schoevers62, TG Schulze29,63,64,65,66, J Shi67, SI Shyn68, JH Smit10, JW Smoller25,26,27, F Streit29, J Strohmaier29, A Teumer69, J Treutlein29, S Van der Auwera31, G van Grootheest10, AM van Hemert70, H Völzke69, BT Webb1, MM Weissman71,72, J Wellmann11, G Willemsen7, SH Witt29, DF Levinson73, CM Lewis14,74, NR Wray5,6, J Flint75,76, PF Sullivan77,78,79 and KS Kendler1,59 on behalf of the CONVERGE consortium and Major Depressive Disorder Working Group of the Psychiatric Genomics Consortium 1Department of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA; 2Department of Psychiatry, Charite Universitatsmedizin Berlin Campus Benjamin Franklin, Berlin, Germany; 3Medical and Population Genetics, Broad Institute, Cambridge, MA, USA; 4Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; 5Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia; 6Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia; 7Department of Biological Psychology, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 8Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich, Germany; 9Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; 10Department of Psychiatry, VU University Medical Center and GGZ inGeest, Amsterdam, The Netherlands; 11Institute of Epidemiology and Social Medicine, University of Muenster, Münster, Germany; 12Division of Psychiatry, University of Edinburgh, Edinburgh, UK; 13King's College London, NIHR BRC for Mental Health, London, UK; 14King's College London, MRC Social Genetic and Developmental Psychiatry Centre, London, UK; 15Department of Clinical Medicine, Translational Neuropsychiatry Unit, Aarhus University, Aarhus, Denmark; 16Department of Biomedicine, University of Basel, Basel, Switzerland; 17Division of Medical Genetics, University of Basel, Basel, Switzerland; 18Institute of Neuroscience and Medicine (INM-1), Research Center Juelich, Jülich, Germany; 19Institute of Human Genetics, University of Bonn, Bonn, Germany; 20Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia; 21Centre for Advanced Imaging, University of Queensland, Brisbane, QLD, Australia; 22Department of Psychological Medicine, Cardiff University, Cardiff, UK; 23EMGO+ Institute, VU University Medical Center, Amsterdam, The Netherlands; 24Department of Genomics, Life and Brain Center, University of Bonn, Bonn, Germany; 25Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; 26Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA; 27Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; 28Department of Psychiatry and Behavioral Sciences, State University of New York Downstate Medical Center, Brooklyn, NY, USA; 29Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany; 30Department of Psychiatry, Trinity College Dublin, Dublin, Ireland; 31Department of Psychiatry and Psychotherapy, University Medicine Greifswald, Greifswald, Germany; 32Department of Psychiatry, Kaiser-Permanente Northern California, San Fransisco, CA, USA; 33Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland; 34Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 35Department of Psychiatry, Washington University in Saint Louis School of Medicine, St Louis, MO, USA; 36Division of Medical Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland; 37Brain and Mind Research Institute, University of Sydney, Sydney, NSW, Australia; 38Human Genomics Research Group, Department of Biomedicine, University of Basel, Basel, Switzerland; 39Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine and Ernst Moritz Arndt University Greifswald, Greifswald, Germany; 40Max Planck Institute of Psychiatry, Munich, Germany; 41Department of Psychiatry and The Behavioral Sciences, University of Southern California, Los Angeles, CA, USA; 42Neuroscience Therapeutic Area, Janssen Research and Development, LLC, Titusville, NJ, USA; 43Department of Psychiatry, Washington University in Saint Louis School of Medicine, St Louis, MO, USA; 44School of Psychology, University of Queensland, Brisbane, QLD, Australia; 45Department of Psychiatry, New York State Psychiatric Institute, Columbia University College of Physicians and Surgeons, New York, NY, USA; 46Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, UK; 47Institute for Molecular Biology, University of Queensland, Brisbane, QLD, Australia; 48Psychosis Research Unit, Aarhus University Hospital, Risskov, Denmark; 49Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK; 50Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Greifswald, Germany; 51Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia; 52MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University School of Medicine, Cardiff, UK; 53Charles E. Received 19 November 2016; accepted 27 November 2016 ORIGINAL ARTICLE Genetic effects inﬂuencing risk for major depressive disorder in Chi d E Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, USA; 54Department of Psychiatry, Harvard Medical School, Boston, MA, USA; 55Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA; 56Medical Genetics Section, CGEM, IGMM, University of Edinburgh, Edinburgh, UK; 57Department of Psychiatry, University of Iowa, Iowa, IA, USA; 58Department of Psychiatry, Washington University in Saint Louis, St Louis, MO, USA; 59Department of Human and Molecular Genetics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA; 60Department of Biochemistry and Molecular Biology II, Institute of Neurosciences, Center for Biomedical Research, University of Granada, Granada, Spain; 61King’s College London, MRC Social Genetic and Developmental Psychiatry Centre, London, UK; 62Department of Psychiatry, University of Groningen, University of Medical Center Groningen, Groningen, The Netherlands; 63Institute of Psychiatric Phenomics and Genomics, Medical Center of the University of Munich, Campus Innenstadt, Munich, Germany; 64Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Göttingen, The Netherlands; 65Department of Psychiatry and Behavioral Sciences, Johns Hopkins University, Baltimore, MD, USA; 66Human Genetics Branch, NIMH Division of Intramural Research Programs, Bethesda, MD, USA; 67Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA; 68Division of Psychiatry, Group Health, Seattle, WA, USA; 69Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany; 70Department of Psychiatry, Leiden University Medical Center, Leiden, The Netherlands; 71Division of Epidemiology, New York State Psychiatric Institute, New York, NY, USA; 72Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, NY, USA; 73Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA; 74King's College London, Department of Medical and Molecular Genetics, London, UK; 75Merton College, University of Oxford, Oxford, UK; 76Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK; 77Medical Epidemiology and Biostatistics, Karolinska Institutet, Solna, Sweden; 78Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA and 79Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Correspondence: Dr TB Bigdeli, Department of Psychiatry, Virginia Commonwealth University School of Medicine, Virginia Institute for Psychiatric and Behavioral Genetics, 800 E. Leigh Street, Biotech One, Suite 101, Richmond, VA 23219-1534, USA. E-mail: tim.bigdeli@gmail.com R i d 19 N b 2016 t d 27 N b 2016 Trans-ancestry genomic analysis of depression TB Bigdeli et al Trans-ancestry genomic analysis of depression d l l 2 Major depressive disorder (MDD) is a common, complex psychiatric disorder and a leading cause of disability worldwide. Trans-ancestry genetic correlation The recently developed popcorn software30 allows for estimation of the trans-ancestry genetic effect correlation (ρg) using GWAS summary statistics. Cross-ancestry reference scores, representing SNP-wise estimates of the similarity of LD (with neighboring SNPs) between populations, were calculated for East Asian (N = 286) and European (N = 379) subjects from the 1000 Genomes Project (v3).28 For computational efﬁciency and consistency with previously reported estimates of genetic correlation, these calculations were based on ~ 1.2 M common SNPs present in HapMap3 (ref. 31) following study-wise exclusion of SNPs with INFOo0.9 or minor allele frequencyo0.01%. ORIGINAL ARTICLE Genetic effects inﬂuencing risk for major depressive disorder in Chi d E Despite twin studies indicating its modest heritability (~30–40%), extensive heterogeneity and a complex genetic architecture have complicated efforts to detect associated genetic risk variants. We combined single-nucleotide polymorphism (SNP) summary statistics from the CONVERGE and PGC studies of MDD, representing 10 502 Chinese (5282 cases and 5220 controls) and 18 663 European (9447 cases and 9215 controls) subjects. We determined the fraction of SNPs displaying consistent directions of effect, assessed the signiﬁcance of polygenic risk scores and estimated the genetic correlation of MDD across ancestries. Subsequent trans-ancestry meta-analyses combined SNP-level evidence of association. Sign tests and polygenic score proﬁling weakly support an overlap of SNP effects between East Asian and European populations. We estimated the trans-ancestry genetic correlation of lifetime MDD as 0.33; female-only and recurrent MDD yielded estimates of 0.40 and 0.41, respectively. Common variants downstream of GPHN achieved genome-wide signiﬁcance by Bayesian trans-ancestry meta-analysis (rs9323497; log10 Bayes Factor = 8.08) but failed to replicate in an independent European sample (P = 0.911). Gene-set enrichment analyses indicate enrichment of genes involved in neuronal development and axonal trafﬁcking. We successfully demonstrate a partially shared polygenic basis of MDD in East Asian and European populations. Taken together, these ﬁndings support a complex etiology for MDD and possible population differences in predisposing genetic factors, with important implications for future genetic studies. Translational Psychiatry (2017) 7, e1074; doi:10.1038/tp.2016.292; published online 28 March 2017 Polygenic risk score proﬁling and binomial sign tests Polygenic risk score proﬁling and binomial sign tests Each data set was ﬁltered on the basis of statistical imputation information (INFO) greater than 0.8 and minor allele frequency greater than 0.01 in both CONVERGE and PGC overall; linkage disequilibrium (LD)-based 'clumping' was used to obtain an approximately independent set of SNPs (r2o0.1) while preferentially retaining the most signiﬁcant SNP within 500- kb windows. We computed weighted polygenic scores (that is, log odds ratio of the associated allele), based on varying P-value thresholds in the 'training set' results (that is, CONVERGE or PGC); P-value thresholds ranged between 10−5 and 0.5. We evaluated the signiﬁcance of case–control differences using logistic regression and covarying ancestry-based principal components and a study indicator variable. The predictive value of these scores is reported in terms of Nagelkerke’s pseudo-R2 (fmsb package in R).29 Whether genetic factors predisposing to MDD are shared across ancestries is not well established, and two replicated genome- wide signiﬁcant associations for MDD in China had markedly lower allele frequencies in Europeans and thus did not replicate.22–24 Allelic heterogeneity and population-speciﬁc genetic effects have been reported for several complex traits;18,25,26 however, the extent of differences across ancestries remains relatively unexplored. p We sought to clarify the extent to which liability to MDD is shared between European and East Asian populations via collaboration between the Psychiatric Genomics Consortium (PGC)22 and CONVERGE6 studies of MDD. We asked whether observed directions of allelic effects are consistent across populations, assessed the signiﬁcance of cross-ancestry polygenic scores and estimated the trans-ancestry genetic correlation of MDD. We attempted to disentangle population differences from those arising from ascertainment or phenotypic deﬁnition through analyses of recurrent MDD and in female subjects. These meta- analyses represent the largest trans-ancestry genetic study of MDD to date. Using the same sets of SNPs and the same P-value thresholds, we applied a binomial sign test to determine whether the number of SNPs demonstrating consistent directions of allelic effects between CONVERGE and PGC was greater than expected by chance (that is, a one-sided test of whether this fraction is greater than 0.5). INTRODUCTION CONVERGE (China, Oxford and Virginia Commonwealth University Experimental Research on Genetic Epidemiology): Brieﬂy, all subjects were Han Chinese women and had two or more episodes of MDD meeting DSM-IV criteria. After applying quality controls modeled after the PGC study, 10 502 samples (5282 cases and 5220 controls) and 6 242 619 SNPs were retained for analysis. Major depressive disorder (MDD) is the most common psychiatric illness and a leading cause of disability worldwide.1,2 MDD is modestly heritable (30–40%), may be genetically complex and likely heterogeneous, complicating efforts to identify replicable risk loci.3,4 The successful detection and interpretation of genetic associations require both increased sample sizes5 and empirically driven efforts to reduce phenotypic heterogeneity.6 PGC MDD: Samples included here comprise Stage 1 of the PGC MDD study.22 Brieﬂy, all subjects were of European ancestry, all cases were assessed using validated methods and met DSM-IV criteria for lifetime MDD, and the majority of controls were screened to exclude lifetime MDD. Available data on number of depressive episodes were used to identify recurrent cases (two or more episodes). Nine studies from the US, Europe and Australia were genotyped using SNP arrays. Imputation was performed with IMPUTE2 (ref. 27) using the 1000 Genomes Project data (v3; GRCh37/hg19),28 resulting in a total of 13 381 627 autosomal and X chromosome SNPs. Underpinning the success of genome-wide association studies (GWAS) of numerous traits has been the emergence of large research consortia.7 In addition to facilitating larger sample sizes, many consortia are increasingly ancestrally diverse, enabling identiﬁcation of novel associations8–10 and independent replica- tion of reported ﬁndings,11,12 as well as improving ﬁne mapping of implicated loci.13,14 Consistent associations at replicated loci have been reported for psychiatric disorders15 and non-psychiatric traits,8,11,16–18 and shared liabilities are often borne out by genome-wide polygenic analyses.19–21 MATERIALS AND METHODS Ascertainment and genotyping Sample ascertainment, SNP genotyping and quality-control procedures for PGC and CONVERGE have been described previously.6,22 Individual sites and sample sizes are presented in Table 1. Translational Psychiatry (2017), 1 – 7 Trans-ancestry genomic analysis of depression TB Bigdeli et al 3 Table 1. Sample sizes by participating study site in discovery and replication phases Phase Study Ancestry No. of controls No. of cases No. of recurrent Discovery Bonn Mannheim EUR 1072 (48.3) 588 (64.1) 408 (66.7) GenRED1 EUR 1097 (42.7) 1020 (70.8) 1020 (70.8) GSK MPIP EUR 859 (67.6) 861 (67.4) 831 (67.3) MPIP MARS 650 EUR 539 (54.5) 373 (55.2) 128 (59.4) NTR/NESDA EUR 1727 (62.0) 1685 (69.2) 834 (71.2) QIMR I317 EUR 960 (55.2) 1017 (61.0) 524 (65.6) QIMR I610 EUR 748 (61.9) 433 (72.3) 250 (72.4) RADIANT EUR 1549 (54.2) 1903 (70.5) 1441 (70.8) RADIANT—Germany EUR 217 (53.9) 327 (65.7) 254 (70.5) STARD EUR 447 (43.8) 1240 (58.6) 912 (59.1) CONVERGEa EAS 5220 5282 5282 Replicationb Edinburgh EUR 285 (48.8) 372 (59.4) — DepGenesNetwork EUR 470 (62.6) 471 (77.5) 471 (77.5) GenRED2 EUR 474 (48.9) 830 (82.8) 830 (82.8) Harvard i2b2 EUR 1067 (50.1) 806 (66.9) 806 (66.9) Janssen EUR 1380 (60.5) 466 (68.2) 466 (68.2) QIMR COEX EUR 526 (57.6) 565 (71.5) — RADIANT—Irish cases EUR 340 (52.4) 109 (82.6) 109 (82.6) RADIANT—US cases EUR 378 (51.9) 223 (78.5) 223 (78.5) RADIANT—Denmark cases EUR 516 (40.7) 133 (69.9) 133 (69.9) SHIP-LEGEND EUR 1087 (44.0) 366 (67.8) — SHIP-TREND-0 EUR 484 (44.6) 163 (71.8) — Totals Discovery 14 435 (71.3) 14 729 (78.4) 11 884 (82.2) Replication 7007 (51.6) 4504 (72.3) 2572 (75.8) Abbreviations: EAS, East Asian; EUR, European. Numbers of female subjects are displayed parenthetically. aAll cases and controls were female . bSee note in Materials and methods section. We attempted to address possible heterogeneity by examining estimates of genetic correlation within- and across-ancestries, and for varying phenotypic deﬁnitions. Brieﬂy, we divided the PGC and CONVERGE studies into approximate halves, performing association analysis in each subsample as described above, and subsequently estimating the genetic correlations between these nonoverlapping halves. Within the PGC, we randomly selected 5 of 10 studies (SEUR1), with the remaining ﬁve studies taken as a comparison sample (SEUR2). We selected N = 30 of a possible 126 paired comparisons for which the sample sizes of each subset were equivalent (~1:1). MATERIALS AND METHODS We followed an analogous procedure in CONVERGE, selecting 12 of 24 sequencing batches (SASN1), with the remaining 12 batches taken as a comparison sample (SASN2). Within-ancestry comparisons were between nonoverlapping subsets (for example, SEUR1 versus SEUR2) and utilized reference scores based on a single population, calculated as described above. Cross-ancestry estimates were based on comparisons of the full set of CONVERGE results to each of N = 60 subsets from the within-PGC analysis. We compared cross-ancestry estimates for lifetime MDD, recurrent MDD and females-only by paired Student’s t-tests. Gene-set enrichment analyses We applied DEPICT34 to identify signiﬁcantly enriched gene sets and pathways in speciﬁc tissues and cell types. Brieﬂy, genes in the vicinity of associated SNPs are tested for enrichment for 'reconstituted' gene sets, comprising curated sets expanded to include co-regulated loci. Tissue and cell-type enrichment analysis is conducted by testing whether genes were highly expressed in any of 209 MeSH annotations based on microarray data for the Affymetrix U133 Plus 2.0 Array platform (Santa Clara, CA, USA).35 Because DEPICT adjusts for potential sources of confounding and multiple testing using precomputed GWAS of randomly distributed phenotypes, we elected to use as input P-values from inverse variance weighted (that is, ﬁxed effects) meta-analysis of PGC and CONVERGE. Recalling that MANTRA is effectively a Bayesian implementation of ﬁxed- effects meta-analysis when allele frequencies are similar between populations, we considered this to be an appropriate strategy, if not somewhat conservative. RESULTS Polygenic risk score proﬁling and binomial sign tests We employed polygenic risk score proﬁling to determine whether ﬁndings from CONVERGE or the PGC are, in aggregate, signiﬁcantly associated with the MDD status in the other study. Scores based on PGC results were nominally associated with MDD in CONVERGE (Figure 1; Supplementary Tables S1–S3), accounting for ~ 0.1% of risk (Nagelkerke’s pseudo-R2 = 7.46 × 10−4; P = 0.02). Scores based on results for female-only yielded similar results (Nagelkerke’s pseudo-R2 = 7.60 × 10 −4; P = 0.0141), whereas scores for recurrent MDD were most strongly associated overall (Nagelkerke’s pseudo-R2 = 0.00201; P = 6.56 × 10 −5). Scores from CONVERGE were nominally associated with MDD status in the PGC data (Nagelkerke’s pseudo-R2 = 6.08 × 10−4; P = 6.66 × 10 −3); these scores yielded similar results when considering female-only (Nagelkerke’s pseudo-R2 = 0.00111; P = 4.15 × 10 −3), and recurrent MDD (Nagelkerke’s pseudo-R2 = 9.13 × 10 −4; P = 2.02 × 10−3). How- ever, only the results based on PGC-trained polygenic scores for recurrent MDD remained signiﬁcant after correction for multiple testing (Supplementary Table S2). Polygenic risk score proﬁling and binomial sign tests Trans-ancestry genetic correlation Table 2 displays the results of the trans-ancestry genetic correlation between East Asian and European populations. For lifetime MDD (ρg = 0.332, 95% conﬁdence interval (CI): (0.270, 0.394)), this was both signiﬁcantly greater than zero (Pρg40 = 7.23 × 10−26) and signiﬁcantly less than one (Pρgo 1 = 1.40 × 10−99), indicating a partially shared polygenic basis of MDD risk between East Asians and Europeans. These ﬁndings remain signiﬁcant after correction for multiple tests. g g By comparison, recurrent MDD and females-only yielded slightly higher estimates of genetic correlation (Table 2). We compared g y We evaluated whether the observed fraction of results displaying the same direction of allelic effects across studies was signiﬁcantly greater than expected by chance (that is, 50%) using binomial sign tests. Supplementary Table S4 gives the number of LD-independent SNPs considered, fraction of these SNPs display- ing the same direction of effect in the other study and a one-sided binomial test P-value. For lifetime MDD, we observed the largest excess of same-direction effects in PGC for SNPs signiﬁcant at Po0.2 in CONVERGE (50.7%; binomial P = 1 × 10−3); this ﬁnding remains signiﬁcant after multiple-testing adjustment (Supplementary Table S4). SNP-based meta-analyses A total of 4504 cases and 7007 controls from 10 independent, European- ancestry cohorts were available for replication (Table 1). These studies represent recent additions to the PGC that were not included in the previously published analysis.22 A brief description of each study site is given in the Supplementary Material. At the time of writing, neither comparable East Asian GWAS data sets nor subject-level data on the number of depressive episodes were readily available. For analyses of recurrent illness, we included those replication studies that speciﬁcally ascertained recurrent cases. Within each study, we tested for association between SNPs and affection status by logistic regression with PLINK,32 using allelic dosages and including ancestry principal components as covariates (plus a site indicator in PGC analyses). Backward-stepwise regression was used to select principal components demonstrating association (Po0.159) with each diagnosis. We excluded SNPs with minor allele frequencyo0.01 or INFOo0.5 in either CONVERGE or PGC (overall), or missing in greater than equal to ﬁve of nine PGC samples. We analyzed the X chromosome as previously described.22 For each phenotype deﬁnition, we identiﬁed independent (pairwise r2o0.1 within 500-kb windows based on European 1000 Genomes Project samples), signiﬁcant autosomal SNPs (log10BF45) from the trans- ancestry meta-analyses (10, 7 and 7 for MDD, female-only and recurrent MDD, respectively). We tested these SNPs for association using logistic regression and including ancestry principal components as covariates. We performed inverse-variance weighted meta-analyses of the replication samples using METAL. We also performed binomial sign tests comparing the directions of allelic effects across discovery and replication stages. We performed Bayesian meta-analyses of PGC and CONVERGE studies using MANTRA.33 By leveraging population differences in local LD structure, MANTRA has greater power to detect genetic effects demon- strating allelic heterogeneity than traditional approaches assuming random effects. When effects are consistent across studies, MANTRA is effectively a Bayesian implementation of ﬁxed-effects meta-analysis. Interstudy genetic distances were calculated from the mean allele frequency differences. We adopted a threshold of log10 Bayes factor (log10BF) 47 for declaring genome-wide signiﬁcance. Translational Psychiatry (2017), 1 – 7 Trans-ancestry genomic analysis of depression TB Bigdeli et al 4 PGC recurrent MDD analysis (51.1%; binomial P = 3.05 × 10 −5); the fraction of same-direction effects in the PGC was largest in the female-only analysis, for SNPs signiﬁcant at Po0.1 in CONVERGE (50.9%; binomial P = 1.11 × 10 −3). SNP-based meta-analyses Although statistically signiﬁcant after correcting for multiple tests (Supplementary Table S4), the observed excess of same-direction effects represents only a very small deviation from expectation under the null hypothesis. SNP-based meta-analyses We observed the strongest overall evidence of association experiment-wide between SNPs upstream of gephyrin (GPHN) at 14q23.3 (rs9323497; log10BF = 8.08) and lifetime MDD (Supple- mentary Figures S3 and S4). Associated SNPs show marked differences in allele frequencies between East Asian and European populations and opposing directions of allelic effect in CONVERGE and PGC (Supplementary Figure 4). This locus encodes a neuronal assembly protein that anchors glycine and GABAA receptors to the postsynaptic density in inhibitory neurons.36 Intriguingly, the gephryin region exhibits an unusual ‘yin-yang’ haplotype structure reﬂecting strong positive selection related to recent, rapid human evolution,37 and has previously yielded suggestive evidence of association with depressive symptoms in the general population.38 We initially considered the simple hypothesis that disease- relevant SNP effects would have similar sizes and directions of effect across European and Han Chinese studies,39 without explicit consideration of population differences arising from genetic drift or divergent genetic architectures. Scores constructed from either PGC or CONVERGE results were signiﬁcantly associated with lifetime MDD in the other study, albeit explaining a diminutive fraction of risk. However, it is commonly observed that polygenic prediction is generally poorer when ‘training’ and ‘testing’ data sets do not originate from a single ancestral population, likely attributable to differences in allele frequencies and patterns of LD.20,21 A total of 10 independent associated SNPs (log10BF45) were prioritized for replication (Supplementary Table S5; Supplementary Figure S4); of these, three were in or near GPHN, two represent previously reported associations in CONVERGE that did not replicate in PGC6 and one was the strongest reported association in the original PGC study.22 No single SNP in either the females-only or recurrent MDD analyses attained genome-wide signiﬁcance (Supplementary Figure S3). From each of these analyses, seven independent associated SNPs were taken forward to the replication stage (Supplementary Tables S6 and S7). Next, we applied the recently developed popcorn method30 to obtain estimates of the genetic effect correlation between these populations. Brieﬂy, the genetic correlation is the correlation coefﬁcient of per-allele SNP effect sizes across populations. We found that the genetic correlation of lifetime MDD was signiﬁcantly different from both zero and one, suggesting that there is substantial but incomplete overlap in common SNP effects predisposing to MDD in Europe and China. RESULTS For the reverse comparison, the largest excess of same-direction effects was observed for SNPS signiﬁcant at Po0.2 in PGC (50.5%; binomial P = 0.016). Table 2. Trans-ancestry genetic correlations between East Asian and European MDD subtypes Traita ρg b Pρg40 Pρgo1 Lifetime MDD 0.332 (0.270, 0.394) 7.23 × 10 −26 1.40 × 10−99 Female-only MDD 0.402 (0.326, 0.477) 2.04 × 10 −25 2.59 × 10 −54 Recurrent MDD 0.410 (0.343, 0.477) 5.40 × 10 −33 2.23 × 10 −66 Abbreviation: MDD, major depressive disorder. aEuropean prevalences of lifetime, females-only and recurrent MDD were assumed to be 0.15, 0.20 and 0.105, respectively; prevalence of recurrent MDD among Chinese women was assumed to be 0.08. bEstimates of ρg are displayed with corresponding 95% conﬁdence intervals. Table 2. Trans-ancestry genetic correlations between East Asian and European MDD subtypes Overall, the greatest excess of same-direction effects in CONVERGE was observed for SNPs signiﬁcant at Po0.1 in the P = 4.04 × 10−3 P = 6.66 × 10−3 P = 2.02 × 10−3 P = 1.41 × 10−2 P = 1.92 × 10−2 P = 6.56 × 10−5 CONVERGE−trained polygenic risk score PGC−trained polygenic risk score 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 0.0012 0.0014 0.0016 0.0018 0.0020 Lifetime MDD Females−only MDD Recurrent MDD Lifetime MDD Females−only MDD Recurrent MDD Diagnosis Variance explained (R2) P−value threshold 0.00001 0.0001 0.001 0.01 0.1 0.2 0.3 0.4 0.5 Figure 1. Trans-ancestry association of polygenic risk scores with major depressive disorder. For scores based on results from PGC or CONVERGE, the variance in risk explained in the other study is shown on the y axis in terms of Nagelkerke’s pseudo-R2; scores based on various P-value inclusion thresholds are displayed as shaded bars. CONVERGE, China, Oxford and Virginia Commonwealth University Experimental Research on Genetic Epidemiology; MDD, major depressive disorder; PGC, Psychiatric Genomics Consortium. Figure 1. Trans-ancestry association of polygenic risk scores with major depressive disorder. For scores based on results from PGC or CONVERGE, the variance in risk explained in the other study is shown on the y axis in terms of Nagelkerke’s pseudo-R2; scores based on various P-value inclusion thresholds are displayed as shaded bars. CONVERGE, China, Oxford and Virginia Commonwealth University Experimental Research on Genetic Epidemiology; MDD, major depressive disorder; PGC, Psychiatric Genomics Consortium. Translational Psychiatry (2017), 1 – 7 Trans-ancestry genomic analysis of depression TB Bigdeli et al 5 Gene-set enrichment analyses these estimates by assuming an approximately normal distribu- tion for ρg and obtaining a Z-score for the difference in values; these differences were found to be nominally signiﬁcant for both recurrent MDD (Pone-sided = 0.023) and females-only (Pone-sided = 0.044). We followed up these results by calculating genetic effect correlation estimates based on comparisons of CONVERGE to N = 60 random subsets of the PGC data (Supplementary Figure S1). Compared with lifetime MDD (ρg = 0.309; 95% CI: (0.290 0.327)), estimates of ρg were signiﬁcantly higher for females-only (ρg = 0.372, 95% CI: (0.344,0.401); t(59) = 7.41, Pone-sided = 2.69 × 10 −10) and recurrent MDD (ρg = 0.375, 95% CI: (0.362, 0.389); t(59) = 15.29, Pone-sided = 1.74 × 10-22). We used DEPICT to investigate whether particular pathways or gene sets were enriched for associations with any of the phenotypic deﬁnitions considered. For SNPs signiﬁcant at Po10−5 in meta-analyses of lifetime, females-only and recurrent MDD (29, 24 and 27 independent loci, respectively), no single pathway or gene set was signiﬁcantly enriched, or contained more signiﬁcant genes than expected by chance, after correction for multiple testing (q ⩾0.20). When we considered a more inclusive threshold (Po10−4), there were 167, 161 and 161 independent loci for lifetime, females-only and recurrent MDD, respectively. Following correc- tion for multiple testing, only central nervous system neuron differentiation (GO:0021953) and axon cargo transport pathways (GO:0008088) were found to be signiﬁcantly enriched (qo0.05) in the analysis of lifetime MDD. An additional 11 gene sets were suggestively enriched (qo0.20) and included several ontology terms related to neurodevelopmental processes (Supplementary Table S8). Finally, no tissue or cell types were enriched for associations with any deﬁnition of MDD (q ⩾0.20), irrespective of the signiﬁcance threshold applied. To aid our interpretation of the cross-ancestry results, we derived analogous within-ancestry estimates for East Asians (ρg = 0.926, 95% CI: (0.967,0.967)) and Europeans (ρg = 0.807, 95% CI: (0.856,0.856); Supplementary Figure S2). Notably, within- ancestry analysis of East Asians yielded signiﬁcantly greater estimates of ρg (t(56.45) = 3.70, Ptwo-sided = 0.0005). However, as CONVERGE represents a single study, actual population differ- ences are confounded here with those arising from ascertainment or heterogeneity in assessment methods and instruments among participating PGC studies. DISCUSSION We have conducted a large, trans-ancestry meta-analysis repre- senting, to our knowledge, the ﬁrst systematic effort to analyze European and Han Chinese studies of MDD. As expected, we identiﬁed a shared, common polygenic basis of MDD between these populations, as exempliﬁed by an excess of same-direction allelic effects, signiﬁcant polygenic risk score proﬁling results and modest estimates of genetic correlation. SNP-based meta-analyses 01ZZ9603, 01ZZ0103 and 01ZZ0403), the Ministry of Cultural Affairs as well as the Social Ministry of the Federal State of Mecklenburg- West Pomerania and the network ‘Greifswald Approach to Individualized Medicine (GANI_MED)’ funded by the Federal Ministry of Education and Research (grant 03IS2061A). Genome-wide data of SHIP have been supported by the Federal Ministry of Education and Research (grant no. 03ZIK012) and a joint grant from Siemens Healthcare, Erlangen, Germany, and the Federal State of Mecklenburg- West Pomerania. The University of Greifswald is a member of the Caché Campus program of the InterSystems GmbH. SHIP LEGEND was funded by the German Research Foundation (grant GR1912/5-1). The Harvard i2b2 project was supported by Award # U54LM008748 from the National Library of Medicine (to ISK) and R01MH086026 and R01MH085542 from the National Institute of Mental Health (to RHP and JWS, respectively). CONFLICT OF INTEREST PFS is a scientiﬁc advisor to Pﬁzer. HJG reports receiving funding from the following: German Research Foundation; Federal Ministry of Education and Research Germany; speakers honoraria from, Eli Lilly and Servier. BM-M consulted for Affectis Pharmaceuticals. RP has received consulting fees from Proteus Biomedical, Concordant Rater Systems, Genomind and RID Ventures. The remaining author declare no conﬂict of interest. Limitations First, the absence of replicable associations with MDD in ancestrally diverse populations precluded more pointed compar- isons of speciﬁc genetic effects. Our attempts to reduce the heterogeneity of MDD, namely by focusing on two particular subtypes of illness, should be regarded as preliminary. Furthermore, questions pertaining to both screen- ing and ascertainment of controls were not addressed in the current study, and could have reduced our power to detect relevant variation. We expect that with larger sample sizes, future studies will be sufﬁciently powered to address these issues. Finally, by having conducted multiple separate analyses for females-only and recurrent MDD, we increased the multiple- testing burden. As these do not represent completely indepen- dent analyses, we have not corrected exhaustively for the total number of tests performed. CONCLUSIONS We have demonstrated a common polygenic basis of MDD that is partially shared between European and Han Chinese populations. Importantly, our ﬁndings appear to reinforce the idea that subtyping of MDD may yield additional insight into its etiology.40 Striking an advantageous balance between phenoty- pically more homogeneous deﬁnitions of illness and sample size represents an ongoing and nuanced challenge for genetic studies of MDD. ACKNOWLEDGMENTS The CONVERGE study was funded by the Wellcome Trust (WT090532/Z/09/Z, WT083573/Z/07/Z, WT089269/Z/09/Z) and by NIH grant MH100549. All authors are part of the CONVERGE consortium (China, Oxford and VCU Experimental Research on Genetic Epidemiology) and gratefully acknowledge the support of all partners in hospitals across China. The PGC is supported by NIH grant U01 MH094421. Statistical analyses were carried out on the Genetic Cluster Computer (http://www.geneticcluster. org), which is ﬁnancially supported by the Netherlands Scientiﬁc Organization (NWO 480-05-003 PI: Posthuma) along with a supplement from the Dutch Brain Foundation and the VU University Amsterdam. We thank TH Pers for support for the DEPICT software. Generation Scotland (GS:SFHS) was supported by a Wellcome Trust Strategic Award 'Stratifying Resilience and Depression Longitudinally' (STRaDL; Reference 104036/Z/14/Z), and received core support from the Chief Scientist Ofﬁce (CSO) of the Scottish Government Health Directorates (grant number CZD/16/6) and the Scottish Funding Council (HR03006). AMM is supported by a Scottish Funding Council Senior Clinical Fellowship and by the Dame Theresa and Mortimer Sackler Foundation. The Bonn/Manheim (BoMa) study was supported by the German Federal Ministry of Education and Research(BMBF) through the Integrated Genome Research Network (IG) MooDS (Systematic Investigation of the Molecular Causes of Major Mood Disorders and Schizophrenia; grant 01GS08144 to Markus M Nöthen and Sven Cichon, grant 01GS08147 to Marcella Rietschel), under the auspices of the National Genome Research Network plus (NGFNplus), and through the Integrated Network IntegraMent (Integrated Understanding of Causes and Mechanisms in Mental Disorders, grant BMBF01ZX1314G), under the auspices of the e:Med Programme. The GenRED GWAS project was supported by NIMH R01 Grants MH061686 (DF Levinson), MH059542 (WH Coryell), MH075131 (WB Lawson), MH059552 (JB Potash), MH059541 (WA Scheftner) and MH060912 (MM Weissman). We acknowledge the contributions of Dr George S Zubenko and Dr Wendy N Zubenko, Department of Psychiatry, University of Pittsburgh School of Medicine, to the GenRED I project. The NIMH Cell Repository at Rutgers University and the NIMH Center for Collaborative Genetic Studies on Mental Disorders SNP-based meta-analyses We thank the twins and their families registered at the Australian Twin Registry for their participation in the many studies that have contributed to this research. Genotyping of STARD was supported by an NIMH Grant MH072802 (SP Hamilton). STARD was funded by the National Institute of Mental Health (contract N01MH90003) to the University of Texas Southwestern Medical Center at Dallas (AJ Rush, principal investigator). SHIP is part of the Community Medicine Research net of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research (grants no. 01ZZ9603, 01ZZ0103 and 01ZZ0403), the Ministry of Cultural Affairs as well as the Social Ministry of the Federal State of Mecklenburg- West Pomerania and the network ‘Greifswald Approach to Individualized Medicine (GANI_MED)’ funded by the Federal Ministry of Education and Research (grant 03IS2061A). Genome-wide data of SHIP have been supported by the Federal Ministry of Education and Research (grant no. 03ZIK012) and a joint grant from Siemens Healthcare, Erlangen, Germany, and the Federal State of Mecklenburg- West Pomerania. The University of Greifswald is a member of the Caché Campus program of the InterSystems GmbH. SHIP LEGEND was funded by the German Research Foundation (grant GR1912/5-1). The Harvard i2b2 project was supported by Award # U54LM008748 from the National Library of Medicine (to ISK) and R01MH086026 and R01MH085542 from the National Institute of Mental Health (to RHP and JWS, respectively). made essential contributions to this project. Genotyping was carried out by the Broad Institute Center for Genotyping and Analysis with support from Grant U54 RR020278 (which partially subsidized the genotyping of the GenRED cases). Collection and quality-control analyses of the control data set were supported by grants from NIMH and the National Alliance for Research on Schizophrenia and Depression. We are grateful to Knowledge Networks (Menlo Park, CA, USA) for assistance in collecting the control data set. We express our profound appreciation to the families who participated in this project, and to the many clinicians who facilitated the referral of participants to the study. The RADIANT studies were funded by the following: a joint grant from the UK Medical Research Council and GlaxoSmithKline (G0701420); the National Institute for Health Research (NIHR) Specialist Biomedical Research Centre for Mental Health at the South London and Maudsley NHS Foundation Trust and King’s College London; and the UK Medical Research Council (G0000647). SNP-based meta-analyses The GENDEP study was funded by a European Commission Framework 6 grant, EC Contract Ref.: LSHB-CT-2003-503428. Max Planck Institute of Psychiatry MARS study was supported by the BMBF Program Molecular Diagnostics: Validation of Biomarkers for Diagnosis and Outcome in Major Depression (01ES0811). Genotyping was supported by the Bavarian Ministry of Commerce, and the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN2 and NGFN-Plus, FKZ 01GS0481 and 01GS08145). The Netherlands Study of Depression and Anxiety (NESDA) and the Netherlands Twin Register (NTR) contributed to GAIN-MDD and to MDD2000. This study was funded by the Netherlands Organization for Scientiﬁc Research (MagW/ ZonMW Grants 904-61-090, 985-10- 002, 904-61-193, 480-04-004, 400-05-717, 912-100- 20; Spinozapremie 56-464-14192; Geestkracht program Grant 10-000-1002); the Center for Medical Systems Biology (NWO Genomics), Biobanking and Biomolecular Resources Research Infrastructure, VU University’s EMGO Institute for Health and Care Research and Neuroscience Campus Amsterdam, NBIC/BioAssist/ RK (2008.024); the European Science Foundation (EU/QLRT-2001-01254); the European Community’s Seventh Framework Program (FP7/2007-2013); ENGAGE (HEALTH-F4-2007-201413); and the European Science Council (ERC, 230374). Genotyping was funded in part by the Genetic Association Information Network (GAIN) of the Foundation for the US National Institutes of Health, and analysis was supported by grants from GAIN and the NIMH (MH081802). Funding for the QIMR samples was provided by the Australian National Health and Medical Research Council (241944, 339462, 389927, 389875, 389891, 389892, 389938, 442915, 442981, 496675, 496739, 552485, 552498, 613602, 613608, 613674 and 619667), the Australian Research Council (FT0991360 and FT0991022), the FP-5 GenomEUtwin Project (QLG2-CT- 2002-01254) and the US National Institutes of Health (AA07535, AA10248, AA13320, AA13321, AA13326, AA14041, MH66206, DA12854 and DA019951) and the Center for Inherited Disease Research (Baltimore, MD, USA). We thank the twins and their families registered at the Australian Twin Registry for their participation in the many studies that have contributed to this research. Genotyping of STARD was supported by an NIMH Grant MH072802 (SP Hamilton). STAR*D was funded by the National Institute of Mental Health (contract N01MH90003) to the University of Texas Southwestern Medical Center at Dallas (AJ Rush, principal investigator). SHIP is part of the Community Medicine Research net of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research (grants no. SNP-based meta-analyses Of particular interest, comparisons based on females-only or recurrent MDD, which better recapitulated the ascertainment strategy in CONVERGE, yielded signiﬁcantly higher estimates of genetic correlation despite an attendant reduction in sample size. We attempted to replicate these single-SNP associations in a collection of independent replication samples (4504 MDD cases and 7007 controls). For lifetime MDD, no single SNP yielded nominally signiﬁcant evidence of association (Po0.05) in ﬁxed- effects meta-analysis of these replication samples (Supplementary Table S5). Replication analyses also failed to generate replication support for SNP associations identiﬁed for females-only or recurrent MDD (Supplementary Tables S6 and S7). Regional association and forest plots for these SNPs are provided in the Supplementary Figures S4–S6. Given the extensive heterogeneity of MDD, and an expected and demonstrable loss of power arising from between-study differences in ancestry and ascertainment, our limited success in identifying novel, replicable evidence of genome-wide signiﬁcant association is perhaps unsurprising. It is well understood that a trait’s heritability—and by extension, a shared polygenic liability— is a less important determinant of successful identiﬁcation of relevant associations than its underlying genetic architecture. Considering the relatively low genetic correlations reported here, we might expect an attenuation of statistical power to detect individual variants, that is, as compared with a similarly sized studies of the same ancestry. A concomitant, statistically For selected SNPs from the trans-ancestry meta-analyses, we assessed the signiﬁcance of the observed fraction of SNPs showing the same direction of effect across discovery and replication phases; these fractions were 0.30 (P = 0.9453), 0.286 (P = 0.9375) and 0.571 (P = 0.5) for lifetime, females-only and recurrent MDD, respectively. Translational Psychiatry (2017), 1 – 7 Translational Psychiatry (2017), 1 – 7 Trans-ancestry genomic analysis of depression TB Bigdeli et al Trans-ancestry genomic analysis of depression TB Bigdeli et al 6 signiﬁcant enrichment of biologically relevant gene sets is taken as an additional support for this interpretation. signiﬁcant enrichment of biologically relevant gene sets is taken as an additional support for this interpretation. made essential contributions to this project. Genotyping was carried out by the Broad Institute Center for Genotyping and Analysis with support from Grant U54 RR020278 (which partially subsidized the genotyping of the GenRED cases). Collection and quality-control analyses of the control data set were supported by grants from NIMH and the National Alliance for Research on Schizophrenia and Depression. SNP-based meta-analyses We are grateful to Knowledge Networks (Menlo Park, CA, USA) for assistance in collecting the control data set. We express our profound appreciation to the families who participated in this project, and to the many clinicians who facilitated the referral of participants to the study. The RADIANT studies were funded by the following: a joint grant from the UK Medical Research Council and GlaxoSmithKline (G0701420); the National Institute for Health Research (NIHR) Specialist Biomedical Research Centre for Mental Health at the South London and Maudsley NHS Foundation Trust and King’s College London; and the UK Medical Research Council (G0000647). The GENDEP study was funded by a European Commission Framework 6 grant, EC Contract Ref.: LSHB-CT-2003-503428. Max Planck Institute of Psychiatry MARS study was supported by the BMBF Program Molecular Diagnostics: Validation of Biomarkers for Diagnosis and Outcome in Major Depression (01ES0811). Genotyping was supported by the Bavarian Ministry of Commerce, and the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN2 and NGFN-Plus, FKZ 01GS0481 and 01GS08145). The Netherlands Study of Depression and Anxiety (NESDA) and the Netherlands Twin Register (NTR) contributed to GAIN-MDD and to MDD2000. This study was funded by the Netherlands Organization for Scientiﬁc Research (MagW/ ZonMW Grants 904-61-090, 985-10- 002, 904-61-193, 480-04-004, 400-05-717, 912-100- 20; Spinozapremie 56-464-14192; Geestkracht program Grant 10-000-1002); the Center for Medical Systems Biology (NWO Genomics), Biobanking and Biomolecular Resources Research Infrastructure, VU University’s EMGO Institute for Health and Care Research and Neuroscience Campus Amsterdam, NBIC/BioAssist/ RK (2008.024); the European Science Foundation (EU/QLRT-2001-01254); the European Community’s Seventh Framework Program (FP7/2007-2013); ENGAGE (HEALTH-F4-2007-201413); and the European Science Council (ERC, 230374). Genotyping was funded in part by the Genetic Association Information Network (GAIN) of the Foundation for the US National Institutes of Health, and analysis was supported by grants from GAIN and the NIMH (MH081802). Funding for the QIMR samples was provided by the Australian National Health and Medical Research Council (241944, 339462, 389927, 389875, 389891, 389892, 389938, 442915, 442981, 496675, 496739, 552485, 552498, 613602, 613608, 613674 and 619667), the Australian Research Council (FT0991360 and FT0991022), the FP-5 GenomEUtwin Project (QLG2-CT- 2002-01254) and the US National Institutes of Health (AA07535, AA10248, AA13320, AA13321, AA13326, AA14041, MH66206, DA12854 and DA019951) and the Center for Inherited Disease Research (Baltimore, MD, USA). 6 CONVERGE consortium. Sparse whole-genome sequencing identiﬁes two loci for major depressive disorder. Nature 2015; 523: 588–591. REFERENCES Biological interpretation of genome-wide association studies using predicted gene func- tions. Nat Commun 2015; 6: 5890. 15 Schizophrenia Working Group of the Psychiatric Genomics, Consortium. Biological insights from 108 schizophrenia-associated genetic loci. 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REFERENCES 20 Ikeda M, Aleksic B, Kinoshita Y, Okochi T, Kawashima K, Kushima I et al. Genome- wide association study of schizophrenia in a Japanese population. Biol Psychiatry 2011; 69: 472–478. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/ by/4.0/ © The Author(s) 2017 Supplementary Information accompanies the paper on the Translational Psychiatry website (http://ww Translational Psychiatry (2017), 1 – 7
https://openalex.org/W2989657509	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/FB5172CFE214C9D686C1382C1E694F9F/S0033291719003374a.pdf/div-class-title-shared-alterations-in-resting-state-brain-connectivity-in-adults-with-attention-deficit-hyperactivity-disorder-and-their-unaffected-first-degree-relatives-div.pdf	English	null	Shared alterations in resting-state brain connectivity in adults with attention-deficit/hyperactivity disorder and their unaffected first-degree relatives	Psychological medicine	2,019	cc-by	11,249	Key words: Attention-deficit/hyperactivity disorder; default mode network; dimensional; endophenotype; functional connectivity; resting state; sustained attention Methods. In a group of adult ADHD probands (n = 20), their unaffected first-degree relatives (n = 20) and typically developing control participants (n = 20), we assessed endophenotypic alterations in the default mode network (DMN) connectivity during resting-state functional magnetic resonance imaging in relation to cognitive performance and clinical symptoms. In an external validation step, we also examined the dimensional nature of this neurocognitive trait in a sample of unrelated healthy young adults (n = 100) from the Human Connectome Project (HCP). Author for correspondence: Deniz Vatansever, E-mail: deniz@fudan.edu.cn Author for correspondence: Deniz Vatansever, E-mail: deniz@fudan.edu.cn Results. The results illustrated reduced anti-correlations between the posterior cingulate cor- tex/precuneus and right middle frontal gyrus that was shared between adult ADHD probands and their first-degree relatives, but not with healthy controls. The observed connectivity alterations were linked to higher ADHD symptoms that was mediated by performance in a sustained attention task. Moreover, this brain-based neurocognitive trait dimensionally explained ADHD symptom variability in the HCP sample. Conclusions. Alterations in the default mode connectivity may represent a dimensional endo- phenotype of ADHD, hence a significant aspect of the neuropathophysiology of this disorder. As such, brain network organisation can potentially be employed as an important neurocog- nitive trait to enhance statistical power of genetic studies in ADHD and as a surrogate efficacy endpoint in the development of novel pharmaceuticals. These authors contributed equally to this work. Cite this article: Pironti VA, Vatansever D, Sahakian BJ (2021). Shared alterations in resting-state brain connectivity in adults with attention-deficit/hyperactivity disorder and their unaffected first-degree relatives. Psychological Medicine 51, 329–339. https:// doi.org/10.1017/S0033291719003374 1Department of Psychiatry, School of Clinical Medicine, University of Cambridge, Cambridge, UK; 2Behavioural and Clinical Neuroscience Institute, University of Cambridge, Cambridge, UK; 3Suno Innova Ltd, Unit 6, 109 Cambridge Road Industrial Estate, Cambridge, UK; 4Institute of Science and Technology for Brain-inspired Intelligence, Fudan University, Shanghai, PR China; 5Division of Anaesthesia, School of Clinical Medicine, University of Cambridge, Cambridge, UK and 6Department of Psychology, University of York, Heslington, York, UK Introduction Attention-deficit/hyperactivity disorder (ADHD) is a childhood onset neurodevelopmental condition, characterised by clinical symptoms of impulsivity, hyperactivity and inattention as well as emotional dysregulation (Kieling et al., 2010) that often persists into adulthood (Faraone, 2007) with far-reaching negative consequences on quality of life (Barkley & Fischer, 2010). In addition to the clinical symptoms and deficits in core cognitive functions (McLean et al., 2004), emerging research now indicates anatomical and neurophysiological abnormalities associated with this disorder. Reductions in grey matter volume and cortical thickness (Carmona et al., 2005; Shaw et al., 2006, 2007), disruptions to white matter tracts (Valera, Faraone, Murray, & Seidman, 2007) and aberrant brain functional network organisa- tion (Konrad & Eickhoff, 2010) have all been suggested to form the neuropathophysiology of ADHD. Collectively, this evidence indicates wide-spread dysfunctions in core neuronal circuits, highlighting the biological underpinnings of ADHD symptomatology. © The Author(s) 2019. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2019. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2019. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. g g g g p g y p gy In this regard, a recent review on the genetic basis of ADHD indicates the mean heritability across 37 twin studies as 74% (Faraone & Larsson, 2019). Furthermore, in a population-based family sample, greater genetic relatedness was linked to greater familial aggregation of ADHD with a heritability estimate of around 80% that was heightened with the persistence of ADHD into adulthood (Chen et al., 2017). However, despite this reported heritability (Franke et al., 2012; Mick & Faraone, 2008; Thapar, O’Donovan, & Owen, 2005), genetic studies for the iden- tification of ADHD susceptibility genes have so far produced disparate results (Hinney et al., Valentino Antonio Pironti1,2,3,, Deniz Vatansever1,4,5,6,* and Barbara Jacquelyn Sahakian1,2,4 Valentino Antonio Pironti1,2,3,, Deniz Vatansever1,4,5,6, and Barbara Jacquelyn Sahakian1,2,4 Valentino Antonio Pironti1,2,3,, Deniz Vatansever1,4,5,6, and Barbara Jacquelyn Sahakian1,2,4 These authors contributed equally to this work. Abstract Received: 27 January 2019 Revised: 15 September 2019 Accepted: 4 November 2019 First published online: 26 November 2019 Background. Attention-deficit/hyperactivity disorder (ADHD) is a developmental condition that often persists into adulthood with extensive negative consequences on quality of life. Despite emerging evidence indicating the genetic basis of ADHD, investigations into the familial expression of latent neurocognitive traits remain limited. Original Article Original Article https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press 330 Valentino Antonio Pironti et al. Williams, Gatt, & Grieve, 2014). Nevertheless, endophenotype- based research studies into the neuropathophysiology of this net- work across ADHD probands and their first-degree relatives in comparison with typically developing controls, as well as direct investigations into its cognitive and clinical correlates, have not been previously conducted. 2011). This might possibly be due to the heterogeneity of the dis- order, intrinsic limitations of the commonly employed noso- graphic systems, experimental focus that is largely confined to young children, and limited reports on endophenotype-based research, which requires further investigation. q g Defined as covert, quantitative traits, endophenotypes are assumed to be highly heritable and closely related to the expres- sion of genes linked to a disorder (Cannon & Keller, 2006). Furthermore, the number of genes associated with such basic neu- rocognitive components are expected to be fewer as compared with those linked to the overt clinical phenotype, which enhances the statistical power in studies aimed at discovering susceptibility genes (Gottesman & Gould, 2003). Thus, similar to the strategies employed in assessing ‘at-risk’ individuals in other mental health disorders such as schizophrenia (Whitfield-Gabrieli et al., 2009), further research into familial endophenotypes that incorporate parents, full siblings or children who share 50% of their genetic makeup with the patients, may provide a more fruitful route to identifying genes that will ultimately improve translational research and drug discovery (Castellanos & Tannock, 2002). This better understanding of the neuropathophysiology of ADHD should in turn improve success rates in the search for novel pharmaceutical agents (Doyle et al., 2005). To this end, here we investigated alterations in brain functional network connectivity within the DMN during rs-fMRI and tested the suitability of this measure as a potential neurocognitive endo- phenotype in a cohort of 20 adult ADHD probands, their 20 unaffected first-degree relatives and 20 typically developing con- trol participants (n = 60). Given prior research, we hypothesised that alterations in the connectivity of DMN to the frontal brain regions would be shared between adults with ADHD and their first-degree relatives, which would significantly correlate with deficits in sustained attention performance and severity of ADHD symptoms. Finally, based on recent reports that highlight ADHD symptomatology as dimensional traits across a continuum of normal to abnormal functioning (Chabernaud et al., 2012; Elton, Alcauter, & Gao, 2014), in a separate dataset from the Human Connectome Project (HCP) (n = 100) we aimed to assess the generalisability of our findings by examining the potential utility of this neurocognitive trait in explaining dimensional vari- ation in ADHD symptomatology in a cohort of unrelated healthy young adults. To this end, emerging familial investigations have recently illu- strated shared abnormalities in sustained attention performance (Gau & Huang, 2014) and brain morphology (Pironti et al., 2014) between ADHD patients and their first-degree relatives that may potentially make up vital endophenotypes of ADHD. In line with these findings, another latent neurocognitive trait may be rooted in the intrinsic brain connectivity dynamics as mea- sured via resting-state functional magnetic resonance imaging (rs-fMRI). Termed functional connectivity (Friston, 1994), inves- tigations into the temporal correlations of spontaneous blood- oxygen-level-dependent (BOLD) signal fluctuations from distinct brain regions (Biswal, Yetkin, Haughton, & Hyde, 1995) have been largely popularised for their utility in identifying modifica- tions to the intrinsic brain functional network organisation across mental health disorders (Fox & Greicius, 2010). Specifically, one particular large-scale system, namely the default mode network (DMN), has been recognised as a new locus of dysfunction in ADHD, with studies reporting on its altered levels of integrity and connectivity during wakeful states of rest (Castellanos, Kelly, & Milham, 2009; Konrad & Eickhoff, 2010; Posner, Park, & Wang, 2014). Participants and clinical assessment Ethical approval for this experiment was obtained from the Cambridgeshire 3 Research Ethics Committee (REC: 09/H0306/ 38). All individuals gave informed consent prior to their partici- pation. ADHD probands and their first-degree relatives were recruited from the Adult ADHD Research Clinic, Addenbrooke’s Hospital, Department of Psychiatry, University of Cambridge, whereas typically developing controls were recruited from the local community. y For the patient cohort, the diagnosis of ADHD (DSM-IV Text Revision) was based on a full clinical interview with the patient and an informant who had known the patient since childhood (e.g. parents). The clinical assessment included Barkley Adult ADHD Rating Scale Version IV (BAARS-IV) for childhood and adult symptoms, provided by both the patient and the informant (Barkley, 2011). In addition, the Mini International Neuropsychiatric Interview (MINI) (Sheehan et al., 1998) was administered to screen for DSM-IV Axis I disorders, and the National Adult Reading Test (NART) (Bright, Jaldow, & Kopelman, 2002) was used to estimate full scale IQ. Eligible ADHD patients were then asked to contact a first-degree relative (e.g. parents, full siblings). Both the first-degree relatives of the ADHD probands and the typically developing control partici- pants were screened for undiagnosed adult ADHD or a comorbid disorder using the same clinical assessments, except for only the self-report BAARS-IV (Barkley & Murphy, 2006) for adult symp- toms was administered in these groups, without the informant. g, ) Comprising the anterior/posterior midline as well as the posterior parietal cortices, the DMN has been associated with internal, self-referential thoughts during unconstrained resting- state conditions (Andrews-Hanna, Smallwood, & Spreng, 2014) with further implications in executive functions commonly impaired in ADHD such as sustained attention, working memory and cognitive flexibility (Bozhilova, Michelini, Kuntsi, & Asherson, 2018; McLean et al., 2004). Across both adults (Castellanos et al., 2008) and adolescents (Sun et al., 2012) with ADHD, previous research has shown significant reductions in anti-correlations (or negative connectivity) of a core DMN hub, namely the posterior cingulate cortex and precuneus (PCC/ PCUN) (Fransson & Marrelec, 2008), and regions commonly implicated in cognitive control. https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press Participants and clinical assessment In combination with these reports, the significant heritability estimate of DMN in the general population (Glahn et al., 2010) suggests the potentially utility of this large-scale network as putative brain-based endophenotype under neurodevelopmental conditions (Korgaonkar, Ram, The exclusion criteria were: NART full scale IQ score equal to or less than 90, current or past history of developmental, neuro- logical or psychiatric disorders based on a formal DSM-IV Text Revisions diagnosis and contraindication to an MRI scan. The final group of ADHD participants recruited for this study was 20 (mean age = 32.20 years, S.D. = 10.31, 3/17 female to male https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press 331 Psychological Medicine ratio), comprising 16 patients with combined type and four with inattentive type; 16 of which were medicated with methylphenid- ate, while four were not receiving any medication for their ADHD diagnosis. In addition to the patient group, a total of 20 age and gender-matched first-degree relatives (mean age = 38.85 years, S.D. = 15.31, 10/10 female to male ratio) of the ADHD probands and 20 typically developing control participants (mean age = 32.5 years, S.D. = 5.8, 7/13 female to male ratio) were recruited for this study. Neither the first-degree relatives, nor the typically developing control participants showed ADHD symptoms meeting the DSM-IV Text Revision diagnostic threshold for ADHD, or a diag- nosis of any other comorbid disorder. With the aim of minimising the potential impact of psychotropic medication on cognitive per- formance and brain dynamics, ADHD participants were asked to withhold taking their medication 24 h before (Gualtieri et al., 1982; Turner, Blackwell, Dowson, McLean, & Sahakian, 2005) and to refrain from consuming alcohol or caffeine-containing drinks on the day of the testing. The same request was made for the first-degree relatives and control participants. imaging sequence parameters for the 8.73-min eyes-closed resting-state functional data acquisition were as follows: 32 slices in each volume, 3.0 mm slice thickness, 3.0 × 3.0 × 3.0 voxel size, TR = 2000 ms, TE = 30 ms, flip angle = 78 degrees, 262 volumes. Cognitive assessment All participants completed the Rapid Visual Information Processing (RVP) test of sustained attention from the Cambridge Neuropsychological Test Automated Battery (CANTAB) (Robbins, James, Owen, & Sahakian, 1994; Sahakian & Owen, 1992). In this task, participants were asked to pay atten- tion to a computer screen in which pseudo-random digits [2–9] were presented one at a time, at a frequency of 100 per min, next to a list of three-digit sequences. Participants had to press a button any time they detected one of the three-digit target sequences. Requiring both sustained attention and working mem- ory, performance in this task has been previously shown to be lower in ADHD patients and their siblings as compared to typic- ally developing controls (Gau & Huang, 2014). Univariate analysis of covariance (ANCOVA) was used to assess group differences in performance, with group membership (i.e. ADHD probands, their first-degree relatives, healthy controls) entered as fixed factor and total hits in the RVP task as the dependent variable. Age, gen- der and NART full scale IQ scores were included as covariates and post-hoc t tests were corrected for multiple comparisons using the Bonferroni method. y The functional connectivity analysis was carried out using the Conn functional connectivity toolbox (Version 17.f) (https://www. nitrc.org/projects/conn) (Whitfield-Gabrieli & Nieto-Castanon, 2012). At first, a strict pipeline of denoising procedure was employed that included CompCor components attributable to the signal from white matter and cerebrospinal fluid (Behzadi, Restom, Liau, & Liu, 2007) as well as a linear detrending term, eliminating the need for global signal normalisation that report- edly enhances spurious anti-correlations (Chai, Castanon, Ongur, & Whitfield-Gabrieli, 2012; Murphy, Birn, Handwerker, Jones, & Bandettini, 2009). The subject-specific six realignment parameters, the main effect of rest-condition and their second- order derivatives were also included in the analysis as potential confounds (Fair et al., 2007). In an additional step to remove potential motion artefacts, the data were motion scrubbed using the composite motion score. The settings of a global signal greater than z = 9 and head motion greater than 2 mm were used to identify volumes with high composite motion score which were then added as potential confounds to the linear regression. Furthermore, mean framewise displacement, denoting scan-to-scan motion, was also calculated to be used as a covariate in our subsequent correlation analyses. Moreover, a temporal filter of 0.009 and 0.08 Hz was applied to focus on low frequency fluctuations (Fox et al., 2005). Functional connectivity analysis Seed-based functional connectivity analyses were shown to be influenced by the definition of seed regions of interest (ROI) (Smith et al., 2011), which can be partially alleviated by the use of large-scale meta-analyses. For this reason, we employed an objective approach to define our seed ROI using Neurosynth (http://neurosynth.org/) (Yarkoni, Poldrack, Nichols, Van Essen, & Wager, 2011). Following the meta-analysis performed on fMRI brain activity data for three search terms stored in the Neurosynth database, namely ‘default (n = 519), default mode (n = 419), default network (n = 80)’, three false-positive discovery rate corrected, statistical co-activation maps (P term\|activation) were created. The three maps corresponding to the three search terms were binarised, overlapped and masked with an anatomical automated labelling atlas-based PCC/PCUN template obtained from the Wake Forest University PickAtlas toolbox (http://fmri. wfubmc.edu/software/pickatlas) (Maldjian, Laurienti, Kraft, & Burdette, 2003). This procedure provided us with a PCC/PCUN seed that was previously utilised in a resting-state study (Vatansever, Manktelow, Sahakian, Menon, & Stamatakis, 2016). PCC, together with PCUN (Fransson & Marrelec, 2008) is considered to be one of the most robust DMN hubs (Leech & Sharp, 2014), hence providing a reliable seed region to assess DMN connectivity. In order to ensure matching of demographic information between groups, age, gender and NART full scale IQ scores were entered into multivariate and univariate analyses of variance (ANOVA) and post-hoc t tests (online Supplementary Table S1) using the SPSS Statistics software package (Version 23.0) (https://www.ibm.com/products/spss-statistics). The self-report BAARS-IV current total symptom scores as well as the inattentive and hyper-impulsive sub-scales were also assessed for group dif- ferences with the same statistical procedures, correcting for age, gender and NART full scale IQ scores (online Supplementary Table S2). Participants and clinical assessment The MRI data preprocessing pipeline included slice-time and motion (six degrees of freedom) corrections, co-registration to the high resolution T1-weighted structural image, normalisation to the Montreal Neurological Institute (MNI) space through the unified segmentation–normalisation algorithm (Ashburner & Friston, 2005) and smoothing with an 8 mm FWHM Gaussian kernel using the SPM software package (Version 12.0) (http:// www.fil.ion.ucl.ac.uk/spm/), based on the MATLAB platform (Version 15a) (http://www.mathworks.co.uk/products/matlab/). MRI data acquisition and preprocessing Participants were scanned using a Siemens MAGNETOM Tim Trio 3T scanner (32-channel head coil) at the Wolfson Brain Imaging Centre, Cambridge. The brain imaging session started with a high resolution T1-weighted, magnetisation-prepared rapid gradient-echo (MPRAGE) structural scan (TR = 2300 ms, TE = 2.98 ms, slice thickness = 1.00 mm). The echo planar https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press 332 Valentino Antonio Pironti et al. Valentino Antonio Pironti et al. The high-quality HCP data were acquired with a very short, 0.72 s TR meaning each slice in a given volume was obtained highly close together, thus eliminating the need for slice-time cor- rection (Glasser et al., 2013). In addition, previous connectivity studies using HCP data have shown that spatial smoothing did not alter the results and thus no smoothing was used (Finn et al., 2015). Finally, instead of the typical anterior–posterior or posterior–anterior phase encoding directions, the HCP rs-fMRI images were acquired with left–right (LR) and right–left (RL) phase encoding directions using an asymmetric acquisition matrix with the purpose of reducing signal loss and distortions that result from high-resolution data acquisition. Hence, functional data from both the LR and RL phase-encoding runs were used to cal- culate average functional connectivity maps (Glasser et al., 2013; Van Essen et al., 2013). Following this procedure, seed-based functional connectivity analyses were performed for each subject using the average BOLD signal from the binarised ROI map described above. One-sample t-statistics were used to obtain PCC/PCUN connect- ivity maps representing the DMN and its anti-correlations for each group. All reported clusters were multiple-comparison cor- rected using the family-wise error detection technique at the 0.05 level of significance (voxel-level 0.001 uncorrected). Moreover, group-level ANOVA was carried out using F-statistics to examine changes in DMN connectivity (PCC/PCUN seed) across ADHD probands, their first-degree relatives and the typic- ally developing control group. The individual connectivity values (z-scores) extracted from the cluster that showed significant alterations in connectivity to the chosen seed were then entered into a univariate ANCOVA (correcting for age, gender and NART full scale IQ score) to further examine the fixed-effects of group membership on DMN connectivity. Post-hoc t tests were carried out to assess pair-wise group differences that were corrected using the Bonferroni method. Subsequently the same data denoising procedures (except for scrubbing) and functional connectivity analyses from the main experimental study was employed for the HCP functional data. Brain, cognition and symptom correlation analysis Across the whole study cohort, the association between PCC/ PCUN connectivity to the brain regions which showed a signifi- cant group interaction (z-scores), BAARS-IV current total symp- tom scores and the number of total hits from the RVP sustained attention task was examined using bivariate non-parametric par- tial Spearman’s correlations (correcting for age, gender, NART full scale IQ score and mean framewise displacement). All mea- sures were assessed for normality using the Shapiro–Wilk test and Q–Q plots, and the correlations were Bonferroni corrected for multiple-comparisons. The results were visualised using the Seaborn package (Waskom et al., 2018) from Python library. In addition, a mediation analysis was performed with the RVP task (total hits) sustained attention scores as the mediator using PROCESS macro Version 3 (Hayes, 2018) with a percentile bootstrap estimation approach (10 000 samples) (Shrout & Bolger, 2002). Finally, the cluster of brain regions showing significant alter- ation in its connectivity to the chosen seed from the main experi- ment was decoded for cognitive terms using Neurosynth meta-analysis toolbox (Rubin et al., 2017). The top 100 terms were chosen, from which we removed anatomical terms such as ‘prefrontal cortex’, leaving behind only the cognitive terminology. These terms were then entered into a word cloud with the asso- ciated weights. A total of 23 cognitive terms (after removal of ana- tomical terms) most associated with activity in this cluster of brain regions was used to create a word cloud to aid in the cog- nitive interpretation of our results. MRI data acquisition and preprocessing A one-sample t test was performed in order to assess DMN con- nectivity, and a binarised mask for the region that showed signifi- cant alterations in connectivity between groups from the main experiment was used to extract connectivity values (z-scores). We then employed bivariate non-parametric partial Spearman’s correlation to assess the link between DMN connectivity and Achenbach Adult Self-Report (ASR) DSM-oriented Attention Deficit/Hyperactivity (AD/H) scale (Achenbach & Rescorla, 2003) within the upper 50th percentile of the full cohort, who scored relatively high on ADHD symptoms (corrected for age, gender and mean framewise displacement). This cut-off point was chosen to avoid floor effects in this neurotypical cohort (mean age = 28.98 years, S.D. = 3.583, female to male ratio = 22/28). External validation dataset and analysis An independent dataset from the HCP was used to provide exter- nal validation to our results from the main experiment, to assess generalisability of our findings and to test the potential utility of the identified neurocognitive traits in explaining dimensional variation in ADHD symptomatology across a group of unrelated healthy young adult participants. A total of 100 participants’ high-quality, openly available data were downloaded from the HCP Q5 Release (mean age = 29.11 years, S.D. = 3.676, 54/46 female to male ratio). Full details on the HCP dataset, including the acquisition parameters are provided in the literature (Van Essen et al., 2013). The output from the HCP minimal preproces- sing pipeline was used for the analyses. Specifically, T1-weighted images from the output of the HCP PreFreeSurfer pipeline that was biased corrected and normalised to the MNI space were uti- lised as high-resolution structural images. Similarly, the func- tional data output of the fMRIVolume pipeline from two-runs of 15-min rs-fMRI sessions that was artefact/motion corrected and normalised to the MNI space was used for the seed-based functional connectivity analyses. This preprocessing pipeline largely matched the one used for the main experimental data besides few exceptions mentioned below. https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press Default mode connectivity alterations In line with previous studies, our PCC/PCUN seed-based func- tional connectivity analysis (Fig. 1a) revealed an intact DMN across all three groups, covering regions of the posterior cingulate, medial prefrontal cortices, bilateral angular gyri, anterior/medial temporal lobes and superior frontal gyri (Fig. 1b). However, an ANCOVA across the three groups with DMN connectivity as the dependent variable highlighted a cluster of brain regions centred on the right middle frontal gyrus (MFG) [extending into the inferior frontal gyrus (IFG)] [MNI peak: +48 + 48 + 16, cluster size = 414 voxels] as showing significant alterations in its connectivity to the chosen seed ROI (F(2,54) = 14.402, p < 0.001, partial η2 = 0.348) (Fig. 1b). Post-hoc t tests on the connectivity values extracted from this cluster showed a significant difference (i.e. reduced anti-correlation) between ADHD probands and con- trols [t(54) = 5.288, 95% confidence interval (CI) (0.163–0.363), https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press 333 Psychological Medicine Fig. 1. Group differences in resting-state default mode connectivity in ADHD probands, their unaffected first-degree relatives and typically developing control par- ticipants. (a) An overlap of the meta-analytic activation maps related to the terms ‘default, default mode and default network’ from the Neurosynth database was used to create an objective seed of the PCC/PCUN for seed-based functional connectivity analysis of the DMN. (b) Group-level one sample t tests and an F-contrast revealed consistent yet altered DMN connectivity across the three groups (displayed on an inflated brain surface from the Montreal Neurological Institute – MNI). Specifically, the group difference was centred on the right middle frontal gyrus (MFG) [MNI peak: +48 + 48 + 16, cluster size = 414 voxels], extending into the inferior frontal gyrus (IFG). Fig. 1. Group differences in resting-state default mode connectivity in ADHD probands, their unaffected first-degree relatives and typically developing control par- ticipants. (a) An overlap of the meta-analytic activation maps related to the terms ‘default, default mode and default network’ from the Neurosynth database was used to create an objective seed of the PCC/PCUN for seed-based functional connectivity analysis of the DMN. (b) Group-level one sample t tests and an F-contrast revealed consistent yet altered DMN connectivity across the three groups (displayed on an inflated brain surface from the Montreal Neurological Institute – MNI). Generalisability to an independent dataset Similar to the results obtained from our main experimental study, the PCC/PCUN seed-based functional connectivity analysis revealed a DMN covering brain regions commonly associated with this network in the literature (Andrews-Hanna et al., 2014) (Fig. 3a). In the upper 50th percentile of this group of unrelated participants, who scored relatively high on ADHD symptoms, the same brain connection identified in our main experiment showed a significant correlation with DSM-oriented AD/H scores [partial rs = 0.341, 95% CI (0.07–0.565), p = 0.019], corrected for age, gen- der and mean framewise displacement (Fig. 3b). In other words, reduced anti-correlation between the PCC/PCUN and right MFG was associated with greater ADHD symptoms. Moreover, further analysis using the ASR DSM-oriented AD/H sub-scales indicated that the observed relationship was mainly originating from the inattentiveness scores [partial rs = 0.397, 95% CI (0.134–0.608), p = 0.006], whereas the hyperactivity scores showed no significant correlation with DMN connectivity [partial rs = 0.109, 95% CI (−0.174 to 0.375), p = 0.465]. Finally, a meta-analytic inquiry into the right MFG on the Neurosynth database revealed terms strongly associated with working memory, attentional load, task demands and other higher executive functions (Fig. 3c). ( ) p p η ] ( g ) In contrast, an ANCOVA between groups on the BAARS-IV current symptom scores showed a significant group difference (F(2,54) = 62.328, p < 0.001, partial η2 = 0.698) that diverged away from brain connectivity and sustained attention task performance results (Table 1, Fig. 2c). Post-hoc t tests revealed that the ADHD probands scored significantly higher than both their first-degree relatives [t(54) = 8.982, 95% CI (21.061–33.165), p < 0.001, partial η2 = 0.599] and typically developing controls [t(54) = 10.229, 95% CI (24.816–36.916), p < 0.001, partial η2 = 0.660]; however, the latter two groups did not show a significant difference to each other [t(54) = 1.241, 95% CI (−2.304 to 9.811), p = 0.220, partial η2 = 0.028]. Similar results were obtained for the inattentive and hyperactive-impulsive sub-scales (online Supplementary Table S2). Default mode connectivity alterations Specifically, the group difference was centred on the right middle frontal gyrus (MFG) [MNI peak: +48 + 48 + 16, cluster size = 414 voxels], extending into the inferior frontal gyrus (IFG). 95% CI (−0.563 to −0.117), p = 0.006] (Fig. 2e), which was in turn negatively correlated with BAARS-IV current total symptom scores [partial rs = −0.456, 95% CI (−0.636 to −0.229), p < 0.001] (Fig. 2f, online Supplementary Table S3), corrected for age, gender and mean framewise displacement. Findings from the inattentive and hyperactive-impulsive sub-scales were in line with the main results (online Supplementary Tables S4 and S5). In addition, a mediation analysis indicated that there was a significant indirect effect of PCC/PCUN to right MFG connectivity on BAARS-IV total current symptom scores through the participants’ perform- ance (total hits) on the RVP sustained attention task [b = 8.054, 95% CI (0.920–19.3727)]. In other words, DMN connectivity was linked to ADHD symptom scores that were approximately 8.1 points higher as mediated by attentional performance. p < 0.001, partial η2 = 0.341] as well as between first-degree relatives and controls [t(54) = 3.440, 95% CI (0.071–0.271), p = 0.001, partial η2 = 0.180]; but there were no significant differences between the ADHD probands and their first-degree relatives [t(54) = 1.844, 95% CI (−0.008 to 0.192), p = 0.071, partial η2 = 0.059] (Table 1, Fig. 2a). Association of brain connectivity, cognition and ADHD symptomatology Across all groups, the BAARS-IV current total symptom scores showed a significant correlation with PCC/PCUN to right MFG connectivity [partial rs = 0.405, 95% CI (0.168–0.598), p = 0.002] (Fig. 2d). Moreover, there was a significant negative correlation between the PCC/PCUN to right MFG connectivity and total hits in the RVP sustained attention task [partial rs = −0.360, Cognitive and clinical assessments In parallel to the DMN connectivity results, ANCOVA for the sustained attention scores showed a significant difference in the RVP total hits across the three experimental groups (F(2,54) = 6.300, p = 0.003, partial η2 = 0.189), in which both the ADHD probands [t(54) = −3.037, 95% CI (−7.477 to −1.531), p = 0.004, partial η2 = 0.146] and their unaffected first-degree relatives [t(54) = −3.110, 95% CI (−7.595 to −1.641), p = 0.003, partial η2 = 0.152] performed significantly worse than the typically developing control participants, whereas the former two groups showed no significant difference to each other [t(54) = −0.077, 95% CI (−3.088 to 2.861), p = 0.939, partial η2 < 0.001] (Table 1, Fig. 2b). In parallel to the DMN connectivity results, ANCOVA for the sustained attention scores showed a significant difference in the RVP total hits across the three experimental groups (F(2,54) = 6.300, p = 0.003, partial η2 = 0.189), in which both the ADHD probands [t(54) = −3.037, 95% CI (−7.477 to −1.531), p = 0.004, partial η2 = 0.146] and their unaffected first-degree relatives [t(54) = −3.110, 95% CI (−7.595 to −1.641), p = 0.003, partial η2 = 0.152] performed significantly worse than the typically developing control participants, whereas the former two groups showed no significant difference to each other [t(54) = −0.077, 95% CI (−3.088 to 2.861), p = 0.939, partial η2 < 0.001] (Table 1, Fig. 2b). In contrast, an ANCOVA between groups on the BAARS-IV current symptom scores showed a significant group difference (F(2,54) = 62.328, p < 0.001, partial η2 = 0.698) that diverged away from brain connectivity and sustained attention task performance results (Table 1, Fig. 2c). Post-hoc t tests revealed that the ADHD probands scored significantly higher than both their first-degree relatives [t(54) = 8.982, 95% CI (21.061–33.165), p < 0.001, partial η2 = 0.599] and typically developing controls [t(54) = 10.229, 95% CI (24.816–36.916), p < 0.001, partial η2 = 0.660]; however, the latter two groups did not show a significant difference to each other [t(54) = 1.241, 95% CI (−2.304 to 9.811), p = 0.220, partial η2 = 0.028]. Similar results were obtained for the inattentive and hyperactive-impulsive sub-scales (online Supplementary Table S2). Valentino Antonio Pironti et al. 334 Table 1. Statistical comparison of resting-state brain connectivity, cognitive performance and symptom scores across ADHD probands, their unaffected first-degree relatives and typically developing control participants, corrected for age, gender and NART full scale IQ score. ADHD probands First-degree relatives Healthy controls ANCOVA Mean S.D. Mean S.D. Mean S.D. F p partial η2 Brain connectivity PCC/PCUN to rMFG (z) −0.02 0.14 −0.10 0.13 −0.27 0.15 14.40 <0.001 0.348 Cognition RVP (total hits) 15.95 5.22 15.35 5.67 22.0 3.57 6.30 0.003 0.189 ADHD symptomatology BAARS-IV (current total symptoms) 36.15 12.39 10.00 7.38 5.20 4.29 62.33 <0.001 0.698 PCC/PCUN, posterior cingulate cortex/precuneus; rMFG, right middle frontal gyrus; RVP, rapid visual information processing; BAARS-IV, Barkley Adult ADHD Rating Scale Version IV. Fig. 2. Shared neurocognitive impairments in adults with ADHD and their unaffected first-degree relatives in relation to cognitive performance and ADHD symp- tomatology. (a) Default mode functional connectivity as characterised by PCC/PCUN to right MFG interactions and (b) cognitive assessment on the RVP task of sustained attention revealed neurocognitive endophenotypes i.e. traits that are potentially closer to the genetic expression as opposed to the clinical outcome. (c) All groups were also assessed for ADHD symptoms using Barkley Adult ADHD Rating Scale Version IV (BAARS-IV) self-reports. In line with the clinical diagnosis, the ADHD probands scored higher than both their first-degree relatives and typically developing control participants, whereas no significant difference was observed between the latter two groups. (d–f ) Non-parametric bivariate partial Spearman’s correlations (rs) across the three experimental groups illustrated sig- nificant links between brain connectivity, cognitive performance and ADHD symptom scores (corrected for age, gender, NART full scale IQ scores and mean frame- wise displacement). In other words, participants with higher BAARS-IV current total symptom scores showed reduced DMN anti-correlation and a smaller number of total hits on the RVP sustained attention task, which in turn was linked to reduced DMN anti-correlation. The violin plots illustrate the kernel density estimation of the underlying distribution, whereas the boxplots show the interquartile range. denotes p < 0.001 and denotes p < 0.01. The shaded areas represent 95% CIs. Fig. 2. Shared neurocognitive impairments in adults with ADHD and their unaffected first-degree relatives in relation to cognitive performance and ADHD symp- tomatology. (a) Default mode functional connectivity as characterised by PCC/PCUN to right MFG interactions and (b) cognitive assessment on the RVP task of sustained attention revealed neurocognitive endophenotypes i.e. Discussion The primary goal of this investigation was to assess whether the default mode functional interactions at rest would show shared https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press Valentino Antonio Pironti et al. Valentino Antonio Pironti et al. traits that are potentially closer to the genetic expression as opposed to the clinical outcome. (c) All groups were also assessed for ADHD symptoms using Barkley Adult ADHD Rating Scale Version IV (BAARS-IV) self-reports. In line with the clinical diagnosis, the ADHD probands scored higher than both their first-degree relatives and typically developing control participants, whereas no significant difference was observed between the latter two groups. (d–f ) Non-parametric bivariate partial Spearman’s correlations (rs) across the three experimental groups illustrated sig- nificant links between brain connectivity, cognitive performance and ADHD symptom scores (corrected for age, gender, NART full scale IQ scores and mean frame- wise displacement). In other words, participants with higher BAARS-IV current total symptom scores showed reduced DMN anti-correlation and a smaller number of total hits on the RVP sustained attention task, which in turn was linked to reduced DMN anti-correlation. The violin plots illustrate the kernel density estimation of the underlying distribution, whereas the boxplots show the interquartile range. * denotes p < 0.001 and denotes p < 0.01. The shaded areas represent 95% CIs. alterations in adults with ADHD and their unaffected first-degree relatives as compared to typically developing controls, thus consti- tuting a brain-based endophenotype of this disorder. Our main results revealed significantly reduced anti-correlation (or negative connectivity) of the chosen PCC/PCUN seed, a main hub of the DMN, with a cluster of brain regions centred on the right MFG extending into the IFG that is commonly associated with cognitive control. Moreover, across the whole cohort of participants, the identified functional connection was linked to ADHD symptom severity with performance in the sustained attention task significantly mediating this relationship. Finally, in an external validation step, we illustrated that the same brain connection identified in our main study also explained dimensional variabil- ity in ADHD symptoms in an independent cohort of unrelated healthy young adults, providing evidence for the generalisability of our findings. Taken together, such results collectively highlight the central importance of the identified default mode functional interaction as a familial neurocognitive trait in adults with ADHD, hence meeting the criteria for an endophenotype of this disorder. https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press 335 Psychological Medicine Fig. 3. Generalisability of the identified neurocognitive link as a dimensional trait in an independent sample of unrelated healthy young adults. Valentino Antonio Pironti et al. (a) Default mode con- nectivity as revealed by PCC/PCUN seed-based functional interactions across two runs of 15-min resting-state ses- sions in a sample of 100 unrelated participants from the HCP showed a similar pattern of connectivity as iden- tified in the main experimental study. (b) In the upper 50th percentile of high symptom scorers, Achenbach Adult Rating Scale (ASR) DSM-oriented AD/H scores and the PCC/PCUN anti-correlation with the right MFG cluster showed a significant link, in which reduced anti- correlation was associated with greater AD/H symptoms (rs = partial Spearman’s correlation corrected for age, gender and mean framewise displacement). (c) Cognitive terms that were most commonly associated with activation in the right MFG was extracted from the Neurosynth database. This right hemispheric cluster depicted strong association with working memory, atten- tional load, task demands and other higher executive functions. While the font size of the terms indicates strength, the colour represents directionality of the load- ings (red = positive, blue = negative). The shaded areas represent 95% CIs. Previous studies investigating resting-state brain functional connectivity alterations have highlighted DMN connectivity, spe- cifically that of the PCC/PCUN, as a potential new locus of dys- function in ADHD (Castellanos et al., 2009; Sonuga-Barke & Castellanos, 2007). For example, in comparison with age and gender-matched healthy volunteers, Castellanos and colleagues reported on significantly reduced anti-correlations between the PCC/PCUN and dorsal anterior cingulate cortex in adults with ADHD (Castellanos et al., 2008), which was further replicated in a group of male adolescents (Sun et al., 2012). Though gener- ally in line, our results instead revealed an impairment in the PCC/PCUN interaction with a cluster centred on the right MFG that is typically associated with a large-scale cognitive con- trol system (Dosenbach, Fair, Cohen, Schlaggar, & Petersen, 2008). This is further evidenced by this cluster’s strong association with terms linked to higher cognitive processes in our meta- analytic decoding. Importantly, such connectivity alterations mirrored the observed group differences in performance during a sustained attention task, but also showed a stark contrast to the overt clinical phenotype. This ‘exophenotype’ remained in line with the existing diagnosis, revealing heightened symptoms in ADHD probands, which did not reach a diagnostic threshold in either the unaffected first-degree relatives or the typically devel- oping controls, yet it still showed significant links with the iden- tified neurocognitive endophenotypes. a potential endophenotype of ADHD. https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press Valentino Antonio Pironti et al. Such developmental differences may underlie the sustained attention deficits observed in adults with ADHD (Martinussen, Hayden, Hogg-Johnson, & Tannock, 2005), which seem to benefit from methylphenidate administra- tion (Turner et al., 2005) that also normalises DMN alterations (Silk, Malpas, Vance, & Bellgrove, 2016). However, more extensive investigations linking physiological and behavioural endopheno- types with susceptibility genes will be required to find causal interactions. To this end, further research aiming to integrate large-scale datasets (Milham, Fair, Mennes, & Mostofsky, 2012) will be necessary to further characterise the potential endopheno- types of ADHD. On the other hand, our group has recently shown DMN engagement during the stable application of learned rules in an externally-oriented, attention-demanding cognitive flexibility task (Vatansever, Menon, & Stamatakis, 2017), which highlights the vital role of this network in core cognitive processes that include, yet transcend beyond internal mentation (Vatansever, Manktelow, Sahakian, Menon, & Stamatakis, 2018). Called the ‘autopilot hypothesis’, the potential role that the DMN might play in the maintenance of contextual-goals for fast and efficient processing while avoiding distractors, even that of our thoughts (Bozhilova et al., 2018), might provide a fresh perspective on the DMN’s dysfunction in ADHD. In fact, along these lines, we recently provided evidence for the link between impaired context-regulation of thoughts, brain connectivity and ADHD symptom severity (Vatansever, Bozhilova, Asherson, & Smallwood, 2018), highlighting the heterogeneity of this associ- ation. In sum, resting-state aberrations found in this study might represent altered DMN contribution to core cognitive and attentional processes. Supplementary material. The supplementary material for this article can be found at https://doi.org/10.1017/S0033291719003374 Acknowledgements. We thank Mrs Victoria Lupson and the rest of the staff in the Wolfson Brain Imaging Centre (WBIC) at Addenbrooke’s Hospital for their assistance in MRI scanning of the original participant cohort. In addition, we thank all the participants for their contribution to this study. All raw data for the main experiment is archived at the Wolfson Brain Imaging Centre, Cambridge and Division of Anaesthesia, School of Clinical Medicine, University of Cambridge, Cambridge, UK. Furthermore, data were provided (in part) by the Human Connectome Project, WU-Minn Consortium (Principal Investigators: David Van Essen and Kamil Ugurbil; 1U54MH091657) funded by the 16 NIH Institutes and Centers that support the NIH Blueprint for Neuroscience Research; and by the McDonnell Center for Systems Neuroscience at Washington University. In relation to the findings of this study, a few limitations should be highlighted. Valentino Antonio Pironti et al. Called the ‘autopilot hypothesis’, the potential role that the DMN might play in the maintenance of contextual-goals for fast and efficient processing while avoiding distractors, even that of our thoughts (Bozhilova et al., 2018), might provide a fresh perspective on the DMN’s dysfunction in ADHD. In fact, along these lines, we recently provided evidence for the link between impaired context-regulation of thoughts, brain connectivity and ADHD symptom severity (Vatansever, Bozhilova, Asherson, & Smallwood, 2018), highlighting the heterogeneity of this associ- ation. In sum, resting-state aberrations found in this study might represent altered DMN contribution to core cognitive and attentional processes. Originally described as a network of brain regions that showed task-induced deactivations during goal-oriented, attention- demanding paradigms (Mazoyer et al., 2001; Raichle et al., 2001; Shulman et al., 1997) and increased activity in autobio- graphical memory-based tasks (Addis, Wong, & Schacter, 2007), the role of the DMN in human cognition has long been confined to self-referential internal mentation processes such as mind- wandering or daydreaming (Andrews-Hanna et al., 2014). Combined with evidence relating lapses in attention to DMN activity (Weissman, Roberts, Visscher, & Woldorff, 2006), and alterations in the core DMN regions in ADHD (Castellanos et al., 2008), the overarching hypothesis for the contribution of this large-scale network to ADHD symptomatology has been the exces- sive generation of mental content that potentially leads to the observed impairments in sustained attention (Posner et al., 2014). g y p Overall, this study suggests that the abnormal DMN connect- ivity is a significant aspect of the adult ADHD neuropathophy- siology that could be employed as a latent neurocognitive trait to enhance statistical power of genetic studies in adult ADHD and as a surrogate efficacy endpoint in the research and develop- ment of novel pharmaceuticals for treatment. In addition to pre- vious research showing volumetric differences in cortical grey matter and aberrant white matter connections in ADHD (Pironti et al., 2014) the identification of candidate genes linked to neuronal growth, myelination, maintenance and protection (Franke et al., 2012) has strengthened the hypothesis that the observed deficits in ADHD may arise from developmental abnor- malities in cortical maturation and pruning that persist into adult- hood (Shaw et al., 2007). Conflict of interest. BJS is a consultant for Cambridge Cognition, PEAK and Mundipharma. Valentino Antonio Pironti et al. Moreover, further medi- ation analysis revealed a significant indirect effect of DMN con- nectivity on ADHD symptom severity through the participants’ performance in the sustained attention task. In a watershed model of biological pathways that link upstream genes to down- stream observable phenotypes, Cannon and Keller have argued for the potential utility of identifying intermediary endopheno- types such as neural circuits and more broader cognitive processes in order to improve our understanding of the mechanisms behind complex mental health disorders (Cannon & Keller, 2006). In light of this analogy, our results indicate that alterations in the connectivity of the main DMN hub, namely the PCC/PCUN, may represent an upstream biological dysregulation. This aberrant connectivity in turn may lead to and be further influenced by downstream cognitive dysfunctions such as attentional problems that result in heightened ADHD symptoms. However, taken together with prior reports (Posner et al., 2014), it is clear that fur- ther research will be required to decipher the exact neural mechanisms behind the DMN functional interactions that are impaired in ADHD, specifically the relevance of anti-correlations between default mode and frontal brain regions to the neuro- pathophysiology of this disorder. Since the initial report by Fox et al., (2005), anti-correlation (or negative connectivity) in the spontaneous BOLD signal fluctua- tions in rs-fMRI data has been an important topic of debate in the neuroimaging literature. Specifically, the biological basis of this relationship remains largely unknown, with some reports advocat- ing for its foundation in the employed computational techniques (Chai et al., 2012; Murphy et al., 2009). Nevertheless, emerging evi- dence now highlights the potential contribution of this functional interaction to healthy and adaptive brain processing (Fox, Zhang, Snyder, & Raichle, 2009; Uddin, Kelly, Biswal, Castellanos, & Milham, 2009), with important modifications observed across ageing (Spreng, Stevens, Viviano, & Schacter, 2016) and in mental g p yp Notably, the PCC/PCUN to MFG connectivity at rest showed a positive correlation with the current total symptoms and a nega- tive correlation with sustained attention task performance, sug- gesting a close link between the DMN’s functional interactions at rest and both inattention and overall symptomatology, which we replicated in an independent cohort of healthy young adults. Collectively, these functional and behavioural links emphasise the significance of the identified disconnection between the PCC/PCUN and right MFG as a neurocognitive trait, and thus https://doi.org/10.1017/S0033291719003374 Published online by Cambridge University Press Valentino Antonio Pironti et al. Valentino Antonio Pironti et al. 336 health disorders (Whitfield-Gabrieli et al., 2009). In parallel, the observed modifications to the segregation between default mode and cognitive control networks have been suggested as a potential mechanism behind the attentional lapses in ADHD (Mills et al., 2018; Posner et al., 2014). However, direct behavioural and func- tional evidence will be required to not only decipher the biological basis and cognitive relevance of default mode anti-correlations, but also their contribution to the attentional deficits in this disorder. directly assess the contribution of default mode interactions to attentional task performance in ADHD. Finally, it is important to note that although all of our analyses were corrected for factors such as age and gender, prior studies do indicate certain charac- teristic differences related to the subtypes and comorbidities in children with ADHD, that are suggested to normalise in adults (Rucklidge, 2010; Williamson & Johnston, 2015). Hence, future research should explicitly test the potential age and gender effects on the brain-based endophenotypes of ADHD. Notwithstanding, the results of our study highlight shared alterations in resting-state DMN connectivity between ADHD probands and their unaffected first-degree relatives that might have a role into modu- lating both overall symptoms and inattention. also their contribution to the attentional deficits in this disorder. Originally described as a network of brain regions that showed task-induced deactivations during goal-oriented, attention- demanding paradigms (Mazoyer et al., 2001; Raichle et al., 2001; Shulman et al., 1997) and increased activity in autobio- graphical memory-based tasks (Addis, Wong, & Schacter, 2007), the role of the DMN in human cognition has long been confined to self-referential internal mentation processes such as mind- wandering or daydreaming (Andrews-Hanna et al., 2014). Combined with evidence relating lapses in attention to DMN activity (Weissman, Roberts, Visscher, & Woldorff, 2006), and alterations in the core DMN regions in ADHD (Castellanos et al., 2008), the overarching hypothesis for the contribution of this large-scale network to ADHD symptomatology has been the exces- sive generation of mental content that potentially leads to the observed impairments in sustained attention (Posner et al., 2014). 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Although we used an objective approach to define this seed and focused on a DMN hub that was previously implicated in ADHD, in light of recent evidence indicating functional sub- specialisation within this network (Kernbach et al., 2018), there might be broader ADHD related alterations in the functional interaction of the DMN that should be further considered. For that purpose, other analysis techniques such as independent com- ponent analysis (Beckmann, Mackay, Filippini, & Smith, 2009) or the assessment of multivariate interactions of the DMN within the wider complex brain network architecture via graph theoretical analyses might prove important avenues for future research (Fornito & Bullmore, 2015). In addition, our study focused on resting-state alterations in brain connectivity that was related to cognitive performance outside the scanner with a computerised task. 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https://openalex.org/W2741974463	https://europepmc.org/articles/pmc5535263?pdf=render	English	null	Usefulness of Intravital Multiphoton Microscopy in Visualizing Study of Mouse Cochlea and Volume Changes in the Scala Media	Frontiers in neurology	2,017	cc-by	7,923	Usefulness of Intravital Multiphoton Microscopy in Visualizing Study of Mouse Cochlea and Volume Changes in the Scala Media Hyun Mi Ju1,2, Sun Hee Lee1,2, Tae Hoon Kong1,2, Seung-Hae Kwon3, Jin Sil Choi1,2 and Young Joon Seo1,2* 1 Laboratory of Smile Snail, Yonsei University Wonju College of Medicine, Wonju, South Korea, 2 Department of Otorhinolaryngology, Yonsei University Wonju College of Medicine, Wonju, South Korea, 3 Department of Bio-imaging, Korea Basic Science Institute, Chuncheon, South Korea Conventional microscopy has limitations in viewing the cochlear microstructures due to three-dimensional spiral structure and the overlying bone. But these issues can be over- come by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM). By using near-infrared lasers for multiphoton excitation, intravital MPM can detect endog- enous fluorescence and second harmonic generation of tissues. In this study, we used intravital MPM to visualize various cochlear microstructures without any staining and non-invasively analyze the volume changes of the scala media (SM) without removing the overlying cochlear bone. The intravital MPM images revealed various tissue types, ranging from thin membranes to dense bone, as well as the spiral ganglion beneath the cochlear bone. The two-dimensional, cross-sectional, and serial z-stack intravital MPM images also revealed the spatial dilation of the SM in the temporal bone of pendrin- deficient mice. These findings suggest that intravital MPM might serve as a new method for obtaining microanatomical information regarding the cochlea, similar to standard histopathological analyses in the animal study for the cochlea. Given the capability of intravital MPM for detecting an increase in the volume of the SM in pendrin-deficient mice, it might be a promising new tool for assessing the pathophysiology of hearing loss in the future. Edited by: Toshihisa Murofushi, Teikyo University, Japan Reviewed by: Chisato Fujimoto, University of Tokyo, Japan Yi-Ho Young, National Taiwan University, Taiwan *Correspondence: Young Joon Seo okas2000@hanmail.net Specialty section: This article was submitted to Neuro-otology, a section of the journal Frontiers in Neurology Received: 24 May 2017 Accepted: 26 June 2017 Published: 31 July 2017 Keywords: cochlea, anatomy, Meniere’s disease, multiphoton microscopy, scala media INTRODUCTION Despite its small size, the cochlea has a complex three-dimensional helical structure, consisting of the modiolus, a central pillar of spongy bone, and the spiral lamina winding around it. These structures include spaced filled with fluid [scala vestibuli, scala media (SM), and scala tympani] and the organ of Corti. The traditional microscopic methods used for examining these cochlear microstructures involve the creation of paraffin-embedded tissue sections. However, the three-dimensional complex microstructure of the cochlea requires precise tissue manipulation for histological processing and viewing technically challenging (1). And many embedding sections may result in the loss of tissue antigenicity. In addition, those make it difficult to conduct immunochemical studies on sections. Original Research published: 31 July 2017 doi: 10.3389/fneur.2017.00332 Mice Six male C57BL/6 mice for ototoxicity testing were involved and allowed free access to water and a regular mouse diet. They were kept at room temperature under a standard 12-h light/dark cycle for 1 week of acclimatization before the experiments. For comparing the changes of the SM, two normal (Pds+/+) and two pendrin-deficient mice (i.e., Pds-knockout; Pds−/−) were obtained from colonies bred from heterozygous mice kindly provided by Choi et al. (16). The animals were 5 weeks old and weighed approximately 18–25 g. The mice were anesthetized by intraperitoneal injection of 30 mg/kg tiletamine–zolazepam (Zoletil, 500 mg/vial; Virbac, Carros, France) and 10 mg/kg xylazine (Rompun; Rompun, Bayer Korea, Ansan, Korea). Animals underwent cardiac perfusion with phosphate-buffered saline (PBS: Dulbecco’s Formula Modified, ICN Biochemicals, England) before tissue harvest. After temporal bones were dissected, the bony shells of the cochlea and vestibule were removed in Cl-free physiologic saline. All experimental pro- cedures performed in this study followed ethical guidelines for animal studies and were approved by the Institutional Animal Care and Use Committee of Yonsei University College of Medicine (IACUC No. YWC-150728-1). All experiments were performed in accordance with relevant guidelines and regulations. In 1990, Denk et al. (3) at Cornell University pioneered two-photon microscopy, later referred to as intravital MPM. It proved to be a useful tool for detailed visualization of the microstructures of various unprocessed tissues, even at the subcellular levels, by using only intrinsic two-photon-excited fluorescence (TPEF) and SHG. The intrinsic endogenous fluorescence and SHG from tissues provide subcellular- resolution images with sufficient morphological details that render them diagnostically useful, as has been demonstrated with unprocessed biopsy specimens obtained from the human urinary bladder (4), ovary (5), gastrointestinal tract (6), lungs (7), and prostate (8). Moreover, the intravital MPM images provide information about tissue architecture, similar to that obtained using established histopathological diagnostic methods. Ablation of the overlying bone is required to access the cochlear microstructures, especially the SM, which is located deep within the cochlear bone. Although conventional fluorescence microscopy provides excellent spatial resolu- tion and allows selective quantification of cochlear changes, it requires the ablation of the overlying bone to access the cochlear structures and preparatory thinning of the cochlear lateral wall. Intravital MPM is a light microscopy technique that allows in vivo imaging up to a depth of 1 mm from the surface of some tissue specimens (9). Citation: Ju HM, Lee SH, Kong TH, Kwon S-H, Choi JS and Seo YJ (2017) Usefulness of Intravital Multiphoton Microscopy in Visualizing Study of Mouse Cochlea and Volume Changes in the Scala Media. Front. Neurol. 8:332. doi: 10.3389/fneur.2017.00332 July 2017 \| Volume 8 \| Article 332 Frontiers in Neurology \| www.frontiersin.org 1 Ju et al. Intravital MPM in the Cochlea Frontiers in Neurology \| www.frontiersin.org MATERIALS AND METHODS Frozen sections can retain the antigenicity of most tissues and can provide thicker sections that allow some depth of field (2). However, these issues can be overcome by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM), which allows the visualization of cochlear microstructures by determin- ing the exited endogenous fluorescence and second harmonic generation (SHG) of the explant cochlea. Frozen sections can retain the antigenicity of most tissues and can provide thicker sections that allow some depth of field (2). However, these issues can be overcome by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM), which allows the visualization of cochlear microstructures by determin- ing the exited endogenous fluorescence and second harmonic generation (SHG) of the explant cochlea. Ototoxic Drug Administrationhi Ototoxic Drug Administration The first injection for each mouse was given at the beginning of the daily light cycle. The three mice in the ototoxicity group received a subcutaneous injection of kanamycin (550 mg/kg; Sigma-Aldrich, Oakville, ON, Canada) dissolved in PBS, fol- lowed by an intraperitoneal injection of furosemide (130 mg/kg; Sigma-Aldrich, Oakville, ON, Canada) via the tail vein 30 min later (17). The three mice in the control group received a subcu- taneous injection of saline, and followed by another saline injec- tion 30 min later intraperitoneally. Mice showing signs of severe dehydration or other serious illness were killed. All animals were monitored by trained animal care technologists supervised by a veterinarian. All animals survived the drug administration. Mice Moreover, it overcomes the constraints posed by the scattering and absorption of photons in light microscopy by using as its excitation source near-infrared light at frequencies that have superior scattering characteristics. The application of two simultaneous photons with high frequency and low energy allows for more focused imaging confined to small volumes (10). Thus, intravital MPM eliminates the need for invasive tissue sampling and complex preparation, while enabling the volumetric visualization of a specimen. Various methods, such as laser-Doppler flowmetry (11, 12), micro-computed tomography (CT) (13, 14), and mag- netic resonance imaging (15), have been applied to visualize the microstructures over the cochlear bone. However, these techniques reveal only the blood vessels over the cochlear bone or require the invasive use of contrast media to identify the microstructure of the SM. Intravital MPM can be used to visualize the SM over the cochlear bone without any contrast injection.i Hearing Testingh The thresholds of Pre-test auditory brainstem response (ABR) were measured 24 h prior to the first drug injection. Each animal was gently anesthetized with an intraperitoneal injection of ketamine (100 mg/kg; Yuhan Corporation, Seoul, Korea) and xylazine (1 mg/kg; Rompun; Korea Bayer, Ansan, Korea) and kept warm by using a heating pad. Under effective anesthesia, subdermal needle electrodes were positioned on the scalp ver- tex, ipsilateral posterior bulla, and contralateral posterior bulla for recording ABR. The clicks were used as a test stimuli using BioSigRP (Tucker-Davis Technologies). The stimulus intensity was decreased gradually in 5-dB steps, and the lowest sound level that caused this waveform was defined as the ‘threshold’. Post- test ABR thresholds were measured 14 days after kanamycin and furosemide administration. We confirmed that all three mice in In this study, we first describe a new morphometric method for various cochlear microstructures by using intravital MPM without any contrast medium, and compare it with light micros- copy and confocal microscopy. We then applied intravital MPM to visualize the volume changes of the SM without removing the overlying cochlear bone in the pendrin-deficient mouse, a patho- logic model for endolymphatic hydrops. To our knowledge, this is the first report on the use of this approach for cochlear imaging. July 2017 \| Volume 8 \| Article 332 2 Ju et al. Intravital MPM in the Cochlea FIGURE 1 \| (A) Experimental setup to microscopy based on an upright microscope and LSM-780 NLO confocal scan head; AOM, acousto-optic modulator; T1, telescope; λ/4, quarter waveplate; G1/G2, galvanometer mirrors. (B) Ti:Sa Laser provides pulsed mode 80 MHz. (C) Pulses out of the laser typically have a <70 fs (D) of 8.7 nm. (E) Schematic showing ex vivo temporal bone imaging (left) and ex vivo imaging of the cryosections of the mouse cochlea (right) by using water- immersion lens. FIGURE 1 \| (A) Experimental setup to microscopy based on an upright microscope and LSM-780 NLO confocal scan head; AOM, acousto-optic modulator; T1, telescope; λ/4, quarter waveplate; G1/G2, galvanometer mirrors. (B) Ti:Sa Laser provides pulsed mode 80 MHz. (C) Pulses out of the laser typically have a <70 fs (D) of 8.7 nm. (E) Schematic showing ex vivo temporal bone imaging (left) and ex vivo imaging of the cryosections of the mouse cochlea (right) by using water- immersion lens. Histological Assessmenth The temporal bones were removed, and the apex of the coch- lea, the round window, and the oval window were punctured. A fixative was perfused through the cochlear apex with 4% paraformaldehyde (Biosesang Seongnam, Korea), and then the sample was immersed in the fixative for 24 h at 4°C. Cochleas were decalcified by immersion in Calci-Clear Rapid (National Diagnostics, Atlanta, GA, USA) for 24 h, dehydrated in 30% sucrose (Sigma-Aldrich, Gillingham, UK) for 24 h, embedded in optimal cutting temperature compound (Leica, Bensheim, Hearing Testingh FIGURE 1 \| (A) Experimental setup to microscopy based on an upright microscope and LSM-780 NLO confocal scan head; AOM, acousto-optic modulator; T1, telescope; λ/4, quarter waveplate; G1/G2, galvanometer mirrors. (B) Ti:Sa Laser provides pulsed mode 80 MHz. (C) Pulses out of the laser typically have a <70 fs (D) of 8.7 nm. (E) Schematic showing ex vivo temporal bone imaging (left) and ex vivo imaging of the cryosections of the mouse cochlea (right) by using water- immersion lens the ototoxicity group showed higher thresholds over 50 dB than did the control mice (Figure S1 in Supplementary Material). Germany), and sectioned at 5- to 2-µm thickness in a cryostat (Leica CM1850 Cryostat; Leica, Wetzlar, German). A standard hematoxylin and eosin (H&E) staining protocol was followed, with a 1- to 3-min incubation in hematoxylin and 30- to 60-s staining with eosin, before mounting the samples. Ex Vivo Intravital MPM Imaging on the Cryosections of Mouse Cochleah The laser operating power was kept below 10 mW to minimize phototoxicity. with a confocal scan head (LSM-780 NLO, Carl Zeiss Microscopy GmbH, Germany). The excitation beam was provided by a mode- locked Ti: Sapphire laser (Spectra-Physics. Irvine, CA, USA) emitting <70 fs width pulses at the wavelength of 690–1,040 nm with the 80 MHz repetition rate. This laser system worked with a DeepSee for group velocity dispersion compensation and an acousto-optic modulator (AOM) for laser power attenuation. And the beam was coupled to the scan head after a collimating telescope (T1). Circular polarized light was used to avoid ani- sotropies for different fibril’s directions. To do this, we placed a broadband quarter wave plate (λ/4-Newport) in the laser beam in front of the microscope scanning head and moved the plate until the optical power after the objective lens no longer changes due to the rotation of the polarizer. The beam was focused onto the sample by a 40×/1.20 NA water immersion. The sample was exposed to an excitation wavelength ranging from 800 to 900 nm, and the output power of the laser was about 10 mW. Images were acquired using the ZEN software (Carl Zeiss). Spectral images were acquired using a 32-channel GaAsP detector, which col- lected photon signals in the selected wavelength range from 415 to 686 nm at an interval of 8.7 nm. The signals were then converted to a pseudo-colored image in which the color of each pixel matched the corresponding photoluminescence spectrum. Ex Vivo Intravital MPM Imaging on the Cryosections of Mouse Cochleah The cryosections of mouse cochlea were visualized under the spectral imaging mode of a multiphoton laser microscope (LSM- 780 NLO; Carl Zeiss, Jena, Germany) equipped with a MaiTai (690–1,040 nm) laser (Figure 1). Figure 1A shows an experimen- tal apparatus. The system showed an upright microscope equipped July 2017 \| Volume 8 \| Article 332 Frontiers in Neurology \| www.frontiersin.org 3 Ju et al. Intravital MPM in the Cochlea FIGURE 2 \| Intravital multiphoton microscopy (MPM) imaging of the microstructures in the sections of the normal cochlea. (A) A schematic cross section of the cochlea showing the three compartments (the scala vestibuli, scala tympani, and scala media), which are separated by two elastic partitions: Reissner’s membrane and the basilar membrane (BM). (B) A micrograph of a 2-mm-thick cryosection slide under light microscopy (200×) does not show the cochlear microstructures. (C) The intravital MPM image without any staining clearly shows the different cochlear microstructures. (D) A schematic of the organ of Corti, which is located above the BM, showing the inner and outer hair cells, as well as hairs attached to the flap called the tectorial membrane, which sits over the organ of Corti. (E) A micrograph of a 2-mm thick cryosection slide under light microscopy (400×). (F) At a higher magnification (400×), the intravital MPM image shows the organ of Corti in detail with green-endogenous autofluorescence and second harmonic generation. FIGURE 2 \| Intravital multiphoton microscopy (MPM) imaging of the microstructures in the sections of the normal cochlea. (A) A schematic cross section of the cochlea showing the three compartments (the scala vestibuli, scala tympani, and scala media), which are separated by two elastic partitions: Reissner’s membrane and the basilar membrane (BM). (B) A micrograph of a 2-mm-thick cryosection slide under light microscopy (200×) does not show the cochlear microstructures. (C) The intravital MPM image without any staining clearly shows the different cochlear microstructures. (D) A schematic of the organ of Corti, which is located above the BM, showing the inner and outer hair cells, as well as hairs attached to the flap called the tectorial membrane, which sits over the organ of Corti. (E) A micrograph of a 2-mm thick cryosection slide under light microscopy (400×). (F) At a higher magnification (400×), the intravital MPM image shows the organ of Corti in detail with green-endogenous autofluorescence and second harmonic generation. Frontiers in Neurology \| www.frontiersin.org RESULTS to 900 nm from an argon ion laser with a power level of up to 10 mW and measurements were taken at the back focal plane of the objective. However, the peaks of intensity were different for five microstructures (Figure 3; region 1: spiral ganglion (SG); region 2: stria vascularis; region 3: TM; region 4: spiral ligament; and region 5: cochlear bone). The TM (blue line) had two high- intensity peaks of 65,353 at 546 nm and 23,496 at 573 nm (Table S1 in Supplementary Material). Although the other cochlear regions had their own peaks, most wavelengths were in the range of 530–610 nm. Discriminating the borders between the stria vascularis, spiral ligament, and cochlear bone was difficult. Using the pseudo-coloring option in the ZEN software (Carl Zeiss) according to the characteristics of different intensities, we could clearly discriminate the borders and even visualize the nuclei of cells in the stria ligaments (Figure 4). Intravital MPM Imaging of Microstructures in the Sections of the Normal Cochlea In the normal cochlea, the space of the SM contains the organ of Corti, surrounded by the spiral ligament, stria vascularis, Reissner’s membrane (RM), and basilar membrane, and attached to the cochlear nerve tract and modiolus (Figure 2A). The organ of Corti consist of the tectorial membrane (TM), outer hair cells (OHC), inner hair cells (IHC), and supporting cells (Deiter cells, Hensen cells, and Claudius cells) (Figure 2D). We could not iden- tify these microstructures in the views for the cryosection slides of the cochlea without H&E staining (Figures 2B,E). Moreover, in the confocal microscopy images, the unstained sections showed very weak autofluorescence. No cochlear microstructures could be visualized by confocal microscopy. Nevertheless, intravital MPM imaging allowed for the easy identification of cochlear microstructures without any staining. Autofluorescence gener- ated over the range of 500 to 650 nm in two different channels as red and green fluorescence was recorded. Although autofluores- cence provides enough low magnification (200×, Figure 2C) to discriminate the microstructures, a higher magnification (400×) could be more beneficial, as shown in Figure 2F.if Ex Vivo Intravital MPM Imaging of the Temporal Boneh (C) Although the five regions have different spectra, the combined spectral image stacks of the five microstructures explain why most of the tissues in the cochlea show similar distribution of green autofluorescence. Ex Vivo Intravital MPM Imaging of the Temporal Boneh p The extracted temporal bones were placed in 2-ml microcentri- fuge tubes containing 250–500 µl 4% paraformaldehyde diluted with 10 mM PBS at pH 7.4 and incubated at room temperature for 24 h. The sample was then placed between the bottom of the confocal glass bottom culture dish from which the cover and cover slip had been removed. Bone wax was filled in the gap between the cover and the bottom of the culture dish and the dish was filled with water (18). The temporal bone was placed on a microscopy examination table filled with water; the water- immersion lens was dipped into this water-containing dish for imaging. The apical and second turns of the cochlea were imaged using the spectral imaging mode of the LSM-780 multiphoton microscope at an excitation wavelength range of 800–900 nm. In situ spectral deconvolution imaging, in which ZEN software (Carl Zeiss) was used to acquire an image in spectral imaging mode and immediately deconvolved it into a signal correspond- ing to the reference spectrum. Background autofluorescence was discarded during the image acquisition period. July 2017 \| Volume 8 \| Article 332 4 Intravital MPM in the Cochlea Ju et al. FIGURE 3 \| Different fluorescence emission spectra of each tissue in the cochlea. (A) The peaks of intensity are different for five microstructures: Region 1 (red), spiral ganglion; region 2 (green), stria vascularis; region 3 (blue), tectorial membrane; region 4 (yellow), spiral ligament; and region 5 (sky-blue), cochlear bone. (B) Excitation spectra of the five different tissues show that irrespective of their own peaks, most wavelengths exist in the range of 530–610 nm. (C) Although the five regions have different spectra, the combined spectral image stacks of the five microstructures explain why most of the tissues in the cochlea show similar distribution of green autofluorescence. FIGURE 3 \| Different fluorescence emission spectra of each tissue in the cochlea. (A) The peaks of intensity are different for five microstructures: Region 1 (red), spiral ganglion; region 2 (green), stria vascularis; region 3 (blue), tectorial membrane; region 4 (yellow), spiral ligament; and region 5 (sky-blue), cochlear bone. (B) Excitation spectra of the five different tissues show that irrespective of their own peaks, most wavelengths exist in the range of 530–610 nm. Frontiers in Neurology \| www.frontiersin.org Intravital MPM Imaging of Microstructures in the Sections of the Cochlea with Ototoxicityh The images of the H&E-stained sections and corresponding intravital MPM images revealed similar ototoxicity-induced morphological changes (Figure 5). The loss of OHC in the organ of Corti was higher in the cochlea exposed to the ototoxicity than the normal cochlea (Figures 5B,H). Extensive destruction of the OHC in the three-layer arrays, normal appearance of the IHC, and decreased count and vacuolization of the SG cells were i Intravital MPM did not show significantly different spectra of intrinsic fluorescence even in the excitation range from 800 July 2017 \| Volume 8 \| Article 332 5 Ju et al. Intravital MPM in the Cochlea FIGURE 4 \| Using the pseudo-coloring option in the ZEN software (Carl Zeiss) according to the characteristics of different intensities, we could compare the green-endogenous autofluorescence and second harmonic generation (SHG) with intravital multiphoton microscopy. (A) (i) SHG is directional depending on the distribution and orientation of the non-linear dipoles, whereas general fluorescence is isotropically emitted. (ii) A diagram illustrating the different reactions in the absorption of two photons to excite the fluorescent molecule and the visible fluorescence emitted during relaxation. (iii) Detection of spectral ranges for excitation at 415–686 nm: half the excitation wavelength for SHG and two spectral bands for two-photon-excited fluorescence (TPEF). (B) Combined SHG/TPEF images of the cochlea clearly show the borders and even the nuclei of cells in the stria ligaments with pseudo-coloring; however, it is not easy to discriminate the borders between the stria vascularis, spiral ligament, and cochlear bone. SHG (violet), cochlear bone; TPEF (green), stria ligaments. FIGURE 4 \| Using the pseudo-coloring option in the ZEN software (Carl Zeiss) according to the characteristics of different intensities, we could compare the green-endogenous autofluorescence and second harmonic generation (SHG) with intravital multiphoton microscopy. (A) (i) SHG is directional depending on the distribution and orientation of the non-linear dipoles, whereas general fluorescence is isotropically emitted. (ii) A diagram illustrating the different reactions in the absorption of two photons to excite the fluorescent molecule and the visible fluorescence emitted during relaxation. (iii) Detection of spectral ranges for excitation at 415–686 nm: half the excitation wavelength for SHG and two spectral bands for two-photon-excited fluorescence (TPEF). Intravital MPM Imaging of Microstructures in the Sections of the Cochlea with Ototoxicityh (B) Combined SHG/TPEF images of the cochlea clearly show the borders and even the nuclei of cells in the stria ligaments with pseudo-coloring; however, it is not easy to discriminate the borders between the stria vascularis, spiral ligament, and cochlear bone. SHG (violet), cochlear bone; TPEF (green), stria ligaments. typical initial characteristics of the cochlea exposed to ototoxicity (Figures 5C,I). Unlike light microscopy and confocal micros- copy, intravital MPM imaging without staining allowed for the identification of changes in the various microstructures of the cochlea exposed to ototoxicity. The morphological features of RM in mice with endolymphatic hydrops were clearly shown in the intravital MPM images.h The bone of apical turn in cochlea was an optical area in Figure 6, but we could confirm the space in both apical and sec- ond turns of cochlea. The dilation of the SM could be clearly seen even at a relative shallow depth in the apical turn. The penetra- tion depth of this wavelength in turbid media (such as biological tissues) varies from approximately 0.5 to 7 mm. Several linear structures in the apical turns exhibit high signal magnification (400×), which indicates the basilar and RMs. The stria vascularis and spiral ligament were well visualized as a distinct crescent- shaped structure adjacent to the promontory bone. The boundary between the bony labyrinths and the membrane labyrinths was clearly identified and a spirall ligament was observed. Clear identification of this structure made it possible to distinguish the overlying stria vascularis. Ex Vivo Intravital MPM Imaging of the SM Space in the Bone Overlying the Cochlea of the Normal and Pendrin-Deficient Mice Figures 6 and 7 show the two-dimensional cross-sectional and serial z-stack intravital MPM images of the internal structures of the extracted bone covering the intact cochlea. We compared the volumetric changes of the SM in normal and pendrin-deficient mice. Pendrin-knockout mice are models of endolymphatic hydrops, which shows spatial dilation of the SM. In the normal cochlea, the SM appears as a right-angled triangle (Figure 6A; Video S1 in Supplementary Material). However, in the cochlea of pendrin-knockout mouse, relatively large endolymphatic fluid-filled spaces are seen in different sections of the SM (Figures 6B,C; Video S2 in Supplementary Material). Reissner’s membrane with a swollen sharp turn bends into the scala vestibuli. We were able to visualize various tissue types from Reissner’s membrane to high density bone and SG. However, the organ of Corti, which contains remarkably delicate hair cells, could not be discriminated from the surrounding tissues.h The original images (Figures 6A–C) could be reconstructed differently by using pseudo-coloring at various wavelengths July 2017 \| Volume 8 \| Article 332 Frontiers in Neurology \| www.frontiersin.org 6 Ju et al. Intravital MPM in the Cochlea FIGURE 5 \| Comparison between intravital multiphoton microscopy (MPM) and conventional light microscopy in the sections of the cochlea exposed to o We compared the intravital MPM images without any staining in the cryosections from normal mice (A–C) and mice exposed to ototoxicity (G–I) with hem and eosin (H&E) staining of normal mice (D–F) and mice exposed to ototoxicity (J–L). The images acquired after H&E staining and the intravital MPM imag imilar morphological changes caused by ototoxicity in the cochlea, organ of Corti, and spinal ganglion. The organ of Corti (B,E,H,K) shows a clear cytoa Note the appearance of the organ of Corti in a normal mouse (B,E) and the injuries to the IHC in the basal turn (H,K): the arrows indicate the area without upporting cells, pillar elements, or hair cells [(H,K) IHC, open arrowheads; OHC, arrowheads]. Note the appearance of the SG in a normal mouse (C,F). he nerve fibers show diminished staining and decreased neuronal packing density in the SG (I,L) of a mouse exposed to ototoxicity. Intravital MPM can b uccessfully used to image the cochlea, and the cochlear structures show good correlation between the intravital MPM images without any staining and conventional light microscopic images with H&E staining. Ex Vivo Intravital MPM Imaging of the SM Space in the Bone Overlying the Cochlea of the Normal and Pendrin-Deficient Mice SV, scala vestibuli; SM, scala media; ST, scala tympani; RM, Reissner’s membrane; TM, tectoria membrane; BM, basilar membrane; SG, spiral ganglion; OHC, outer hair cells; IHC, inner hair cells. FIGURE 5 \| Comparison between intravital multiphoton microscopy (MPM) and conventional light microscopy in the sections of the cochlea exposed to ototoxicity. We compared the intravital MPM images without any staining in the cryosections from normal mice (A–C) and mice exposed to ototoxicity (G–I) with hematoxylin and eosin (H&E) staining of normal mice (D–F) and mice exposed to ototoxicity (J–L). The images acquired after H&E staining and the intravital MPM images reveal similar morphological changes caused by ototoxicity in the cochlea, organ of Corti, and spinal ganglion. The organ of Corti (B,E,H,K) shows a clear cytoarchitecture. Note the appearance of the organ of Corti in a normal mouse (B,E) and the injuries to the IHC in the basal turn (H,K): the arrows indicate the area without supporting cells, pillar elements, or hair cells [(H,K) IHC, open arrowheads; OHC, arrowheads]. Note the appearance of the SG in a normal mouse (C,F). In contrast, the nerve fibers show diminished staining and decreased neuronal packing density in the SG (I,L) of a mouse exposed to ototoxicity. Intravital MPM can be successfully used to image the cochlea, and the cochlear structures show good correlation between the intravital MPM images without any staining and conventional light microscopic images with H&E staining. SV, scala vestibuli; SM, scala media; ST, scala tympani; RM, Reissner’s membrane; TM, tectorial membrane; BM, basilar membrane; SG, spiral ganglion; OHC, outer hair cells; IHC, inner hair cells. July 2017 \| Volume 8 \| Article 332 Frontiers in Neurology \| www.frontiersin.org Ju et al. Intravital MPM in the Cochlea FIGURE 6 \| The two-dimensional cross-sectional (A–C) intravital multiphoton microscopy images (32-channel GaAsP) showing the internal structures of the extracted bone covering the intact temporal bone in the normal and pendrin-deficient mice. (A) In the normal cochlea, the scala media (SM) appears as a right-angled triangle. (B,C) The cochlea of a pendrin-deficient mouse shows a relatively large endolymphatic fluid-filled space, or dilated SM, in the different sections. The images in (A–C) could be reconstructed differently with pseudo-coloring according to different wavelengths. FIGURE 6 \| The two-dimensional cross-sectional (A–C) intravital multiphoton microscopy images (32-channel GaAsP) showing the internal structures of the extracted bone covering the intact temporal bone in the normal and pendrin-deficient mice. Ex Vivo Intravital MPM Imaging of the SM Space in the Bone Overlying the Cochlea of the Normal and Pendrin-Deficient Mice (A) In the normal cochlea, the scala media (SM) appears as a right-angled triangle. (B,C) The cochlea of a pendrin-deficient mouse shows a relatively large endolymphatic fluid-filled space, or dilated SM, in the different sections. The images in (A–C) could be reconstructed differently with pseudo-coloring according to different wavelengths. FIGURE 7 \| The serial z-stack intravital multiphoton microscopy images (32-channel GaAsp) of the internal structures of extracted bone covering intact cochlea. (A) The normal cochlea has the scala media (SM) shown as a right-angled. On the contrary. (B) The cochlea of pendrin knockout mouse confirms relatively large endolymphatic fluid-filled space, SM, at each different section. FIGURE 7 \| The serial z-stack intravital multiphoton microscopy images (32-channel GaAsp) of the internal structures of extracted bone covering intact cochlea. (A) The normal cochlea has the scala media (SM) shown as a right-angled. On the contrary. (B) The cochlea of pendrin knockout mouse confirms relatively large endolymphatic fluid-filled space, SM, at each different section. (Figure 7). While the normal cochlea showed strong intensi- ties at wavelengths ranging from 450 to 500 nm, the cochlea of pendrin-deficient mice showed strong intensities at wavelengths ranging from 580 to 620 nm. These differences could determine the different pseudo-coloring between the cochlea of normal and pendrin-deficient mice. the same region before. Following tissue processing, it was shown that two-photon excitation intrinsic fluorescence and SHG provide similar microstructural information, as in stand- ard histopathology. Although the conventional histopathology could be cheap and an easy access tool, it could not evaluate the whole 3D spiral structure of cochlea. And the reissner’s membrane would be ruptured during manipulating the speci- men. Definitely, there might be several advantages in cochlear study with using the intravital MPM. Our results demonstrate that, similar to H&E stating, two-photon-excited intrinsic fluo- rescence and SHG can provide enough structural and spectral contrast to identify the occurrence of ototoxicity in the cochlea. Intravital MPM could detect typical ototoxicity-related patho- logical changes in mouse microstructures, without the need for tissue processing. Furthermore, intravital MPM could be used to observe the space of the SM and to compare the pathologic changes occurring in this space, without having to remove the Frontiers in Neurology \| www.frontiersin.org DISCUSSION We describe a new method for microscopic analysis of the mammalian cochlea that offers several advantages over alter- native histological preparations and allows for non-invasive spatial visualization of the SM without removing the cochlear bone. First, we performed ex vivo intravital MPM imaging on cryosections of unprocessed cochlea from normal mice and those exposed to ototoxicity. A comparison of the images acquired after H&E staining and intravital MPM images from July 2017 \| Volume 8 \| Article 332 Frontiers in Neurology \| www.frontiersin.org 8 Intravital MPM in the Cochlea Ju et al. bone covering the unprocessed cochlea in pendrin-deficient mice.t possible to discriminate the microstructures in the cochlear cryo- sections by pseudo-coloring. However, discriminating the stria ligaments between the stria vascularis and the cochlear bone can be difficult because these layers are closely attached to each other. Different spectra of wavelengths in different tissues (vessels, ligaments, bone, etc.) could be easily shown as different colors by using the ZEN software. We presume the spectra would differ according to differences in the density and laser absorption ability of cells. Intrinsic emissions from living tissues can yield different details that might ultimately prove useful in clinical diagnosis. Fluorescence is the emission of visible light after the substance has absorbed light or other electromagnetic radiation. Normally, the absorbed light gets a shorter wavelength and higher energy than the emitted light. This is known as single photon fluorescence applied in traditional fluorescence microscopy. In conventional confocal microscopy, upon excitation, the fluorophore molecule absorbs energy from a single photon and then releases energy as emitted photons. In contrast, intravital MPM excite the fluoro- phores in the specimen with pulsed longer wavelength light (19). In two-photon excitation, two photons must fill the phosphor simultaneously (within 10–18 s) in order to excite the phosphor to emit fluorescent photons of shorter wavelength and higher energy. This phenomenon is rare and can occur only in high photon density regions. This phenomenon makes intravital MPM more advantageous than conventional confocal microscopy (3, 20, 21). Intravital MPM is based on non-linear optical effects such as SHG. Owing to the intensity of the laser passing through a highly polarized material, SHG is generated at precisely half the wavelength of the original light. By using near-infrared lasers for two-photon excitation, the SHG can be kept in the range of the visible wavelength. DISCUSSION Because many intrinsic biological structures, including collagen fiber, muscle, brain, and bone, induce this kind of effect, these structures can be visualized without labeling them with exogenous probes. Using intravital MPM, Pena et al. (22) provided significant insights into lung fibrosis; in their study, intravital MPM allowed for the separation of the inflammatory and fibrotic steps in the pathophysiology of lung fibrosis. In brain research, we studied nerve structure and function using intravital MPM imaging. Dendrites and spine can be imaged to study changes in shape and size. Changes in the axonal morphology of deep brain tissue could be determined by visualizing the activity of the neuronal population (23, 24). Our findings in the mouse cochlea show that the excited endogenous autofluorescence and SHG in intravital MPM images can provide useful information similar to histopathological analyses, without the need for tissue processing. We also demonstrate the utility of replacing histo- pathological analysis of the cochlea with intravital MPM without staining. Leake et al. (25) studied the effect of aminoglycoside ototoxicity, the first row of OHC first degenerated, then the second row, then the third row; the last cells to degenerate in a given region were the IHC. Moreover, the SG displayed different patterns of degeneration, early cell loss occurred in the middle of the cochlea. In our study, Intravital MPM images revealed these ototoxicity-induced changes in the cochlear organ of the mouse cochlea, the stria vascularis and the organ of the SG. Another advantage of intravital MPM is the ability to obtain bright, high-resolution images from deep areas of tissues and organs. In this study, we have referred to these images as intra- vital MPM images for discriminating them from the cryosection images acquired using intravital MPM. The intravital MPM can spatially limit the excitation region to the focal point of the objective lens and the objective lens concentrates photons in a very small region (9). This enables the visualization of structures without fixation or thin sectioning. The near-infrared laser used in the intravital MPM can absorb more or less penetrate than the visible light or UV light used in confocal microscopy and can deeply penetrate, so it has a depth of 100–1,000 µm in the body MPM. Depths less than 100 µm can be visualized using confocal microscopy (21). DISCUSSION Visualizing deep cochlear bone tissue has inherent limitations because both visible light and infrared light are easily scattered by crystallized calcium phosphate in the bone matrix. However, the cochlear bone of the mouse is only 32–100 µm thick, which is thin enough to fall within the penetration range of the MPM in the body. Intravital MPM overcomes a number of problems associated with substantial histological artifacts due to fixation, decalcification, dehydration, and cochlear implantation. An entire cochlea can be imaged using intravital MPM within a couple of minutes, whereas the fixation, decalcification, paraffin embedding, sectioning, and imaging in histological analysis would typically take a week or more to complete. In addition, excitation with near-infrared lasers can minimize photobleaching, i.e., the destruction of fluorophores and phototoxicity-induced tissue damage. Optical coherence tomography could identify the histological confirma- tion of the cochlea (27). OCT is an imaging technique that utilizes the principle of interferometry to obtain images of tomographic images such as ultrasound and CT. Though the OCT is capable of depth-resolved imaging and its axial resolution is good, the scattering contrast of OCT lacks the molecular and biochemical specificity, while intravital MPM could detect the other fluores- cent proteins in the live animal study. We thought that intravital MPM and OCT have complementary characteristics which make them good candidates for viewing the microstructures of cochlea in the future. As long wavelength and low energy excitation light reduces photodamage and increases penetration depth, thereby enabling imaging of live specimens, intravital MPM has revolutionized development in biological imaging, it is thought (26). The most commonly used fluorophores in intravital MPM have excitation spectra in the range of 400–500 nm, whereas the laser used to produce TPEF emits in the wavelength range of 700–1,000 nm. In our study, we collected photon signals in the selected wavelength range from 415 to 686 nm after excitation using wavelengths ranging from 800 to 900 nm. By using these wavelengths, it was Pds−/− mice develop early-onset, profound deafness, and hence serve as a valuable model for studying auditory dysfunc- tions associated with pendrin deficiency. The lack of pendrin in Pds−/− mice leads to excessive dilation of the SM inside the cochlea. This is secondary to a change in the osmotic pressure environment of the endolymph caused by the absence of the anion transport function of pendlin (28). Frontiers in Neurology \| www.frontiersin.org AUTHOR CONTRIBUTIONS VIDEO S1 \| The serial z-stack views in the scala media of the normal mice cochlea. VIDEO S1 \| The serial z-stack views in the scala media of the normal mice cochlea. YS carried out design of the study and performed the statisti- cal analysis. HJ and TK participated in performing the animal VIDEO S2 \| The serial z-stack in the scala media of the pendrin-knockout mice cochlea. FUNDING This research was supported by Basic Science program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF- 2015R1C1A1A02036354) and by the Technology Innovation Program (or Industrial Strategic Technology Development Program) (10070232) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea). This research was supported by Basic Science program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF- 2015R1C1A1A02036354) and by the Technology Innovation Program (or Industrial Strategic Technology Development Program) (10070232) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea). 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https://openalex.org/W2996474877	http://evu.encyclopedia.kyiv.ua/volume-10/articles/entsyklopedychnyi-visnyk-ukrainy-2018.pdf#page=31	Ukrainian	null	Subject specific encyclopedias as an important stage in the development of scientific disciplines and terminology systems (on example of The Mining Encyclopedia as a work in mining industry and geology)	Enciklopedičnij vìsnik Ukraïni	2,018	cc-by	29,854	Адреса редакції: Україна, 01024, м. Київ-4, вул. Терещенківська, 3 e-mail: esu@esu.com.ua www.evu.encyclopedia.kiev.ua Україна, 01024, м. Київ-4, вул. Терещенківська, 3 e-mail: esu@esu.com.ua www.evu.encyclopedia.kiev.ua НАЦІОНАЛЬНА АКАДЕМІЯ НАУК УКРАЇНИ ІНСТИТУТ ЕНЦИКЛОПЕДИЧНИХ ДОСЛІДЖЕНЬ _______________________________________________________________ ЕНЦИКЛОПЕДИЧНИЙ ВІСНИК УКРАЇНИ Число 10 2018 РЕДАКЦІЙНА КОЛЕГІЯ Головний редактор Микола Железняк Відповідальний секретар Олександр Іщенко Сергій Бортник Іван Дзюба Тетяна Добко Станіслав Кульчицький Константин Манолаке (Молдова) Ірина Матяш Любомир Пиріг Роман Сенькусь (Канада) Ярослав Яцків Адреса редакції: Україна, 01024, м. Київ-4, вул. Терещенківська, 3 e-mail: esu@esu.com.ua www.evu.encyclopedia.kiev.ua Засновано у жовтні 2009 року 2018 © Інститут енциклопедичнних досліджень НАН України НАЦІОНАЛЬНА АКАДЕМІЯ НАУК УКРАЇНИ ІНСТИТУТ ЕНЦИКЛОПЕДИЧНИХ ДОСЛІДЖЕНЬ ЕНЦИКЛОПЕДИЧНИЙ ВІСНИК УКРАЇНИ ЕНЦИКЛОПЕДИЧНИЙ ВІСНИК УКРАЇНИ Число 10 2018 EDITORIAL BOARD Editor-in-Chief Executive Secretary Herald contacts: Ukraine, 01024, Kyiv-4, 3, Тereshchenkivska str. e-mail: esu@esu.com.ua www.evu.encyclopedia.kiev.ua Ukraine, 01024, Kyiv-4, 3, Тereshchenkivska str. e-mail: esu@esu.com.ua www.evu.encyclopedia.kiev.ua Засновано у жовтні 2009 року 2018 © Інститут енциклопедичнних досліджень НАН України NATIONAL ACADEMY OF SCIENCES OF UKRAINE INSTITUTE OF ENCYCLOPEDIC RESEARCH _______________________________________________________________ ЕNTSYKLOPEDYCHNYI VISNYK UKRAINY (THE ENCYCLOPEDIA HERALD OF UKRAINE) Volume 10 2018 EDITORIAL BOARD Editor-in-Chief Mykola Zhelezniak Executive Secretary Oleksandr Ishchenko Serhiy Bortnyk Ivan Dziuba Tetiana Dobko Stanislav Kulchytskyi Constantin Manolache (Moldova) Iryna Matiash Lubomyr Purih Roman Senkus (Canada) Yaroslav Yatskiv Herald contacts: Ukraine, 01024, Kyiv-4, 3, Тereshchenkivska str. e-mail: esu@esu.com.ua www.evu.encyclopedia.kiev.ua Founded in October 2009 2018 © NASU Institute of Encyclopedic Research NATIONAL ACADEMY OF SCIENCES OF UKRAINE INSTITUTE OF ENCYCLOPEDIC RESEARCH ЕNTSYKLOPEDYCHNYI VISNYK UKRAINY Founded in October 2009 2018 © NASU Institute of Encyclopedic Research Спецвипуск Спецвипуск до 100-річчя Національної академії наук України Special volume devoted to the 100th anniversary of National Academy of Sciences of Ukraine Special volume Special volume devoted to the 100th anniversary of National Academy of Sciences of Ukraine p devoted to the 100th anniversary of National Academy of Sciences of Ukrain devoted to the 100th anniversary of National Academy of Sciences of Ukraine ЗМІСТ Микола Железняк Енциклопедична справа в Національній академії наук України: історія та сьогодення.................................................................................................................6 Олександр Іщенко Про порушення і захист «екології» енциклопедичних знань (огляд звіту Центру досліджень Європейського парламенту про національні онлайн-енциклопедії в країнах Європи)..........................................................................19 Володимир Білецький, Геннадій Гайко Галузева енциклопедія як важливий етап розвитку наукової дисципліни та її терміносистеми (на прикладі циклу праць із гірництва й геології «Гірнича енциклопедія»).....................................................................................................31 Микола Железняк, Олександр Іщенко Академічні онлайн-енциклопедії в контексті розвитку сучасного інформаційного простору.................................................................................39 Українська енциклопедична справа в термінах Матеріали до словника енциклопедичних термінів.............................................50 Українська енциклопедична справа в подіях Тетяна Березюк, Оксана Давиденко Енциклопедична хроніка............................................................................................83 ..83 4 Researches in encyclopedia science Mykola Zhelezniak The encyclopedia publishing in the National Academy of Sciences of Ukraine: history and present ..........................................................................................................6 Oleksandr Ishchenko On violation and protection of the information ecosystem in national encyclopedias (review of the report on national online encyclopedias in Europe by the European Parliamentary Research Service) ................................19 Volodymyr Biletskyi, Hennadii Haiko Subject specific encyclopedias as an important stage in the development of scientific disciplines and terminology systems (on example of The Mining Encyclopedia as a work in mining industry and geology).........................................31 Mykola Zhelezniak, Oleksandr Ishchenko Academic online encyclopedias in wiev of the recent information space developing....................................................39 Ukrainian encyclopedia science in terms Materials to Glossary for Encyclopedia Science .........................................................50 Events of the encyclopedia publishing in Ukraine Tetiana Bereziuk, Oksana Davydenko Encyclopedic newsreel....................................................................................................83 Mykola Zhelezniak The encyclopedia publishing in the National Academy of Sciences of Ukraine: history and present ..........................................................................................................6 Ukrainian encyclopedia science in terms Materials to Glossary for Encyclopedia Science .. Енциклопедичний вісник України. 2018. Число 10 Events of the encyclopedia publishing in Ukraine Tetiana Bereziuk, Oksana Davydenko Encyclopedic newsreel............................. 5 ДОСЛІДЖЕННЯ З ЕНЦИКЛОПЕДИСТИКИ ЕНЦИКЛОПЕДИЧНА СПРАВА В НАЦІОНАЛЬНІЙ АКАДЕМІЇ НАУК УКРАЇНИ: ІСТОРІЯ ТА СЬОГОДЕННЯ* Микола Железняк Інститут енциклопедичних досліджень НАН України, Київ, Україна У дослідженні схарактеризовано українську академічну енциклопедичну спра- ву. Увагу акцентовано на особливостях розвитку енциклопедистики в Націо- нальній академії наук України: окреслено її зародження та найважливіші ака- демічні енциклопедичні видання. Проаналізовано спектр енциклопедичних проектів за науковими напрямами та зроблено висновок про значне кількісне переважання представлення знань у форматі енциклопедій суспільно-гумані- тарних наук над природничими й технічними. Запропоновано власне бачення подальшого розвитку академічної енциклопедистики. Ключові слова: національна енциклопедія, академічна енциклопедистика, Національна академія наук України, «Енциклопедія Сучасної України», Науко- ве товариство імені Шевченка. Українська енциклопедистика, на думку багатьох дослідників, почина ється 1914 року, коли з’явився перший том синтетичної праці «Украинскій народъ в его прошломъ и настоящемъ», у створенні якої взяли участь най- * Дослідження оприлюднено на круглому столі «Енциклопедистика в діяльності Націо нальної академії наук України та Наукового товариства імені Шевченка», приуроченого до 100-річчя Національної академії наук України (www.roundtable2018.encyclopedia.kiev.ua). Енциклопедичний вісник України. 2018. Число 10 © Інститут енциклопедичних досліджень НАН України © Інститут енциклопедичних досліджень НАН України Про енциклопедичну справу в Національній академії ... визначніші науковці того часу – Федір Вовк, Михайло Грушевський, Агатан гел Кримський, Михайло Туган-Барановський, Олександр Русов, Степан То машівський, Петро Єфименко, Степан Рудницький та багато інших. Хоча за родження традицій створення довідкової літератури, матеріали яких характе ризуються енциклопедичністю, сягають ще часів Київської Русі – тоді писали ізборники, найвідомішим серед яких є «Ізборник Святослава» (1073 рік); зго дом – України Середніх віків, коли укладали лексикони, зокрема «Лексіконъ славенорωсскїй альбо Именъ тлъкованїє» П. Беринди (1627 рік). Упродовж 20 століття енциклопедистика пройшла етапи становлення й поступу. За радянської доби розвиток енциклопедичної справи пов’язаний, з одного боку, з видавництвом УРЕ (згодом реорганізувалося в Головну ре дакцію УРЕ (1957–1989), створену 1930 року для підготовки «Української радянської енциклопедії»), що стала найвідомішим енциклопедичним ви данням в радянській Україні; з іншого боку – з Науковим товариством імені Шевченка, що здійснювало підготовку національної енциклопедії в емігра ції – «Енциклопедії українознавства», а до цього – «Української загальної ен цикльопедії», що з’явилася в 3-х томах у 1930–1935 роках. Розвиток енциклопедичної справи в НАН України починається зі здобут тя Україною незалежності, а з нею, як зазначає С. Кульчицький, і «права влас ності на власну історію» [7, с. 39], це уможливило в 1990-х роках появу ідеї про створення сучасної національної енциклопедії, яка згодом почала реалізову ватися під назвою «Енциклопедія Сучасної України». ЕНЦИКЛОПЕДИЧНА СПРАВА В НАЦІОНАЛЬНІЙ АКАДЕМІЇ НАУК УКРАЇНИ: ІСТОРІЯ ТА СЬОГОДЕННЯ* Так, спочатку Комітет науки і культури при АН України (нині – Український міжнародний комітет з питань науки і культури при НАН України) у співпраці з Науковим това риством імені Шевченка, передусім А. Жуковським, мав на меті до 80-річчя заснування Академії створити осучаснену версію «Енциклопедії українознав ства», яка формально була б її продовженням – ЕУ-3 (докладніше див.: [3]). Втім, під час підготовчих робіт стало зрозуміло, що цю ідею реалізувати фактично неможливо, або це дуже компліковано й створить багато незруч ностей користувачеві, оскільки він повинен обов’язково мати можливість паралельно користуватися «Енциклопедією українознавства», що лише в останні роки стала доступною для українського читача, натомість матері ал, який почала отримувати редакційна колегія ЕУ-3, показав, що можливо створити цілком нову українську неабияк актуальну для утвердження нової держави національну енциклопедію, і «розпорядженням Президії НАН Укра їни № 329 від 5 березня 1996 року було сформовано Координаційне бюро з питань підготовки та видання “Енциклопедії Сучасної України” на правах структурного підрозділу Українського міжнародного комітету з питань науки і культури при НАН України» [1, с. 50]. Координаційне бюро згодом реорга нізували в Інститут енциклопедичних досліджень НАН України (Постанова Президії НАН України № 121 від 14 квітня 2004 року), основне завдання яко го – підготовка «Енциклопедії сучасної України», окрім цього, Інститут було визначено координаційним центром в Україні з питань енциклопедистики. 7 Микола Железняк Звичайно, варто пам’ятати, що розвиток академічної енциклопедисти ки тісно пов’язаний передусім з «Українською радянською енциклопедією», адже перше 17-томне видання цієї енциклопедії (за ред. М. Бажана, 1959– 1965) було підготовлено, коли Головна редакція УРЕ перебувала у складі АН УРСР (1957–1974); від 1974 вона була в підпорядковуванні Держком видаву УРСР, саме тоді видано друге 12-томне видання «Української радян ської енциклопедії» та його російськомовна версія – «Украинская советская энциклопедия». Дослідники зауважують, що друге видання здійснювалося в суттєво гірших політичних умовах, аніж перше. Виведення УРЕ зі складу АН УРСР і диктат партійного керівництва, особливо вороже ставлення до на ціональних енциклопедій М. Суслова, негативно вплинуло на рівень переви даної УРЕ: багато матеріалів з першого видання не потрапило через цензуру на його сторінки [6]. У період перебування видавництва УРЕ в структурі Академії, окрім «Української радянської енциклопедії», вийшла друком також низка фунда ментальних енциклопедичних праць: перше видання «Українського радян ського енциклопедичного словника» у 3-х томах (за ред. М. Бажана, 1966– 1968), «Енциклопедія народного господарства УРСР» у 4-х томах (відп. ред. С. Ямпольський, 1969–1972), «Радянська енциклопедія історії України» у 4-х томах (відп. ред. А. ЕНЦИКЛОПЕДИЧНА СПРАВА В НАЦІОНАЛЬНІЙ АКАДЕМІЇ НАУК УКРАЇНИ: ІСТОРІЯ ТА СЬОГОДЕННЯ* Скаба, 1969–1972), перша в світі «Енциклопедія кібер нетики» у 2-х томах (відп. ред. В. Глушков, 1973) та інші відомі галузеві й регіональні енциклопедичні одно- й багатотомники, до створення яких так чи інакше були причетні науковці академічних установ. Однак характерною особливістю статей у цих виданнях, особливо суспільного й історичного спря мувань, є заідеологізований підхід у викладенні фактів, оцінці подій чи харак теристиці осіб. Загалом у той час енциклопедична справа була радше части ною (складовою) державної політики, ніж фундаментальної науки. Хоча все ж необхідно наголосити на тому, що підготовка зазначених вище енциклопедій з науково-методичної точки зору була на високому рівні, а ідеологічне наша рування стосувалося на той час майже всіх соціогуманітарних наукових на прямів, особливо історичних галузей. Академічна енциклопедистика за роки незалежності держави здійснила вагомий внесок у подолання інтелектуальної ізольованості, ознайомлення зі світовим досвідом у всіх сферах життя, відтворення повної картини історії, науки, культури. Наголосимо, що особлива роль у цьому належить згаданій вище багатотомній енциклопедії національного значення – «Енциклопедії Сучасної України». Це видання про сучасне політичне, економічне, наукове, культурне та духовне життя в Україні, яке ґрунутється на кращих традиціях як вітчизняного, так і зарубіжного енциклопедознавства. Серед авторів енци клопедії – відомі у світі дослідники, визнані авторитети у своїй галузі – про відні фахівці НАН України, галузевих академій, науковці вищої школи, мис тецтвознавці, краєзнавці, видатні науковці діаспори. Зауважимо, що натепер вона складається з 20-ти томів і є найбільшим в Україні енциклопедичним 8 Про енциклопедичну справу в Національній академії ... виданням, оскільки кількісно вже переважає найбільшу донедавна 17-томну «Українську радянську енциклопедію». Ще одна важлива особливість цієї ен циклопедії – представлення її в мережі Інтернет як мультимедійної онлайн- енциклопедії1. Великим науково-культурним досягненням стало видання різними академічними установами низки галузевих енциклопедій, серед яких фундаментальна «Енциклопедія історії України» в 10-ти томах (відп. ред. В. Смолій, 2003–2013), «Шевченківська енциклопедія» в 6-ти то мах (відп. ред. М. Жулинський, 2012–2015), «Юридична енциклопедія» в 6-ти томах (відп. ред. Ю. Шемчушенко), «Політична енциклопедія» (відп. ред. Ю. Левенець, 2012), «Енциклопедія етнокультурознавства» (відп. ред. Ю. Римаренко, 2000–2002), 4 видання енциклопедії «Україн ська мова» (відп. ред. В. Русанівський, 2004–2013), 5 томів «Української музичної енциклопедії» (відп. ред. Г. Скрипник), «Філософський енци клопедичний словник» (відп. ред. В. Шинкарук), «Астрономічний ен циклопедичний словник» (відп. ред. І. Климишин, А. Корсунь, 2003), 2-томний енциклопедичний словник «Фізика твердого тіла» (відп. ред. В. Бар’яхтар, 1996–1998). Нещодавно опубліковано «Українську нафтога зову енциклопедію» (відп. ред. В. Іванишин, 2016) та «Українську фоль клористичну енциклопедію» (відп. ред. В. 1 Режим доступу: http://esu.com.ua ЕНЦИКЛОПЕДИЧНА СПРАВА В НАЦІОНАЛЬНІЙ АКАДЕМІЇ НАУК УКРАЇНИ: ІСТОРІЯ ТА СЬОГОДЕННЯ* Сокіл), а також перші томи «Франківської енциклопедії» в 7-ми томах (відп. ред. М. Жулинський, 2016) та перший том енциклопедії «Країни світу і Україна», запланованої в 5-ти томах (відп. ред. А. Кудряченко, 2017). Загальний перелік енци клопедій НАН України (зокрема й тих, що підготовлено в радянські часи) наводимо окремо (див. додаток). Варто також зауважити про участь різних установ НАН України у підго товці низки українських енциклопедичних видань, що публікувалися поза Академією. Серед них – енциклопедії, підготовлені Науковим товариством імені Шевченка, наприклад, енциклопедія «Наукове товариство імені Шев ченка» (відп. ред. О. Купчинський) – за участі Інституту енциклопедичних досліджень НАН України; проект «Гірнича енциклопедія» (кер. В. Білець кий, містить «Тлумачний гірничий словник», «Гірничий енциклопедичний словник» і «Малу гірничу енциклопедію», 1998–2013) – за участі Інституту геохімії, мінералогії та рудоутворення імені М. Семененка НАН України, Ін ституту фізико-органічної хімії й вуглехімії імені Л. Литвиненка НАН Укра їни. А також енциклопедії, підготовлені різними компетентними видав ництвами, наприклад, «Українська літературна енциклопедія» (відп. ред. І. Дзеверін, М. Жулинський, 1988–1995) – за участі Інституту літератури імені Т. Шевченка НАН України; «Енциклопедія Львова» у 4-х томах (відп. ред. А. Козицький, І. Підкова, 2007–2010) – за участі Інституту українознав ства імені І. Крип’якевича НАН України та Інституту народознавства НАН 9 9 Микола Железняк України; «Екологічна енциклопедія» у 3-х томах (відп. ред. А. Толстоухов, 2006–2008) – за участі Інституту ботаніки імені М. Холодного та Інституту зоології імені І. Шмальгаузена; «Українська дипломатична енциклопедія» у 2-х томах (відп. ред. Л. Губерський, 2004) – за участі Інституту держави і права імені В. Корецького. Нині фахівців НАН України залучено до створен ня універсальної вітчизняної багатотомної енциклопедії – «Великої україн ської енциклопедії», яку готує державна наукова установа «Енциклопедич не видавництво». Згадаємо також і про те, що чимало українських видань, що творяться поза академічними інститутами, часто застосовують принципи й методич ні рекомендації, запропоновані для укладання академічних енциклопедій. Наприклад, у передмові до «Тернопільського енциклопедичного словника» зазначено, що його готують на засадах укладання «Енциклопедії Сучасної України». На основі згаданого вище переліку пропонуємо кількісний аналіз акаде мічних енциклопедій. Загальна кількість енциклопедичних видань, підготов лених установами НАН України, – 49. Із них відповідно до типології2 енци клопедичних видань (рис. 1): • загальні енциклопедії – 3 (ЕСУ, УРЕ, УРЕС) і і 6 • загальні енциклопедії – 3 (ЕСУ, УРЕ, УРЕС) • галузеві енциклопедичні видання – 46. галузеві енциклопедичні видання 46. галузеві енциклопедії загальні енциклопедії 94% 6% Рис. 1. Типологічний розподіл енциклопедичних видань у НАН України Рис. 1. Галузеві енциклопедії репрезентують такі наукові напрями (рис. 2): • суспільно-гуманітарні науки – 35 видань, • фізико-математичні науки – 6 видань, • хімічно-біологічні науки – 5 видань. 2 Питання типології українських енциклопедичних видань висвітлено в: [9]. • хімічно-біологічні науки – 5 видань. • фізико-математичні науки – 6 видань, ЕНЦИКЛОПЕДИЧНА СПРАВА В НАЦІОНАЛЬНІЙ АКАДЕМІЇ НАУК УКРАЇНИ: ІСТОРІЯ ТА СЬОГОДЕННЯ* Типологічний розподіл енциклопедичних видань у НАН України • суспільно-гуманітарні науки – 35 видань, Рис. 1. Типологічний розподіл енциклопедичних видань у НАН України Галузеві енциклопедії репрезентують такі наукові напрями (рис. 2): • суспільно-гуманітарні науки – 35 видань, • фізико-математичні науки – 6 видань, • хімічно-біологічні науки – 5 видань. 2 Питання типології українських енциклопедичних видань висвітлено в: [9]. 10 Для порівняння: представлення наукових напрямів установами Н їни (згідно з даними – [2]; рис. 3): секція суспільних і гуманітарні наук – 35 установ, секція фізико-технічних і математичних наук – 75 установ, секція хімічних та біологічних наук – 43 установи. суспільно-гуманітарні науки фізико-математичні науки хімічно-біологічні науки 11% 13% 76% Рис. 2. Розподіл енциклопедичних видань за науковими напрямами (в межах НАН України) енциклопедії установи суспільно-гуманітарні науки фізико-математичні науки хімічно-біологічні науки Рис. 3. Представлення академічних знань в енциклопедичному форматі Про енциклопедичну справу в Національній акад Про енциклопедичну справу в Національній академії ... Для порівняння: представлення наукових напрямів установами НАН України (згідно з даними – [2]; рис. 3): • секція суспільних і гуманітарні наук – 35 установ, • секція фізико-технічних і математичних наук – 75 установ, • секція хімічних та біологічних наук – 43 установи. суспільно-гуманітарні науки фізико-математичні науки хімічно-біологічні науки 11% 13% 76% Рис. 2. Розподіл енциклопедичних видань за науковими напрямами (в межах НАН України) Рис. 2. Розподіл енциклопедичних видань за науковими напрямами (в межах НАН України) енциклопедії установи суспільно-гуманітарні науки фізико-математичні науки хімічно-біологічні науки Рис. 3. Представлення академічних знань в енциклопедичному форматі енциклопедії установи суспільно-гуманітарні науки фізико-математичні науки хімічно-біологічні науки Рис. 3. Представлення академічних знань в енциклопедичному форматі Рис. 3. Представлення академічних знань в енциклопедичному форматі Викладені статистичні дані наштовхують на певні міркування. 1. Бажано, щоб кожна чи декілька установ НАН України, що відповіда ють за науковий напрямок, підготували видання, що представлятиме на укові досягнення й здобутки академічних працівників і загалом науковців України, які є фахівцями відповідної галузі знань, у форматі енциклопедії. 11 Микола Железняк Такі видання, окрім наукового значення, будуть дієвим засобом популяри зації в суспільстві науки загалом і НАН України зокрема. 2. Як видно з поданих діаграм, у форматі енциклопедичних видань пред ставлені передусім наукові знання, що створюються установами суспіль но-гуманітарного профілю, однак попри порівняно велику їхню кількість, відзначаємо відсутність ґрунтовних енциклопедичних видань про: еконо міку, соціологію, всесвітню історію, археологію, сходознавство, мистецтво знавство тощо. Можемо констатувати, що перед українською енциклопе дистикою лежить неоране поле не лише в галузі природничих чи техніч них наук, а й гуманітраних. До речі, нам є, з кого брати приклад: у сусідній Білорусі виданнями енциклопедичного формату охоплено майже всі сфери життя (див.: [5]). 2. Рис. 1. Типологічний розподіл енциклопедичних видань у НАН України Нині в авторській базі ЕСУ – понад 4000 осіб, які брали чи беруть участь у підготовці статей для енциклопедії, 12 Про енциклопедичну справу в Національній академії ... велика частина з яких – науковці, зокрема працівники академічних інститу тів. Загалом редакція енциклопедії має налагоджені контакти з переважною більшістю науково-дослідних установ Академії, а також закладів вищої осві ти, обласних, районних і міських державних адміністрацій, творчих спілок, музеїв, спортивних товариств, громадських організацій. Широка консолідація фахівців над створенням національної енциклопедії – це запорука успішності видання, умова його об’єктивності й науковості. Рис. 1. Типологічний розподіл енциклопедичних видань у НАН України Як видно з поданих діаграм, у форматі енциклопедичних видань пред ставлені передусім наукові знання, що створюються установами суспіль но-гуманітарного профілю, однак попри порівняно велику їхню кількість, відзначаємо відсутність ґрунтовних енциклопедичних видань про: еконо міку, соціологію, всесвітню історію, археологію, сходознавство, мистецтво знавство тощо. Можемо констатувати, що перед українською енциклопе дистикою лежить неоране поле не лише в галузі природничих чи техніч них наук, а й гуманітраних. До речі, нам є, з кого брати приклад: у сусідній Білорусі виданнями енциклопедичного формату охоплено майже всі сфери життя (див.: [5]). 3. Очевидно, найбільшим надбанням досвіду підготовки таких видань, як «Енциклопедія Сучасної України», «Енциклопедія українознавства» чи «Українська радянська енциклопедія», є практика залучення до написання енциклопедичних статей відомих науковців, наукових колективів, установ, товариств тощо. 3. Очевидно, найбільшим надбанням досвіду підготовки таких видань, як «Енциклопедія Сучасної України», «Енциклопедія українознавства» чи «Українська радянська енциклопедія», є практика залучення до написання енциклопедичних статей відомих науковців, наукових колективів, установ, товариств тощо. 4. У контексті залучення вчених до створення енциклопедичних видань варто згадати про важливість проведення попередньої роботи: затверджен ня концептуальних і методичних засад, на яких буде створюватися енци клопедичне видання, укладання слóвника, узгодження низки питань із під готовки матеріалів. Зазвичай редакція видає методичні рекомендації, роз робляє схеми чи зразки статей, укладає бібліографічні покажчики й часто започатковує публікування періодичного чи неперіодичного збірника (жур налу, вісника, бюлетеня). «З метою налагодження тісних зв’язків із читача ми та для ширшого залучення наукових установ і громадських організацій до підготовки статей УРЕ редбюро енциклопедії на своєму засіданні 23 січня 1931 р. ухвалило рішення створити “Бюлетень УРЕ”» – про це говориться в першому номері цього видання (див.: [4]). Н. Черниш зауважує, що на його сторінках мали на меті оприлюднювати інструктивні та науково-методичні матеріали, призначені для авторів і редакторів енциклопедії [10, с. 153]. Подібне інформаційне видання мала й редакція «Енциклопедії україно знавства» – «Вісті із Сарселю», в якому обговорювалися біжучі проблеми створення ЕУ, провадили дискусії, друкували відгуки та оцінки як окремих статей, так і енциклопедії загалом. Ці видання є важливим і цікавим джере лом відомостей про творення українських енциклопедій. Започаткував такий часопис й Інститут енциклопедичних досліджень НАН України під назвою «Енциклопедичний вісник України». Окрім цього, для створення «Енциклопедії Сучасної України» важливим було попереднє напрацювання методичних інструкцій щодо підготовки статей, які спочатку існували для вузького кола осіб (редакторів, авторів), а згодом їх опублі ковано науковим виданням (див.: [8]). Секція суспільних і гуманітарних наук 1. Енциклопедія етнокультурознавства: У 2 ч. / Редкол.: Ю. Римаренко, Ю. Шемшученко та ін. та ін.; Інститут держави і права ім. В. Корецького НАН України Київ, 2000–2002. 2. Енциклопедія історії України: У 10 т. / Редкол.: В. Смолій та ін.; Інститут історії України НАН України. Київ, 2003–2013. 3. Енциклопедія м. Часів Яр / Редкол.: В. Гірчак та ін. ; Центр пам’яткознавства НАН України. Часів Яр; Київ, 2010. 4. Енциклопедія морських катастроф України: затонулі пам’ятки антич ності, середньовіччя і нової, історії: залишки міст і поселень, військові ко раблі, цивільні судна, авіа- та бронетехніка / Укл.: С. Воронов; Інститут археології НАН України. Київ, 2008. 5. Енциклопедія народного господарства Української РСР: У 4 т. / Відп. ред. С. Ямпольський. Київ: Головне видавництво УРЕ АН УРСР, 1969– 1972. 6. Енциклопедія Сучасної України: В 30-ти т. Т. 1–20 / Редкол.: І. Дзюба, А. Жуковський, М. Железняк та ін.; Інститут енциклопедичних дослід жень НАН України. Київ, 2001–2018. 7. Звід пам’яток історії та культури України / Редкол.: В. Смолій, Г. Боряк та ін.; Інститут історії України НАН України. Київ, 1999–2018; 8. Дипломатія сучасної України: Енциклопедичний довідник / Редкол.: М. Железняк, І. Дзюба та ін.; Інститут енциклопедичних досліджень НАН України. Київ, 1997. 9. Історія міст і сіл Української РСР: В 26 т. / Гол. ред. П. Тронько. Київ: Го ловна редакція УРЕ АН УРСР, 1968–1973. 10. Країни світу і Україна: У 5 т. Т. 1: Північна Європа. Західна Європа. Пів денна Європа / Редкол.: А. Кудряченко, А. Бульвінський та ін.; Інститут всесвітньої історії НАН України. Київ: Фенікс, 2017. 1. Культура: Ілюстрована енциклопедія України / Укл.: М. Попович. Київ, 2010 13 Микола Железняк 12. Мала енциклопедія етнодержавознавства / Редкол.: Ю. Римаренко та ін.; Інститут держави і права ім. В. Корецького НАН України. Київ, 1996. 13. Мала енциклопедія міжнародного права / Редкол.: Ю. Бошицький та ін.; Київський університет права НАН України. Київ, 2013. 14. Мала енциклопедія теорії держави і права / Редкол.: Ю. Бошицький та ін.; Київський університет права НАН України. Київ, 2010. 15. Мала енциклопедія українського народознавства / Редкол.: С. Павлюк та ін.; Інститут народознавства НАН України. Львів, 2007. 16. Микола Костомаров: Віхи життя і творчості: Енциклопедичний довід ник / Редкол.: В. Смолій, Ю. Пінчук, О. Ясь; Інститут історії України НАН України. Київ, 2005. 17. Наукове товариство імені Шевченка. Енциклопедія. Т. 1–3 / Редкол.: О. Купчинський та ін.; НТШ, Інститут енциклопедичних досліджень НАН України. Львів, 2012–2016. 18. Політична енциклопедія / Редкол.: Ю. Левенець, Ю. Секція суспільних і гуманітарних наук Шаповал та ін.; Інститут політичних і етнонаціональних досліджень ім. І. Кураса. Київ, 2012. 19. Політологічний енциклопедичний словник: У 2-х вид. / Редкол.: Ю. Шем чушенко, В. Бабкін та ін.; Інститут держави і права ім. В. Корецького НАН України. Київ, 1997–2004. 20. Радянська енциклопедія історії України: У 4 т. / Відп. ред. А. Скаба. Київ: Головне видавництво УРЕ АН УРСР, 1969–1972. 21. Тарас Шевченко: енциклопедичний онлайн-портал / Інститут літератури ім. Т. Шевченка НАН України; Інститут телекомунікацій і глобального ін формаційного простору НАН України. Київ, 2014. 22. Україна в міжнародних відносинах: Енциклопедичний словник-довід ник. Вип. 1–4 / Інститут історії України НАН України. Київ, 2009–2013. 23. Українська літературна енциклопедія: У 5 т. Т. 1–3 / Редкол.: І. Дзеверін, М. Жулинський та ін.; Інститут літератури ім. Т. Шевченка НАН України Київ, 1988–1995. 24. Українська мова: Енциклопедія: У 4-х вид. / Редкол.: В. Русанівський та ін.; Інститут української мови НАН України; Інститут мовознавства ім. О. Потебні НАН України К., 2004–2013. 25. Українська музична енциклопедія: У 6 т. Т. 1–5 / Редкол.: Г. Скрипник та ін.; Інститут мистецтвознавства, фольклористики та етнології імені М. Рильського НАН України Київ, 2006–2018. 26. Українська радянська енциклопедія: У 17 т. / За ред. М. Бажана. Київ: Го ловне видавництво УРЕ АН УРСР, 1959–1965. 27. Українська фольклористична енциклопедія / Гол. ред. В. Сокіл; Інститут народознавства НАН України. Львів, 2018. 14 Про енциклопедичну справу в Національній академії ... 28. Український енциклопедичний кінословник / Редкол.: С. Безклубенко та ін.; Інститут мистецтвознавства, фольклористики та етнології ім. М. Риль ського НАН України. Київ, 2006. 29. Український радянський енциклопедичний словник: У 3 т. / За ред. М. Ба жана. Київ: Головне видавництво УРЕ АН УРСР, 1966–1968. 30. Філософський енциклопедичний словник: А–Я / Редкол.: В. Шинкарук та ін.; Інститут філософії ім. Г. Сковороди НАН України. Київ, 2002. 31. Франківська енциклопедія: У 7 т. Т. 1 / Редкол.: М. Жулинський, Є. Нахлік та ін.; Інститут Івана Франка НАН України Львів, 2016. 32. Художня культура західних і південних слов’ян (ХІХ — початок ХХ сто ліття): Енциклопедичний словник / Редкол.: Г. Скрипник та ін.; Інститут мистецтвознавства, фольклористики та етнології ім. М. Рильського НАН України. Київ, 2006. 33. Шевченківська енциклопедія: У 6 т. / Редкол.: М. Жулинський та ін.; Ін ститут літератури ім. Т. Шевченка. Київ, 2012–2015. 34. Шевченківський словник: У 2 т. Редкол.: Є. Кирилюк та ін.; Інститут літе ратури ім. Т. Шевченка. Київ, 1976–1978. 35. Юридична енциклопедія: у 6 т. / Редкол.: Ю. Секція суспільних і гуманітарних наук Шемшученко та ін.; Ін ститут держави і права ім. В. Корецького НАН України. Київ, 1998– 2004. Секція фізико-технічних і математичних наук 1. Астрономічний енциклопедичний словник / Редкол.: І. Климишин, А. Корсунь та ін.; ГАО НАН України, Львівський національний універси тет. Київ; Львів, 2003. 2. Енциклопедія кібернетики: В 2 т. / Редкол.: В. Глушков та ін.; Інститут кібернетики АН УРСР. Київ: Головне видавництво УРЕ, 1973. 3. Імена України в Космосі: Науково-енциклопедичне видання / Редкол.: І. Вавилова, В. Плачинда; ГАО НАН України. Київ, 2003. 4. Надтверді абразивні матеріали в механообробці: Енциклопедичний довідник / Редкол.: М. Новіков та ін.; Інститут надтвердих матеріалів ім. В. Бакуля НАН України. Київ, 2013. 5. Неорганическое материаловедение: Энциклопедическое издание / Сост.: Г. Гнесин и др.; Институт проблем материаловедения им. И. Францевича НАН Украины. Киев, 2007–2008. 6. Физика твердого тела: Энциклопедический словарь: В 2 т. / Редкол.: В. Барьяхтар и др. Киев: НАН Украины, 1996–1998. 15 Микола Железняк Секція хімічних і біологічних наук 1. Ихтиопатологическая энциклопедия / Редкол.: О. Давидов та ін.; Інститут зоології ім. І. Шмальгаузена НАН України. Київ, 2000. 2. Мінералогічна енциклопедія України: онлайн-енциклопедія (Українська мінералопедія) / За ред. О. Пономаренка; Інститут геохімії, мінералогії та рудоутворення ім. М. Семененка НАН України. Київ, 2016 3. Паразитологія та патологія риб: Енциклопедичний словник-довідник / Редкол.: А. Гаєвська та ін.; Інститут проблем міцності ім. Г. Писаренка НАН України; Інститут біології південних морів імені О. Ковалевського НАН України. Київ, 2004. 4. Українська енциклопедія лісівництва: У 2 т. / Редкол.: С. Генсірук та ін.; НТШ, НАН України. Львів, 1999–2007. 5. Українська нафтогазова енциклопедія / Редкол.: В. Іванишин та ін.; Ін ститут геології і геохімії горючих копалин НАН України. Львів, 2016. ЛІТЕРАТУРА 1. Бойко Ю. Інститут енциклопедичних досліджень Національної академії наук України: історія створення і діяльність // Гілея: науковий вісник. 2016. Вип. 109. С. 50–55. 2. Довідкова інформація. Статистичні дані // Національна академія наук України у 2017 році: аналітичний звіт. К.: Академперіодика, 2018. С. 40. 3. Железняк М. Енциклопедія Сучасної України: історія створення // Наука України у світовому інформаційному просторі: зб. наук праць. Вип. 12. Київ, 2015. С. 115–127. 4. Завдання бюлетеню // Бюлетень УРЕ. 1931. № 1. С. 1–4. 5. Імя Петруся Броўкі. Беларуская Энцыклапедыя. Мінск, 2005. 6. Ісаєвич Я., Железняк М. Енциклопедія // Енциклопедія Сучасної України Т. 9 / Редкол.: І. Дзюба, А. Жуковський, М. Железняк та ін.; Інститут ен циклопедичних досліджень НАН України. Київ, 2009. С. 2016–209. 7. Кульчицький С. Вітчизняна історія в енциклопедичному форматі // Ен циклопедичний вісник України. 2009. Ч. 1. С. 39-46. 8. Методичні засади створення паперових і електронних енциклопедичних видань: посібник / Відп. ред. М. Железняк; Інститут енциклопедичних до сліджень. Київ: Академперіодика, 2015. 9. Українські енциклопедії: типологія, стиль, функції: монографія / Відп. ред. Я. Яцків; Інститут енциклопедичних досліджень НАН України. Київ, 2018. 10. Черниш Н. Нариси з історії української енциклопедичної справи. Львів: Піраміда, 2018. 16 Про енциклопедичну справу в Національній академії ... Mykola Zhelezniak NASU Institute of encyclopedic research, Kyiv, Ukraine Mykola Zhelezniak NASU Institute of encyclopedic research, Kyiv, Ukraine The study describes the academic encyclopedias of Ukraine. The main attention is paid to the peculiarities of the development of the encyclopedia publishing at the National Academy of Sciences of Ukraine: its origins and the most important aca demic encyclopedias are outlined. The spectrum of encyclopedic projects is analyzed by the scientific branches and the conclusion is drawn about the significant advantage of social sciences and humanities over others in the contex of presenting knowledge in encyclopedic format. Some vision of further development of the encyclopedia publi shing in Academy is proposed. Keywords: National Encyclopedia, Academic Encyclopeditia publishing, Natio nal Academy of Sciences of Ukraine, Encyclopedia of Modern Ukraine, Shevchenko Scientific Society. nia. Nauka Ukrainy u svitovomu informatsiinomu prostori, 12, 115-127 (in Ukrainian). Zhelezniak, M. (Ed.). (2015). Metodychni zasady stvorennia paperovykh i elektronnykh entsyklopedychnykh vydan. Kyiv: Akademperiodyka (in Ukrainian). nia. Nauka Ukrainy u svitovomu informatsiinomu prostori, 12, 115-127 (in Ukrainian). Zhelezniak, M. (Ed.). (2015). Metodychni zasady stvorennia paperovykh i elektronnykh entsyklopedychnykh vydan. Kyiv: Akademperiodyka (in Ukrainian). REFERENCES Boiko, Y. (2016). Instytut entsyklopedychnykh doslidzhen Natsionalnoi aka demii nauk Ukrainy: istoriia stvorennia i diialnist. Hileia: naukovyi visnyk, 109, 50-55 (in Ukrainian). Boiko, Y. (2016). Instytut entsyklopedychnykh doslidzhen Natsionalnoi aka demii nauk Ukrainy: istoriia stvorennia i diialnist. Hileia: naukovyi visnyk, 109, 50-55 (in Ukrainian). Chernysh, N. (2018). Narysy z istorii ukrainskoi entsyklopedychnoi spravy. Lviv: Piramida (in Ukrainian). Dovidkova informatsiia. Statystychni dani. Natsionalna akademiia nauk Ukrainy u 2017 rotsi: analitychnyi zvit. (2018). Kyiv: Akademperiodyka (in Ukrainian). Imia Petrusia Brovki. Belaruskaia Entsyklapediia. (2005). Minsk (in Bela rusian). Isaievych, Y., Zhelezniak M. (2009). Entsyklopediia. In Entsyklopediia su chasnoi Ukrainy. (Vol. 9, pp. 206-209). Kyiv: NASU Institute of Encyclopedic Research (in Ukrainian). Kulchytskyi, S. (2009). Vitchyzniana istoriia v entsyklopedychnomu formati. Entsyklopedychnyi visnyk Ukrainy, 1, 39-46 (in Ukrainian). Yatskiv, Y. (Ed.). (2018). Ukrainski entsyklopedii: typolohiia, styl, funktsii. Kyiv: NASU Institute of Encyclopedic Research (in Ukrainian). Zavdannia biuleteniu. (1931). Biuleten URE, 1, 1-4 (in Ukrainian). Yatskiv, Y. (Ed.). (2018). Ukrainski entsyklopedii: typolohiia, styl, funktsii. Kyiv: NASU Institute of Encyclopedic Research (in Ukrainian). Kyiv: NASU Institute of Encyclopedic Research (in Ukrainian). Zavdannia biuleteniu. (1931). Biuleten URE, 1, 1-4 (in Ukrainian). Zhelezniak, M. (2015). Entsyklopediia suchasnoi Ukrainy: istoriia stvoren Zavdannia biuleteniu. (1931). Biuleten URE, 1, 1-4 (in Ukrainian). Zhelezniak, M. (2015). Entsyklopediia suchasnoi Ukrainy: istoriia stvoren 17 Микола Железняк nia. Nauka Ukrainy u svitovomu informatsiinomu prostori, 12, 115-127 (in Ukrainian). Zhelezniak, M. (Ed.). (2015). Metodychni zasady stvorennia paperovykh i elektronnykh entsyklopedychnykh vydan. Kyiv: Akademperiodyka (in Ukrainian). 18 18 УДК 031:908(477) УДК 031:908(477) Енциклопедичний вісник України. 2018. Число 10 ПРО ПОРУШЕННЯ І ЗАХИСТ «ЕКОЛОГІЇ» ЕНЦИКЛОПЕДИЧНИХ ЗНАНЬ (ОГЛЯД ЗВІТУ ЦЕНТРУ ДОСЛІДЖЕНЬ ЄВРОПЕЙСЬКОГО ПАРЛАМЕНТУ ПРО НАЦІОНАЛЬНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КРАЇНАХ ЄВРОПИ)* Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна Дослідження присвячено питанням екосистеми енциклопедичних знань. Річ у тім, що довідкову інформацію нині дедалі частіше використовують як засіб мані- пуляції громадською свідомістю (з політичних, ідеологічних, економічних тощо міркувань), що яскраво помітно на прикладі онлайн-енциклопедій з вільним доступом до редагування розміщеної інформації, зокрема таких, як «Вікіпедія». Статтю побудовано на основі опису аналітичного звіту про онлайн-енциклопе- дії в країнах Європи, який підготували фахівці Європейського парламенту. Ключові слова: онлайн-енциклопедія, національна енциклопедія, енцикло- педичні знання, інформаційна екологія, інформаційний вандалізм. Процес глобалізації, що охоплює економічну, політичну, соціальну та духовну сфери життя соціуму, інтенсивно впливає на суспільство загалом та на рівень свідомості людей зокрема, створюючи нові умови та виміри дійснос ті, які треба осягати і до яких необхідно адаптуватися. Глобалізація й істотний розвиток інформаційних технологій спричинив підвищення рівня поширення інформації, доступу до неї та її рецепцію. Унаслідок цього нині в інтернет-про сторі активно розвивається інформаційне суспільство, породжуючи нові яви ща, серед яких виділимо такі (взаємопов’язані), як інформаційне середовище, Дослідження оприлюднено на круглому столі «Енциклопедистика в діяльності Націо нальної академії наук України та Наукового товариства імені Шевченка», приуроченого до 100-річчя Національної академії наук України (www.roundtable2018.encyclopedia.kiev.ua). © Інститут енциклопедичних досліджень НАН України Олександр Іщенко інформаційний вандалізм та інформаційна екологія. Щоправда, треба заува жити, що відповідні терміни з’явилися ще в 20 столітті, коли сучасних техноло гій ще не існувало, але вже тоді провадили дослідження проблем впливу інфор мації на людину (в межах філософських, біологічних чи комп’ютерних наук). Наприклад, інформаційну екологію виокремили в окрему наукову дисципліну, вважаючи її такою, що вивчає закономірності впливу інформації на формуван ня й функціонування людини, людських спільнот, людства загалом, а також на здоров’я як стан психічного й соціального добробуту [4]. інформаційний вандалізм та інформаційна екологія. Щоправда, треба заува жити, що відповідні терміни з’явилися ще в 20 столітті, коли сучасних техноло гій ще не існувало, але вже тоді провадили дослідження проблем впливу інфор мації на людину (в межах філософських, біологічних чи комп’ютерних наук). Наприклад, інформаційну екологію виокремили в окрему наукову дисципліну, вважаючи її такою, що вивчає закономірності впливу інформації на формуван ня й функціонування людини, людських спільнот, людства загалом, а також на здоров’я як стан психічного й соціального добробуту [4]. Сьогодні ж – у час комунікаційно-інформаційних технологій – поняття інформаційної екології набуло дещо іншого значення, хоча й продовжує пе ребувати в загальних рамках наведеного вище тлумачення. 1 Варто зауважити, що багато хто з дослідників не погоджується з визначенням «Вікіпедії» як енциклопедії в традиційному розумінні цього терміна у зв’язку з її спе ПРО ПОРУШЕННЯ І ЗАХИСТ «ЕКОЛОГІЇ» ЕНЦИКЛОПЕДИЧНИХ ЗНАНЬ (ОГЛЯД ЗВІТУ ЦЕНТРУ ДОСЛІДЖЕНЬ ЄВРОПЕЙСЬКОГО ПАРЛАМЕНТУ ПРО НАЦІОНАЛЬНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КРАЇНАХ ЄВРОПИ) Нині її розуміють передусім як захист людей від спотвореної інформації, що здатна впливати на свідомість людських груп, закріплюючи в них певні (хибні) уявлення, переко нання, стереотипи. Інформацію можуть спотворювати навмисне або ж ні (ви падково чи помилково). Із навмисним спотворенням інформації, зокрема яке здійснюється систематично, пов’язане явище інформаційного впливу – «ор ганізоване цілеспрямоване застосування спеціальних інформаційних засобів і технологій для внесення змін у свідомість населення (корекція поведінки) та / або інформаційно-технічну інфраструктуру об’єкта» [5]. На думку окремих фа хівців, питання інформаційної екології, яке сприймається як щось менш істотне порівняно з біологічною екологією, насправді, в сучасному світі потребує наба гато більш активної уваги, зокрема й дослідження її як «доктрини виживання людства в умовах інформаційного середовища» [1, с. 6]. Поряд з інформаційною екологією та інформаційним впливом співіснує поняття інформаційної безпеки, тобто стану чи процесу, коли інформацію за хищено від несанкціонованих дій (отримання, використання, оприлюднення, руйнування, внесення змін, ознайомлення, копіювання чи знищення). Серед несанкціонованих дій варто виокремити так званий інформаційний вандалізм, що безпосередньо стосується сфери енциклопедистики. Це явище характерне для онлайн-енциклопедій вільного типу, тобто таких, у яких відкрито доступ до редагування наявних матеріалів та додавання нових. Найвідомішою вільною онлайн-енциклопедією є «Вікіпедія». За визначенням, розміщеним на одній з її сторінок [8], вандалізмом в енциклопедичній галузі називають шкідницьке додавання, вилучення чи зміну вмісту з метою, що суперечить місії створення енциклопедії. Фундаментальна мета підготовки будь-якої енциклопедії – це пе редусім чітко структуроване й лапідарне подання достовірних (точних, переві рених), об’єктивних (з урахуванням різних точок зору) та неупереджених (тих, що не спрямовані проти когось або чогось; незаангажованих) знань. Відтак опрацювання інформації, внаслідок чого вона набуває ознак оманливості – не точності, суб’єктивності чи упередженості – є інформаційним вандалізмом. У зв’язку з тим, що «Вікіпедія» є 1) енциклопедією1 (тобто жанром літера 1 Варто зауважити, що багато хто з дослідників не погоджується з визначенням «Вікіпедії» як енциклопедії в традиційному розумінні цього терміна у зв’язку з її спе 20 Про порушення і захист «екології» енциклопедичних знань ... тури, який вважають еталонним виданням і надійним джерелом інформації) та 2) найбільш популярною у світі онлайн-енциклопедією, інформаційний ван далізм у ній дедалі частіше стає методом цілеспрямованого інформаційного впливу – поширенню у широкі маси людей певних суджень, пропагандист ських ідей, нав’язування політичних, економічних чи релігійних переконань. Інакше кажучи, «Вікіпедія», яку читають мільйони людей і якій довіряють, є зручним майданчиком для проведення інформаційних війн, протистоянь і конфліктів. Закономірно, що інформаційний захист у «Вікіпедії» – одна з найпоши реніших проблем у наукових дослідженнях з інтернет-енциклопедистики. цифічними особливостями, натомість пропонує вважати її окремим новим видом (жан ром) довідкових джерел – вікіпедією. ПРО ПОРУШЕННЯ І ЗАХИСТ «ЕКОЛОГІЇ» ЕНЦИКЛОПЕДИЧНИХ ЗНАНЬ (ОГЛЯД ЗВІТУ ЦЕНТРУ ДОСЛІДЖЕНЬ ЄВРОПЕЙСЬКОГО ПАРЛАМЕНТУ ПРО НАЦІОНАЛЬНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КРАЇНАХ ЄВРОПИ)* З’ясовано, що наслідки інформаційного вандалізму у «Вікіпедії» можуть бути відносно швидко усунені [2, с. 156]. Та незважаючи на це, читач все одно не захищений від того, що на час ознайомлення з інформацією вона «екологіч на», а не «вандалізована». Викривлення інформації, дезінформація тощо, тобто все те, що порушує екологію інформації (знань) – ці явища в сучасному інформаційному про сторі (мас-медіа, соціальні мережі, відкриті довідкові ресурси) набули загроз ливого зростання. Будь-яку інформацію нині варто перевіряти кілька разів перед тим, як вважати її дійсною. За окремими даними, 80% всієї інформа ції, якою володіє людина, отримано із джерел, що можуть зазнавати інфор маційного впливу: «Треба усвідомити той факт, що ми дізнаємося про світ здебільшого не безпосередньо; переважно йдеться про світ, повідомлений через інформаційні засоби. Те, що людина сьогодні знає, щонайбільше на 20% ґрунтується на її власному досвіді, а 80% передається через пресу, радіо й телебачення» [6, с. 144]. Інформаційні впливи та війни «здатні обернутися для людства тотальною катастрофою, оскільки, як інструмент політики, ін формаційна війна означає існування одного суспільства ціною виключення іншого, за рахунок знищення культурних цінностей та свідомості членів цьо го суспільства» [4, с. 28]. Отже, порушені питання виходять за межі наукових зацікавлень, поза як стосуються безпеки (нехай і інформаційної) кожної людини та людських спільнот (від соціальних груп до народів / націй). Саме тому Європейський парламент, один із найпотужніших законодавчих органів у світі, виявив за непокоєння станом інформаційної екосистеми в глобальному інтернет-про сторі. Зокрема, у зв’язку з дедалі частішим використанням довідкової інфор мації у «Вікіпедії» з метою маніпулювання громадською свідомістю, Центр досліджень Європейського парламенту поставив за мету дослідити в країнах Європи енциклопедичні інтернет-видання, які виявляють стійкість інформа ційної екосистеми, тобто такі, знання в яких, передусім соціогуманітарного характеру, можна вважати екологічними (неспотвореними, надійними) та за хищеними від деструктивного інформаційного впливу. Вибірку таких джерел 21 Олександр Іщенко сформували національні електронні енциклопедії, підготовлені фаховими (науковими) колективами. Результати дослідження представлено в аналітич ному звіті під назвою «Europe’s online encyclopaedias» [7]. сформували національні електронні енциклопедії, підготовлені фаховими (науковими) колективами. Результати дослідження представлено в аналітич ному звіті під назвою «Europe’s online encyclopaedias» [7]. Зазначена розвідка подає основні онлайн-енциклопедії в європейських країнах, характеризує рівень їхньої популярності серед читачів, порівнюючи з «Вікіпедією», аналізує доступність їх в Інтернеті. Прикметно, що в розділі про Україну згадано онлайн-версії «Енциклопедії Сучасної України» (ЕСУ), «Енциклопедії історії України» (ЕІУ) та українську «Вікіпедію». 2 Режим доступу: http://www.alexa.com/siteinfo/esu.com.ua ПРО ПОРУШЕННЯ І ЗАХИСТ «ЕКОЛОГІЇ» ЕНЦИКЛОПЕДИЧНИХ ЗНАНЬ (ОГЛЯД ЗВІТУ ЦЕНТРУ ДОСЛІДЖЕНЬ ЄВРОПЕЙСЬКОГО ПАРЛАМЕНТУ ПРО НАЦІОНАЛЬНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КРАЇНАХ ЄВРОПИ)* У цьому роз ділі, зокрема, так описано про ЕСУ: Електронна енциклопедія ЕСУ містить близько 100 тисяч статей; її створено на основі багатотомної друкованої «Енциклопедії Сучасної України» – сучасної національної енциклопедії, що надає відомості про Україну від початку 20 ст. донині, формуючи цілісний образ країни з акцентом на висвітленні історичних подій, установ, організацій та видатних осіб. ЕСУ укладає Інститут енциклопедичних досліджень Національної академії наук України, який для підготовки енциклопедичних матеріалів згуртував понад 1000 експертів – авторитетних фахівців із тих чи інших галузей знань, пе редусім провідних учених Національної академії наук України та Наукового товариства імені Шевченка. Окрім загальних відомостей про «Енциклопедію Сучасної України», у звіті схарактеризовано рейтинг її сайту, який визначає його позицію стосовно інших у країні, засвідчуючи цим самим доступність / популярність інтернет- ресурсу (автори використовують рейтинг статистичної служби Alexa Internet; він формується на основі обчислення комбінації середнього значення щоден них унікальних користувачів і приблизної кількості переглядів сторінок сайту за останній місяць в країні). За цим показником, як стверджується в дослі дженні, сайт ЕСУ займає 6 322 позицію в Україні (станом на 10 січня 2018 року). Водночас зауважимо, що на сьогодні (червень 2018 року) рейтинг сай ту ЕСУ від служби Alexa Internet2 підвищився до 4300 позиції в українському інтернет-просторі. Варто також зазначити, що відповідно до даних іншої ста тистичної служби (Google Analytics) в середньому за місяць сайт енциклопедії ЕСУ відвідує понад 70 000 тисяч унікальних користувачів, які при цьому пе реглядають понад 100 000 сторінок енциклопедії. Звісно, порівняно з україн ськомовною «Вікіпедією», яка входить до десятки найбільш рейтингових сай тів нашої країни та займає десяте місце серед усіх мовних розділів «Вікіпедії» в Європі [7, с. 12], онлайнову ЕСУ використовують набагато рідше. Автори звіту докладно характеризують і власне «Вікіпедію», зокрема до сліджують різні її аспекти, через які екосистема наявних у ній знань перма нентно зазнає деструктивного втручання. Яскравим прикладом слугує опис ситуації в Туреччині: через загрозу національній безпеці уряд заборонив функціонування «Вікіпедії» в країні. З недовірою до ресурсу поставився й 22 Про порушення і захист «екології» енциклопедичних знань ... уряд Китаю, який у 2017 році запропонував створити китайську національну енциклопедію, найнявши для цього понад 20 тисяч осіб персоналу в споді ванні, що нове джерело довідкових знань буде популярнішим і, головне, на дійнішим в країні за «Вікіпедію». р Описуючи «Вікіпедію» як майданчик інформаційної війни, автори зві ту згадали й про Україну. 3 Режим доступу: http://www.metapedia.org ПРО ПОРУШЕННЯ І ЗАХИСТ «ЕКОЛОГІЇ» ЕНЦИКЛОПЕДИЧНИХ ЗНАНЬ (ОГЛЯД ЗВІТУ ЦЕНТРУ ДОСЛІДЖЕНЬ ЄВРОПЕЙСЬКОГО ПАРЛАМЕНТУ ПРО НАЦІОНАЛЬНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КРАЇНАХ ЄВРОПИ)* Зокрема, навели приклад інформаційної битви в одній російськомовній статті, де подано інформацію про збиття у 2014 році в Донецькій області авіалайнера рейсу MH17 за маршрутом Амстердам – Куала-Лумпур: конфліктним формулюванням було пояснення причини авіа катастрофи – спочатку в статті зазначалося, що літак було збито ракетною установкою «Бук» терористами самопроголошеної ДНР; згодом це тверджен ня відредагували, внаслідок чого причетними до падіння літака стали україн ські солдати. У розвідці підкреслено, що внесені зміни здійснено з IP-адреси однієї з російських державних медіакомпаній. У звіті згадано про ще одну вільну онлайн-енциклопедію – «Метапедію»3, інформація в якій «екологічно» небезпечна. Її позиціоновано як альтернатив ну енциклопедію; вона має різні мовні розділи, технічно діє за принципами «Вікіпедії», функціонуючи на «вікі»-технології. Енциклопедія публікує статті про культуру, мистецтво, науку, філософію і політику. Однак, особливість цього джерела енциклопедичних знань у тому, що статті «забарвлені» ультраправи ми оцінками, пропагандуючи таким чином у Європі нацизм, расизм і шовінізм. Наведені вище факти щодо стану інформаційної безпеки у «Вікіпедії» й спону кали авторів розвідки з’ясувати, чи існують у країнах Європи загальні онлайн- енциклопедії, екосистема знань у яких лишається стійкою. Аналіз європейських національних онлайн-енциклопедій на предмет доступності «екологічних» знань для суспільства призвів до поділу країн на 4 групи (див. рис. 1). Перша група: країни, що не мають базової активної (у якій матеріали ак туалізовані) національної онлайн-енциклопедії (Бельгія, Білорусь, Боснія і Герцеговина, Данія, Ірландія, Мальта, Румунія, Сербія, Словаччина, Словенія, Туреччина, Фінляндія, Чорногорія). Одна з особливостей цієї групи в тому, що жителі відповідних країн (окремих із них) послуговуються енциклопедіями інших мов. Наприклад, бельгійці користуються французькими, німецькими чи голландськими джерелами; шведомовні фіни – шведськими джерелами. Ще одна особливість – мовні розділи відповідних країн у «Вікіпедії» мають низьку кількість статей. Отже, такі країни як правило послуговуються чужою (інших країн) довідковою інформацією. Друга група: енциклопедії, у яких доступ до національних онлайн-енци клопедій обмежений – користування енциклопедіями вимагає реєстрації та / або оплати для доступу до всіх матеріалів (Велика Британія, Естонія, Греція, Португалія). Третя група: країни, у яких доступ до національних онлайн-ен циклопедій цілковито платний (Франція, Іспанія, Німеччина, Нідерланди, 23 Олександр Іщенко Швеція). Четверта група: країни, у яких доступ до національних онлайн- енциклопедій вільний – без реєстрації і оплати (Україна, Польща, Австрія, Болгарія, Норвегія. Чехія, Хорватія, Італія, Латвія, Литва, Росія). Швеція). Четверта група: країни, у яких доступ до національних онлайн- енциклопедій вільний – без реєстрації і оплати (Україна, Польща, Австрія, Болгарія, Норвегія. Чехія, Хорватія, Італія, Латвія, Литва, Росія). Рис. 1. ПРО ПОРУШЕННЯ І ЗАХИСТ «ЕКОЛОГІЇ» ЕНЦИКЛОПЕДИЧНИХ ЗНАНЬ (ОГЛЯД ЗВІТУ ЦЕНТРУ ДОСЛІДЖЕНЬ ЄВРОПЕЙСЬКОГО ПАРЛАМЕНТУ ПРО НАЦІОНАЛЬНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КРАЇНАХ ЄВРОПИ)* Особливості доступу до енциклопедичної інформації в країнах Європи (на прикладі національних онлайн-енциклопедій) Рис. 1. Особливості доступу до енциклопедичної інформації в країнах Європи (на прикладі національних онлайн-енциклопедій) Аналіз рейтингу сайтів національних онлайн-енциклопедій та показників обсягу інформації в них (за кількістю статей) засвідчив про значно нижчу їх популярність та менший обсяг порівняно з «Вікіпедією. Привертає увагу й той факт, що в багатьох країнах користувачі «Вікіпедії» віддають перевагу розділу англійською, а не рідною мовою. Схожа тенденція і в Україні: щоправда, у нас перевагу віддають не англомовному, а російськомовному розділу. У звіті резюмується, що загалом Європа має широку енциклопедичну па літру; її представляють різні енциклопедичні традиції, а знання в європей ських енциклопедіях відображають культурну спадщину держав, їхні цін ності, ознаки самобутності й ідентичності. Автори схиляються до тієї ідеї, що доступ до знань про кожну країну серед європейської спільноти має бути вільним, передусім із мовної точки зору (йдеться про те, що національним енциклопедіям не вистачає полімовної опції, оскільки переважна більшість із них – одномовні). 24 Про порушення і захист «екології» енциклопедичних знань ... Далі з метою ознайомлення наведемо перелік європейських національ них онлайн-енциклопедій. Австрія: Австрія: • онлайн-енциклопедія «Austria-Forum»4. Створено на основі електронної словникової бази «Annotierbare Elektronische Interaktive Österreichische Universallexikon», яка у свою чергу – на базі «Österreich-Lexikon» («Ав стрійський глосарій»). Велика Британія: • онлайн-енциклопедія «Encyclopaedia Britannica»5. Онлайн-енциклопедія від 2010 року є продовженням найвідомішої у світі енциклопедії «Британіка», яка почала друкувати томи ще у 18 столітті. Білорусь: • оцифрована енциклопедія «Беларуская Энцыклапедыя». Оцифровану ко пію 18-ти томів «Білоруської енциклопедії» розміщено на одному із сай тів Білорусі6. Повноцінна національна онлайн-енциклопедія у Білорусі відсутня. 4 Режим доступу: http://austria-forum.org Нідерланди: • онлайн-енциклопедія «Grote Winkler Prins»16. Онлайн-енциклопедія є продовженням відомої нідерландської енциклопедії «Winkler Prins Ency clopedie», яка почала друкуватися ще в 19 столітті. Німеччина: Німеччина: • онлайн-енциклопедія «Brockhaus»17. Створено на основі відомої універ сальної «Енциклопедії Брокгауз» («Brockhaus Enzyklopädie»). Норвегія: • онлайн-енциклопедія «Brockhaus»17. Створено на основі відомої універ сальної «Енциклопедії Брокгауз» («Brockhaus Enzyklopädie»). Норвегія: • онлайн-енциклопедія «Store Norske Leksikon»18. Створено на основі відповідного 16-томного видання («Велика норвезька енциклопедія»). Особливість цієї онлайн-енциклопедії в тому, що в ній поєднано ен циклопедію вільного й закритого типу. Зокрема, йдеться про те, що електронний ресурс надає опцію редагування статей для будь-якого читача; при цьому запропоновані читачами зміни затверджує (або ж відхиляє) експерт, і лише після цього стаття набуває нової редакції для перегляду. Болгарія: р • онлайн-енциклопедія «Знам»7. Створено на основі універсального од нотомного болгарського видання «Българска енциклопедия А-Я» («Болгарська енциклопедія А-Я»). Греція: р • онлайн-енциклопедія «Επιστημη και Ζωή»8. Створено на основі відпо відної універсальної грецької енциклопедії («Енциклопедія науки і жит тя»). Данія: • онлайн-енциклопедія «Den Store Danske»9. Створено на основі універ сальної данської 20-томної енциклопедії «Den Store Danske Encyklopædiс» («Велика данська енциклопедія»). Від 2017 року онлайн-енциклопедія не оновлюється, проте лишається функціонувати надалі. Естонія: • онлайн-енциклопедія «TEA e-Entsüklopeedia»10. Створено на основі низки друкованих енциклопедичних видань Естонії. Іспанія: • онлайн-енциклопедія «Gran Enciclopedia de España»11. Створено на осно ві універсальної 22-томної енциклопедії «Gran Enciclopedia de España» («Велика іспанська енциклопедія»). • онлайн-енциклопедія «Gran Enciclopedia de España»11. Створено на осно ві універсальної 22-томної енциклопедії «Gran Enciclopedia de España» («Велика іспанська енциклопедія»). 4 Режим доступу: http://austria-forum.org 5 Режим доступу: http://www.britannica.com 6 Режим доступу: http://app.box.com/v/bielaruskaja-encyklapiedyja 7 Режим доступу: http://www.znam.bg 8 Режим доступу: http://www.gnosinet.gr 9 Режим доступу: http://denstoredanske.dk 10 Режим доступу: http://www.ents.ee 11 Режим доступу: http://granenciclopedia.es 25 Олександр Іщенко Італія: Італія: • онлайн-енциклопедія «Тreccani»12. Створено на основі 35-томної націо нальної енциклопедії «Enciclopediaitaliana di scienze, lettere, ed arti» («Іта лійська енциклопедія наук, літератури і мистецтв»). Латвія: • онлайн-енциклопедія «Nacionālā enciklopēdija»13 («Національна енци клопедія»; розпочне функціонування у грудні 2018 року). Заплановано також видрукувати енциклопедію. Литва: • онлайн-енциклопедія «Visuotinė Lietuvių Enciklopedija»14. Створено на основі відповідного 25-томного енциклопедичного видання («Універ сальна литовська енциклопедія»). М і Македонія: • оцифрована енциклопедія «Македонска енциклопедија». Два оцифрова ні томи академічної «Македонської енциклопедії» розміщено на одному з македонських сайтів15. Національна онлайн-енциклопедія в Македонії відсутня. Польща: • онлайн-енциклопедія «Encyklopedia PWN»19. Створено на основі енци 12 Режим доступу: http://www.treccani.it 13 Режим доступу: http://enciklopedija.lv 14 Режим доступу: http://www.vle.lt 15 Режим доступу: http://promacedonia.com/mak_enc 16 Режим доступу: http://www.winklerprins.com 17 Режим доступу: http://brockhaus.de 18 Режим доступу: http://snl.no 19 Режим доступу: http://encyklopedia.pwn.pl 12 Режим доступу: http://www.treccani.it 13 Режим доступу: http://enciklopedija.lv 14 Режим доступу: http://www.vle.lt у у p // 15 Режим доступу: http://promacedonia.com/mak_enc 16 Режим доступу: http://www.winklerprins.com 17 Режим доступу: http://brockhaus.de 18 Режим доступу: http://snl.no 19 Режим доступу: http://encyklopedia.pwn.pl 26 Про порушення і захист «екології» енциклопедичних знань ... клопедичних видань, опублікованих у видавництві PWN («Державне на укове видавництво»); клопедичних видань, опублікованих у видавництві PWN («Державне на укове видавництво»); клопедичних видань, опублікованих у видавництві PWN («Державне на укове видавництво»); у • онлайн-енциклопедія «Wielka Interaktywna Encyklopedia Multimedialna»20 («Велика інтерактивна мультимедійна енциклопедія»). Створено на ос нові енциклопедичного видання «Popularna Encyklopedia Powszechna» («Популярна загальна енциклопедія»). Росія: • онлайн-енциклопедія «Большая российская энциклопедия»21. Створено на основі 35-томного відповідного енциклопедичного видання («Велика російська енциклопедія»). і 20 Режим доступу: http://zapytaj.onet.pl/encyklopedia Румунія: • онлайн-енциклопедія «Enciclopedia Romanieio»22. Енциклопедія вільного типу. Не витримала конкуренції з «Вікіпедією» і припинила свій розвиток (містить лише близько 5000 статей). Угорщина: • онлайн-енциклопедія «Magyar Nagylexikon». Нині не функціонує. Її було створено на основі відповідного 19-томного енциклопедичного видання («Велика угорська енциклопедія»); • онлайн-енциклопедія «Magyar Virtuális Enciklopédia» («Угорська вірту альна енциклопедія»)23. Від 2005 року матеріали не оновлюють і нових не додають. • онлайн-енциклопедія «Magyar Virtuális Enciklopédia» («Угорська вірту альна енциклопедія»)23. Від 2005 року матеріали не оновлюють і нових не додають. Франція: Франція: • онлайн-енциклопедія «Encyclopaedia Universalis»24. Створено на основі відповідного 28-томного енциклопедичного видання («Універсальна ен циклопедія»). Хорватія: Хорватія: • онлайн-енциклопедія «Hrvatska enciklopedija»25. Створено на основі від повідного 11-томного енциклопедичного видання («Хорватська енцикло педія»); • онлайн-енциклопедія «Proleksis enciklopedija»26. Створено на основі від повідного 20-томного енциклопедичного видання «Opća i nacionalna enciklopedija» («Загальна і національна енциклопедія»). Чехія: • онлайн-енциклопедія «Encyklopedie CoJeCo»27; • онлайн-енциклопедія «Encyklopedie Vševěd»28. 20 Режим доступу: http://zapytaj.onet.pl/encyklopedia 21 Режим доступу: http://bigenc.ru у у p g 22 Режим доступу: http://enciclopediaromaniei.ro 23 Режим доступу: http://www.hunfi.hu 24 Режим доступу: http://www.encyclopaedia-universalis.fr 25 Режим доступу: http://www.enciklopedija.hr 26 Режим доступу: http://proleksis.lzmk.hr 27 Режим доступу: http://www.cojeco.cz 28 Режим доступу: http://www.vseved.cz 27 Олександр Іщенко Швейцарія: • онлайн-енциклопедія «Historisches Lexikon der Schweiz»29. Створено на основі відповідної багатотомної краєзнавчої енциклопедії («Історичний словник Швейцарії»), особливість якої в тому, що вона має видання трьо ма мовами – німецькою, італійською та французькою. Відповідно онлайн- енциклопедія так само є тримовною. 29 Режим доступу: http://www.hls-dhs-dss.ch 30 Режим доступу: http://www.ne.se 30 Режим доступу: http://www.ne.se 29 Режим доступу: http://www.hls-dhs-dss.ch ЛІТЕРАТУРА 1. Андрейко Г. Экология информационного пространства: лекционный курс. Харьков, 2015. 112 с. 2. Емельяненко В., Жукова С. Википедия как источник научной информации // Ин формация как двигатель научного прогресса: Зб. науч. статей по итогам меж дунар. научно-практической конференции. Стерлитамак, 2018. С. 154–160. 3. Еремин А. Природа и физиология информационной экологии человека // Экология человека. 2000. № 2. С. 55–60. 3. Еремин А. Природа и физиология информационной экологии человека // Экология человека. 2000. № 2. С. 55–60. 4. Кобільник Б., Гізун А. Роль інформаційно-психологічних впливів у інфор маційній війні // Актуальні задачі та досягнення у галузі кібербезпеки: Матеріали всеукраїнської науково-практичної конференції / КНТУ. Кропив ницький, 2016. С. 28–29. 4. Кобільник Б., Гізун А. Роль інформаційно-психологічних впливів у інфор маційній війні // Актуальні задачі та досягнення у галузі кібербезпеки: Матеріали всеукраїнської науково-практичної конференції / КНТУ. Кропив ницький, 2016. С. 28–29. 5. Остроухов В., Петрик В. До проблеми забезпечення інформаційної безпеки України // Політичний менеджмент. 2008. № 4. С. 135–141. 5. Остроухов В., Петрик В. До проблеми забезпечення інформаційної безпеки України // Політичний менеджмент. 2008. № 4. С. 135–141. 6. Рубан А. Сучасні інформаційні комунікації та їх вплив на масову політичну свідомість і здоров’я людини // Young Scientist. 2017. № 9.1 (49.1). С. 143–147. 7. Europe’s online encyclopaedias. Equal access to knowledge of general interest? / N. Bentzen; European Parliamentary Research Service. Brussels, 2018. 8. Vandalism // Wikipedia. Режим доступу: http://en.wikipedia.org/wiki/Wikipedia: Vandalism. Швеція: ц • онлайн-енциклопедія «Nationalencyklopedin»30. Створено на основі 20-томного енциклопедичного видання («Національна енциклопедія»). • онлайн-енциклопедія «Nationalencyklopedin»30. Створено на основі 20-томного енциклопедичного видання («Національна енциклопедія»). Насамкінець, відповідно до наших спостережень, пропонуємо деякі ви сновки-узагальнення. 1. Переважна більшість європейських національних онлайн-енциклопе дій – це комерційні видання, до створення яких держава не має стосунку. Щоправда, видавець піклується не лише про комерційну вигоду, а й про належну високу якість матеріалу, оскільки статті в таких енциклопедіях готують фахівці. Менша частина онлайн-енциклопедій з’явилася з ініці ативи і за фінансування уряду чи державних наукових або видавничих організацій. 2. Національні онлайн-енциклопедії у країнах Європи створено на основі відповідних фундаментальних друкованих енциклопедичних видань. 2. Національні онлайн-енциклопедії у країнах Європи створено на основі відповідних фундаментальних друкованих енциклопедичних видань. 3. Фактично всі онлайн-енциклопедії – одномовні. 4. Всі онлайн-енциклопедії – захищені від втручання в екосистему ен циклопедичних знань. Водночас окремі з них пропонують зворотний зв’язок з читачами, як, наприклад, національна онлайн-енциклопедія в Норвегії (див. вище). У цьому контексті заслуговує на увагу й електронна «Енциклопедія Сучасної України», яка надає читачам опцію коментуван ня (в коментарях читач має можливість запропонувати свої зміни до тек сту статті, які редакція ЕСУ візьме до уваги та за доцільності зробить у статті відповідні корективи). 5. Аналіз рейтингу сайтів онлайн-енциклопедій, наведений у звіті, свідчить про те, що до них звертаються значно рідше, ніж до «Вікіпедії». На нашу думку, причиною цьому можуть бути такі чинники: 1) менша кількість статей, ніж у «Вікіпедії» (тобто читач не впевнений, що інформацію, яка його цікавить, буде знайдено в національній онлайн-енциклопедії, а тому одразу звертається до «Вікіпедії»); 2) обмежений доступ і комерціалізація (пошук інформації за умови оплати, звісно, читачеві менш привабливе, ніж на вільних засадах); 3) одномовність (по-перше, одномовність об межує кількість читачів за мовною належністю, а багатомовність, навпа ки, розширює читацьку аудиторію; по-друге, у «Вікіпедії» мовні розділи про те саме реєстрове слово часто містять інформацію, яка не збігається; 28 Про порушення і захист «екології» енциклопедичних знань ... це сприяє більшій інформативності); 4) зовнішній вигляд сайту й опції з пошуку інформації та її «споживання» (в цьому сенсі, на нашу думку, більшість сайтів, що представляють європейські онлайн-енциклопедії, не вирізняються високим ґатунком). ON VIOLATION AND PROTECTION OF ECOLOGY OF ENCYCLOPEDIC KNOWLEDGE (REVIEW OF THE REPORT OF THE EUROPEAN PARLIAMENTARY RESEARCH SERVICE ON EUROPE’S NATIONAL ONLINE ENCYCLOPEDIAS) Oleksandr Ishchenko NASU Institute of encyclopedic research, Kyiv, Ukraine The research is devoted to issues of the ecosystem of encyclopedic knowledge. The fact is that a reference information is now increasingly used as means of media manipulation in level of public consciousness (for political, ideological, and eco nomic reasons). This is well traced on the example of online encyclopedias with free 29 Олександр Іщенко access to editing of posted information such as Wikipedia. This article is based on the description of the online analytical report on encyclopedias in Europe written by experts of the European Parliament. Keywords: online encyclopedia, national encyclopedia, encyclopedic know ledge, information ecology, online vandalism. REFERENCES Andreiko, G. (2015). Ekologiia informatsionnogo prostranstva lektsionnyi kurs. Kharkov (in Russian). Andreiko, G. (2015). Ekologiia informatsionnogo prostranstva lektsionnyi kurs. Kharkov (in Russian). Bentzen, N. (2018). Europe’s online encyclopaedias. Equal access to knowledge of general interest? Brussels: European Parliamentary Research Service. Bentzen, N. (2018). Europe’s online encyclopaedias. Equal access to knowledge of general interest? Brussels: European Parliamentary Research Service. Emelianenko, V., Zhukova, S. (2018). Vikipediia kak istochnik nauchnoi infor matsii. In Proceedings of the 3d International Conference «Informatsiia kak dvigatel nauchnogo progressa» (pp. 154-160). Sterlitamak (in Russian). Emelianenko, V., Zhukova, S. (2018). Vikipediia kak istochnik nauchnoi infor matsii. In Proceedings of the 3d International Conference «Informatsiia kak dvigatel nauchnogo progressa» (pp. 154-160). Sterlitamak (in Russian). Kobilnyk, B., Hizun, A. (2016). Rol informatsiino-psykholohichnykh vplyviv u informatsiinii viini. In Proceedings of the Conference «Aktualni zadachi ta dosiah nennia u haluzi kiberbezpeky» (pp. 28-29). Kropyvnytskyi: KNTU (in Ukrainian). Kobilnyk, B., Hizun, A. (2016). Rol informatsiino-psykholohichnykh vplyviv u informatsiinii viini. In Proceedings of the Conference «Aktualni zadachi ta dosiah nennia u haluzi kiberbezpeky» (pp. 28-29). Kropyvnytskyi: KNTU (in Ukrainian). Ostroukhov, V., Petryk V. (2008). Do problemy zabezpechennia informatsiinoi bezpeky Ukrainy. Politychnyi menedzhment, 4, 135-141 (in Ukrainian). Ostroukhov, V., Petryk V. (2008). Do problemy zabezpechennia informatsiinoi bezpeky Ukrainy. Politychnyi menedzhment, 4, 135-141 (in Ukrainian). Ruban, A. (2017). Suchasni informatsiini komunikatsii ta yikh vplyv na masovu po litychnu svidomist i zdorovia liudyny. Young Scientist, 9.1(49.1), 143-147 (in Ukrainian). Vandalism. In Wikipedia. http://en.wikipedia.org/wiki/Wikipedia:Vandalism. Yeremin, A. (2000). Priroda i fiziologiia informatsionnoi ekologii cheloveka. Eko logiia cheloveka, 2, 55-60 (in Russian). 30 30 УДК 622.01 Енциклопедичний вісник України. 2018. Число 10 ГАЛУЗЕВА ЕНЦИКЛОПЕДІЯ ЯК ВАЖЛИВИЙ ЕТАП РОЗВИТКУ НАУКОВОЇ ДИСЦИПЛІНИ ТА ЇЇ ТЕРМІНОСИСТЕМИ (НА ПРИКЛАДІ ЦИКЛУ ПРАЦЬ ІЗ ГІРНИЦТВА Й ГЕОЛОГІЇ «ГІРНИЧА ЕНЦИКЛОПЕДІЯ»)* Володимир Білецький НТУ «Харківський політехнічний інститут», Харків, Україна Геннадій Гайко НТУУ «Київський політехнічний інститут імені І. Сікорського», Київ, Україна На прикладі циклу праць «Гірнича енциклопедія» показано особливості розвит ку окремих наукових дисциплін – гірництва, геології; передусім їх фахового тер- мінологічного забезпечення. Стаття характеризує «Гірничу енциклопедію», її актуальність для технічної освіти в Україні, інтеграції загалом вітчизняної ви- щої освіти в європейський та світовий освітній простір. Ключові слова: галузеві енциклопедії, Гірнича енциклопедія, технічна тер- мінологія, технічна освіта. Вступ. Стан термінологічної справи будь-якої держави значною мірою ві дображає рівень її освіти, науки й культури. Саме тому кожна держава дбає про стан фахової мови, відображеної в освітніх і галузевих стандартах, при чому стандартизація термінології неабияк сприяє утвердженню державності країни. О. Колган справедливо зазначає, що «закріплення Конституцією Укра їни статусу української мови як державної створює умови для формування * Дослідження оприлюднено на круглому столі «Енциклопедистика в діяльності Націо нальної академії наук України та Наукового товариства імені Шевченка», приуроченого до 100-річчя Національної академії наук України (www.roundtable2018.encyclopedia.kiev.ua). © Інститут енциклопедичних досліджень НАН України © Інститут енциклопедичних досліджень НАН України Володимир Білецький, Геннадій Гайко й розвитку національної термінології взагалі та її підсистем, зокрема гірничої термінології, що є одним із найважливіших чинників національного відро дження України в період її незалежності» [4, с. 1]. й розвитку національної термінології взагалі та її підсистем, зокрема гірничої термінології, що є одним із найважливіших чинників національного відро дження України в період її незалежності» [4, с. 1]. Вже в перші роки становлення України як незалежної держави за наказа ми Міністерства освіти й Держстандарту на базі НУ «Львівська політехніка» було створено Технічний комітет стандартизації науково-технічної терміноло гії, який зробив багато корисного для розвитку й розповсюдження української фахової термінології, проте гірничий напрямок не отримав належного забезпе чення. Окремі роботи провадили науковці Національного гірничого універси тету, Івано-Франківського університету нафти і газу, Донецького національного технічного університету, Криворізького національного університету тощо, од нак ці праці не набули системного характеру й значного поширення – велика частина фахівців і студентів, особливо на сході України, продовжувала зберігати орієнтацію на російську термінологічну базу й навчальну літературу. На одній із сесій Наукового товариства імені Шевченка (2016 року) було влучно зауважено: модернізація змісту гірничої освіти потребує сучасної термі нологічної системи та оновленої інформаційної бази енциклопедичного рівня, яка охоплює широкий реєстр понять і термінів, що відображають гірництво й геологію як цілісний феномен, що розвивається. ГАЛУЗЕВА ЕНЦИКЛОПЕДІЯ ЯК ВАЖЛИВИЙ ЕТАП РОЗВИТКУ НАУКОВОЇ ДИСЦИПЛІНИ ТА ЇЇ ТЕРМІНОСИСТЕМИ (НА ПРИКЛАДІ ЦИКЛУ ПРАЦЬ ІЗ ГІРНИЦТВА Й ГЕОЛОГІЇ «ГІРНИЧА ЕНЦИКЛОПЕДІЯ»)* Здобутки науки й техніки по стійно та своєчасно мають входити в освітній простір, забезпечуючи «кругове навчання» (дослівний переклад терміна «енциклопедія»), тобто – системний широкий погляд на пов’язані галузі діяльності. Особливо це актуально для гір ництва, де «коло наук» відрізняється надзвичайно великим розмаїттям і потре бує акумуляції сучасних знань і досягнень [6, с. 197]. Мета і завдання. Мета циклу праць «Гірнича енциклопедія» полягає в створенні термінологічної бази даних та енциклопедичної систематизації су часних знань у природничих науках про Землю і прикладних гірничих дисци плінах. Це має забезпечити підвищення якості гірничої освіти та уможливить перехід освітніх процесів вищої школи (зокрема, викладання гірничо-геологіч них дисциплін і створення відповідної навчальної літератури) від російської до української понятійно-термінологічної системи. Серед завдань проекту «Гірнича енциклопедія» виокремимо такі: і і ї й і ї і і 1. сприяння оновленню змісту гірничої й геологічної освіти та підвищенню якості навчальної й методичної літератури шляхом створення й застосуван ня найбільш повної й сучасної енциклопедичної інформаційної системи; 2. гуманітаризація гірничотехнічної освіти й сприяння єдності інженерних і гуманітарних знань, що забезпечується наведенням численних матеріалів з історії гірництва й геологічної науки, геологічного та гірничого законодав ства, охорони праці й охорони геологічного довкілля під час експлуатації надр; 3. покращення доступу до іноземних освітніх і наукових джерел, розвиток по нятійної комунікації між науковцями різних країн, уможливлення міжна родної уніфікації освітніх і наукових процесів у природничих і гірничотех 3. покращення доступу до іноземних освітніх і наукових джерел, розвиток по нятійної комунікації між науковцями різних країн, уможливлення міжна родної уніфікації освітніх і наукових процесів у природничих і гірничотех 32 Галузева енциклопедія як важливий етап розвитку ... нічних галузях шляхом адаптації створеної термінологічної бази даних до англійських, німецьких і російських аналогів (наведення відповідників до кожного українського терміну); 4. відродження української фахової мови в галузі гірництва та геології завдяки використанню питомо українських понять і термінів, врахуванню змін в реа ліях мовної практики й науки в Україні. 4. відродження української фахової мови в галузі гірництва та геології завдяки використанню питомо українських понять і термінів, врахуванню змін в реа ліях мовної практики й науки в Україні. Стисла характеристика проекту. «Гірнича енциклопедія» – комплекс на, міждисциплінарна праця, що систематизує знання в природничих на уках про Землю, таких як геологія, мінералогія, петрографія, гідрогеологія, гео- хімія, геофізика, географія, екологія; прикладних технічних науках з видобуван ня та первинної переробки твердих, рідинних і газоподібних корисних копалин, шахтного та підземного будівництва, маркшейдерії та геодезії, а також відображає сучасний стан та історію гірничо-геологічної науки й освіти в країнах світу. ГАЛУЗЕВА ЕНЦИКЛОПЕДІЯ ЯК ВАЖЛИВИЙ ЕТАП РОЗВИТКУ НАУКОВОЇ ДИСЦИПЛІНИ ТА ЇЇ ТЕРМІНОСИСТЕМИ (НА ПРИКЛАДІ ЦИКЛУ ПРАЦЬ ІЗ ГІРНИЦТВА Й ГЕОЛОГІЇ «ГІРНИЧА ЕНЦИКЛОПЕДІЯ»)* Крім цього, праця окреслює актуальні питання геологічного та гірничого законодав ства, охорони праці й охорони геологічного довкілля при експлуатації надр. «Гір нича енциклопедія» не має вітчизняних аналогів і випереджає за кількістю статей подібні видання слов’янськими мовами. Цикл праць «Гірнича енциклопедія» охоплює 6 книг на 3832 сторінках фор мату А4 обсягом 658 друкованих аркушів, які складають два фундаментальні енциклопедичні видання – «Гірничий енциклопедичний словник» (ГЕС) [3] і «Мала гірнича енциклопедія» (МГЕ) [5]. Загальний обсяг трьох томів – 1936 сто рінок (371,2 друк. аркушів). «Гірничий енциклопедичний словник» містить 12 700, а «Мала гірнича ен циклопедія» – 17 350 термінологічних та номенклатурних одиниць (частину з них введено у мовний обіг вперше). Статті складаються зі слова-заголовка, піс ля якого наведено відповідники російською, англійською та німецькою мовами. Статті в стислій, зручній для користування формі подають найсучаснішу наукову й науково-технічну інформацію, висвітлюють різні аспекти геологічних наук, роз відки, видобування та первинної переробки корисних копалин, знайомлять із ме тодами наукового пошуку й інженерного проектування, популяризують світові й українські досягнення в зазначених галузях. Проект містить понад півтори тисячі ілюстрацій (рисунків, схем, фотографій), чим суттєво покращує розуміння й за своєння матеріалу. Авторський і редакційний колектив – це понад 100 провідних науковців і практиків у галузі гірництва з понад 30 університетів, науково-дослід них інститутів, наукових спілок і організацій України, Польщі й Росії. ГЕС і МГЕ об’єднані в спільний цикл праць «Гірнича енциклопедія», оскіль ки ГЕС (перші два томи) закладає основи термінологічної бази (стислі визначен ня, дефініції), що була значно збільшена й розвинена в енциклопедичні статті, реалізовані у МГЕ. Крім цього, третій том ГЕС містить додаткові матеріали ен циклопедичного характеру про країни, басейни, родовища, гірничі підприємства, компанії, інститути, університети, наглядові та громадські гірничі організації, які не потрапили до МГЕ. Відтак обидва видання взаємопов’язані й доповнюють одне одного. 33 Володимир Білецький, Геннадій Гайко Під час роботи автори дотримувалися інтегральних принципів термінотво рення, коли проблему номінування того чи іншого поняття розв’язують індивіду ально – через аналіз потенціалу словотворення в українській мові або шляхом ін терпретації вже готового терміна з іншої мови, звідки поняття запозичено і впро ваджено в національну терміносистему (транскрибуванням, прямим перекладом, калькуванням). При цьому враховано й традиції вживання гірничих термінів в Україні, їхнє походження, а також ареал розповсюдження гірничих термінів-си нонімів в Україні та світі. «Гірнича енциклопедія» – піонерська робота, яка передбачала підготовку й опублікування низки похідних видань (дочірніх проектів), що були у взаємодії з основним проектом «Мала гірнича енциклопедія» й розвивали окремі напрям ки гірничо-геологічних знань і технологій. ГАЛУЗЕВА ЕНЦИКЛОПЕДІЯ ЯК ВАЖЛИВИЙ ЕТАП РОЗВИТКУ НАУКОВОЇ ДИСЦИПЛІНИ ТА ЇЇ ТЕРМІНОСИСТЕМИ (НА ПРИКЛАДІ ЦИКЛУ ПРАЦЬ ІЗ ГІРНИЦТВА Й ГЕОЛОГІЇ «ГІРНИЧА ЕНЦИКЛОПЕДІЯ»)* Додатково авторський колектив під готував і опублікував 4 словники-довідники, 12 монографій, 4 підручники й 6 навчальних посібників із грифом МОН України. Наукова новизна й практичне значення. Цикл праць «Гірнича енци клопедія» вперше створює збалансовану українськомовну термінологічну сис тему, охоплюючи 17 350 термінологічних та номенклатурних одиниць, адап тованих до англійської, німецької й російської термінологічних баз даних, і є основою для понятійної комунікації в геологічній та гірничій освіті, навчальній, методичній та науковій літературі, а також у періодичних наукових виданнях України та інших країн світу. Це забезпечує потужний ресурс сприяння розви тку природничих, технічних і гуманітарних наук й істотно впливає на покра щення підготовки майбутніх спеціалістів у гірничо-геологічних галузях. Авторами узагальнено й суттєво розвинуто фахову українську мову, зокрема геологічну, географічну, екологічну, гірничо-технологічну і гірничо-технічну тер мінологію. Введено низку нових термінів, причому поповнення термінологічної лексики гірників разом з освоєнням готових лексем (українських та іншомов них) здійснювалося за допомогою творення нових слів на ґрунті словотвірних моделей сучасної української мови. Під час роботи над циклом «Гірнича енци клопедія» напрацьовано засади творення гірничої терміносистеми, здійснено аналіз її витоків і шляхів формування, семантичної диференціації та особли востей функціонування, що сприятиме розвиткові лінгвоукраїністики в галузі термінології, подальшому унормуванню термінології гірництва та геології. До кладніше про «Гірничу енциклопедію» в контексті термінології див. – [1]. Концепція циклу праць «Гірнича енциклопедія» враховує сучасні тенденції інтеграції галузей знань, зокрема інженерних і гуманітарних наук, що знайшло відображення у матеріалах, присвячених історії гірництва та геологічних наук, традиціям і культурній спадщині гірників, гірничому законодавству, охороні праці та навколишнього середовища тощо. Крім того, гуманітарний напрямок проекту «Гірнича енциклопедія» знайшов втілення у викладанні курсу сучас ної української мови у вищих навчальних закладах (зокрема на спеціальних курсах і семінарах). Усе це є чинником гуманітаризації вітчизняної інженерної освіти й наближення її до європейських стандартів. 34 Галузева енциклопедія як важливий етап розвитку ... До виходу «Малої гірничої енциклопедії» фахівці України користували ся російськомовною «Горной энциклопедией», що вийшла друком у 1984– 1991 рр. Українське видання відрізняється не лише мовою, структурою чи значно більшою кількістю статей (майже вдвічі), а й відображає суттєві зміни, викликані науковими досягненнями і прогресом гірничої техніки й техноло гій за минулі десятиріччя. На цій підставі можна твердити, що МГЕ є найно вітнішою системною базою гірничо-геологічних знань серед подібних видань слов’янськими мовами. Цикл праць «Гірнича енциклопедія» сьогодні активно використовують у кількох освітніх процесах: 1. як навчальне видання для викладання кількох десятків дисциплін для гір ничих і геологічних спеціальностей (бакалаври, спеціалісти і магістри); 2. 1 Режим доступу: http://www.experts.in.ua/baza/doc/download/gorE.pdf ГАЛУЗЕВА ЕНЦИКЛОПЕДІЯ ЯК ВАЖЛИВИЙ ЕТАП РОЗВИТКУ НАУКОВОЇ ДИСЦИПЛІНИ ТА ЇЇ ТЕРМІНОСИСТЕМИ (НА ПРИКЛАДІ ЦИКЛУ ПРАЦЬ ІЗ ГІРНИЦТВА Й ГЕОЛОГІЇ «ГІРНИЧА ЕНЦИКЛОПЕДІЯ»)* як базисне універсальне видання для підготовки підручників, посібників, курсів лекцій, навчально-методичної літератури в гірництві та суміжних галузях і науках – геології, мінералогії, петрографії, гідрогеології, геохімії, геофізики, географії, екології, видобуванні та первинній переробці твер дих, рідких і газоподібних корисних копалин, маркшейдерії та геодезії, шахтному та підземному будівництві, а також історії геологічної й гірничої науки та освіти; 3. як розгалужена система сучасних знань для підвищення професійної ква ліфікації викладачів гірничих, геологічних, екологічних та географічних дисциплін вищих навчальних закладів і як довідник для студентів; 4. як ключ до англійської, німецької й російської термінологічних баз даних для покращення доступу до іноземних освітніх і наукових джерел, розви тку мобільності студентів і освітян в рамах Болонського процесу, умож ливлення міжнародної уніфікації освітніх і наукових процесів у природни чих і гірничотехнічних галузях. р у Апробація проекту. В Україні «Мала гірнича енциклопедія» розміщена у вільному доступі на декількох інформаційних порталах, зокрема престижному «Всеукраїнська експертна мережа»1, де вже зафіксовано декілька тисяч перегля дів. Проект «Гірнича енциклопедія» неодноразово презентувався, обговорювався й отримав схвальну оцінку на багатьох міжнародних конференціях, серед них на звемо такі: ХХI Всесвітній гірничий конгрес (Краків, 2008 р.); Польська «Школа експлуатації підземної» (2005–2011 рр., Краків); Українська «Школа підземної розробки» (2009–2014 рр., Гаспра, Бердянськ); Форум гірників (Національний гірничий університет, Дніпропетровськ, 2013 р.); VI Міжнародна науково-тех нічна конференція «Енергетика. Екологія. Людина» (НТУУ «Київський полі технічний інститут імені І. Сікорського», Київ, 2013 р.); зустрічі зі студентами й викладачами в Національному гірничому університеті (Дніпро), Харківському національному університеті імені В. Каразіна, Донецькому національному тех нічному університеті, Донбаському державному технічному університеті (Ал чевськ), Криворізькому національному університеті, Києво-Могилянській акаде 35 Володимир Білецький, Геннадій Гайко мії, Полтавському національному технічному університеті імені Ю. Кондратюка, Івано-Франківському національному технічному університеті нафти і газу тощо. мії, Полтавському національному технічному університеті імені Ю. Кондратюка, Івано-Франківському національному технічному університеті нафти і газу тощо. Цикл праць «Гірнича енциклопедія» висвітлено в авторитетних фахових і спеціалізованих виданнях (див. – [2]), інформацію про неї широко представ лено в мережі Інтернет, зокрема у «Вікіпедії» (українська, німецька, польська, російська й грузинська версії). «Малу гірничу енциклопедію» підготовлено на замовлення Державно го комітету телебачення і радіомовлення України за Програмою випуску со ціально-значимих видань накладом 1000 примірників і доведено державним коштом до більшості міських бібліотек України та бібліотек наукових і освітніх закладів, що підкреслює високу державну оцінку цього видавничого проекту. Висновки. Цикл праць «Гірнича енциклопедія» позитивно впливає на роз виток вітчизняної гірничої та геологічної освіти й науки та утверджує її високий авторитет у світі. ГАЛУЗЕВА ЕНЦИКЛОПЕДІЯ ЯК ВАЖЛИВИЙ ЕТАП РОЗВИТКУ НАУКОВОЇ ДИСЦИПЛІНИ ТА ЇЇ ТЕРМІНОСИСТЕМИ (НА ПРИКЛАДІ ЦИКЛУ ПРАЦЬ ІЗ ГІРНИЦТВА Й ГЕОЛОГІЇ «ГІРНИЧА ЕНЦИКЛОПЕДІЯ»)* Вперше створено термінологічну базу даних у геології та гірни цтві, що нараховує 17 350 термінологічних та номенклатурних одиниць, та адап товано її до англійської, німецької й російської термінологічних баз даних. Цикл праць «Гірнича енциклопедія» широко використовується в освітніх процесах ви щої школи України; виконує свою мету й призначення, зокрема, відроджує укра їнську фахову мову в галузі гірництва й геології, забезпечуючи перехід освітніх процесів до української понятійно-термінологічної системи; сприяє оновленню змісту гірничої й геологічної освіти та підвищенню якості навчальної й методич ної літератури; робить внесок у наукове забезпечення процесу інтеграції вітчизня ної вищої освіти в європейський та світовий освітній простір. 2. Білецький В. Створення першої вітчизняної «Гірничої енциклопедії» // Вісник НУ «Львівська політехніка». Сер.: Проблеми української терміно ЛІТЕРАТУРА 1. Білецький В. Загнітко А., Манець І. Досвід удосконалення сучасної україн ської гірничої термінології // Донецький вісник НТШ. Донецьк, 2003. Т. 3. С. 97–104; Білецький В., Загнітко А., Манець І., Ященко Ю. Розвиток і функ ціонування у науково-технічній літературі національної гірничої та екологіч ної термінології // Проблеми експлуатації обладнання шахтних стаціонар них установок. Вип. 96. Донецьк, 2003. С. 222–229; Білецький В., Манець І. Проблеми української термінології в гірництві, екології та техногенній без пеці // Проблемы эксплуатации оборудования шахтных стационарных уста новок. Вып. 97. Донецк, 2003. С. 114–117; Білецький В., Ященко Ю., Загнітко Ф., Манець І. Українська гірнича термінологія: становлення, удосконалення і вживання // Уголь Украины. 2003. № 12. С. 48–50; Білецький В. Досвід роз робки національної української терміносистеми в гірничій галузі // Мова і час: збірник наукових праць. Донецьк, 2009. С. 14–20; Зуєвська Н. «Мала гірнича енциклопедія» – внесок в українську гірничу освіту, науку, терміно логію // Матеріали VI Міжнародної науково-технічної конференції «Енерге тика. Екологія. Людина». Вип. 5. Київ, 2014. С. 137–139. 2. Білецький В. Створення першої вітчизняної «Гірничої енциклопедії» // Вісник НУ «Львівська політехніка». Сер.: Проблеми української терміно 2. Білецький В. Створення першої вітчизняної «Гірничої енциклопедії» // Вісник НУ «Львівська політехніка». Сер.: Проблеми української терміно 36 Галузева енциклопедія як важливий етап розвитку ... логії. 2000. № 453. С. 483–487; Білецький В. Проект «Гірнича енциклопе дія» // Новые технологии подземного строительства и добычи полезных ископаемых: Материалы международной научно-технической конференции. Алчевск: ДонДТУ. 2008. С. 207–210; Фик І. «Гірнича енциклопедія» – ваго мий внесок у гірничу науку // Вісник Харківського національного універси тету імені В. Каразіна. 2013. № 1098. С. 149. 3. Гірничий енциклопедичний словник: У 3 т. Т. 1 / Відп. ред. В. Білецький. До нецьк: «Східний видавничий дім», 2001. 512 с.; Гірничий енциклопедичний словник: У 3 т. Т. 2 / Відп. ред. В. Білецький. Донецьк: «Східний видавничий дім», 2002. 632 с.; Гірничий енциклопедичний словник: У 3 т. Т. 3 / Відп. ред. В. Білецький. Донецьк: «Східний видавничий дім», 2004. 752 с. 4. Колган О. Семантична та словотвірна структура української гірничої тер мінології. Автореф. дис. на здобуття наук. ступеня канд. філол. наук, спец.: 10.01.01 – «українська мова» / Запорізький національний університет. Запо ріжжя, 2009. 18 с. 5. Мала гірнича енциклопедія: У 3 т. Т. 1 / Відп. ред. В. Білецький. Донецьк: «Донбас», 2004. 640 с.; Мала гірнича енциклопедія: У 3 т. Т. 2 / Відп. ред. В. Білецький. Донецьк: «Донбас», 2007. 652 с.; Мала гірнича енциклопедія: У 3 т. Т. ЛІТЕРАТУРА 3 / Відп. ред. В. Білецький. Донецьк: «Східний видавничий дім», 2013. 644 с. 644 с. 6. Соболь В. Львів, 26.03.2016: ХХVII наукова сесія наукового товариства ім. Шевченка в Україні // Studia Polsko-Ukrainskie. 2016. T. 3. C. 193–200. SUBJECT SPECIFIC ENCYCLOPEDIAS AS AN IMPORTANT STAGE IN THE DEVELOPMENT OF SCIENTIFIC DISCIPLINES AND TERMINOLOGY SYSTEMS (ON EXAMPLE OF THE MINING ENCYCLOPEDIA AS A WORK IN MINING INDUSTRY AND GEOLOGY) Volodymyr Biletskyi Kharkiv Polytechnic Institute, Kharkiv, Ukraine Hennadii Haiko Igor Sikorsky Kyiv Polytechnic Institute, Kyiv, Ukraine 6. Соболь В. Львів, 26.03.2016: ХХVII наукова сесія наукового товариства ім. Шевченка в Україні // Studia Polsko-Ukrainskie. 2016. T. 3. C. 193–200. AS AN IMPORTANT STAGE IN THE DEVELOPMENT OF SCIENTIFIC DISCIPLINES AND TERMINOLOGY SYSTEMS (ON EXAMPLE OF THE MINING ENCYCLOPEDIA AS A WORK IN MINING INDUSTRY AND GEOLOGY) Hennadii Haiko Igor Sikorsky Kyiv Polytechnic Institute, Kyiv, Ukraine Hennadii Haiko Igor Sikorsky Kyiv Polytechnic Institute, Kyiv, Ukraine The peculiarities of the development of some scientific disciplines (such as mining, geology), primarily the professional terminology system are shown on the example of the project The Mining Encyclopedia (ukr .: Hirnycha Entsyklopediia). The study describes The Mining Encyclopedia, its relevance for technical education 37 Володимир Білецький, Геннадій Гайко in Ukraine, as well as for the integration of national higher education in the European and world educational space in general. in Ukraine, as well as for the integration of national higher education in the European and world educational space in general. p g Keywords: subject specific encyclopedias, The Mining Encyclopedia, technical terminology, technical education. Sobol, V. (2016). Lviv, 26.03.2016: 27th naukova sesiia naukovoho tovarystva im. Shevchenka v Ukraini. Studia Polsko-Ukrainskie, 3, 193-200 (in Ukrainian). REFERENCES Biletskyi, V. (Red.). (2001). Hirnychyi entsyklopedychnyi slovnyk (Vol. 1). Donetsk: Skhidnyi vydavnychyi dim (in Ukrainian); Biletskyi, V. (Red.). (2002). Hirnychyi entsyklopedychnyi slovnyk (Vol. 2). Donetsk: Skhidnyi vydavnychyi dim (in Ukrainian); Biletskyi, V. (Red.). (2004). Hirnychyi entsyklopedychnyi slovnyk (Vol. 3). 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Rozvytok i funktsionuvannia u naukovo-tekhnichnii literaturi natsionalnoi hirnychoi ta ekolohichnoi terminolohii. Problemy ekspluatatsii obladnannia shakhtnykh statsionarnykh ustanovok, 96, 222-229 (in Ukrainian); Biletskyi, V., Manets, I. (2003). Problemy ukrainskoi terminolohii v hirnytstvi, ekolohii ta tekhnohennii bezpetsi. Problemy ekspluatatsii obladnannia shakhtnykh statsionarnykh ustanovok, 97, 114-117 (in Ukrainian); Biletskyi, V., Yashchenko, Yu., Zahnitko, F., Manets, I. (2003). Ukrainska hirnycha terminolohiia: stanovlennia, udoskonalennia i vzhyvannia. Ugol Ukrainy, 2, 48-50 (in Ukrainian); Biletskyi, V. (2009). Dosvid rozrobky natsionalnoi ukrainskoi terminosystemy v hirnychii haluzi. In Mova i chas (pp. 14-20). Donetsk (in Ukrainian); Zuievska, N. (2014). «Mala hirnycha entsyklopediia» – vnesok v ukrainsku hirnychu osvitu, nauku, terminolohiiu. In Proceedings of the 6th International Conference «Enerhetyka. Ekolohiia. Liudyna» (pp. 137-139). Kyiv (in Ukrainian). Biletskyi, V., Zahnitko, A., Manets, I. (2003). Dosvid udoskonalennia suchasnoi ukrainskoi hirnychoi terminolohii. REFERENCES Donetskyi visnyk Naukovoho tovarystva imeni Shevchenka, 3, 97-104 (in Ukrainian); Biletskyi, V., Zahnitko, A., Manets, I., Yashchenko, Yu. (2003). Rozvytok i funktsionuvannia u naukovo-tekhnichnii literaturi natsionalnoi hirnychoi ta ekolohichnoi terminolohii. Problemy ekspluatatsii obladnannia shakhtnykh statsionarnykh ustanovok, 96, 222-229 (in Ukrainian); Biletskyi, V., Manets, I. (2003). Problemy ukrainskoi terminolohii v hirnytstvi, ekolohii ta tekhnohennii bezpetsi. Problemy ekspluatatsii obladnannia shakhtnykh statsionarnykh ustanovok, 97, 114-117 (in Ukrainian); Biletskyi, V., Yashchenko, Yu., Zahnitko, F., Manets, I. (2003). Ukrainska hirnycha terminolohiia: stanovlennia, udoskonalennia i vzhyvannia. Ugol Ukrainy, 2, 48-50 (in Ukrainian); Biletskyi, V. (2009). Dosvid rozrobky natsionalnoi ukrainskoi terminosystemy v hirnychii haluzi. In Mova i chas (pp. 14-20). Donetsk (in Ukrainian); Zuievska, N. (2014). «Mala hirnycha entsyklopediia» – vnesok v ukrainsku hirnychu osvitu, nauku, terminolohiiu. In Proceedings of the 6th International Conference «Enerhetyka. Ekolohiia. Liudyna» (pp. 137-139). Kyiv (in Ukrainian). Kolhan, O. (2009). Semantychna ta slovotvirna struktura ukrainskoi hirnychoi terminolohii. (Thesis abstract). Zaporizhzhia National University, Zaporizhzhia (in Ukrai nian). Kolhan, O. (2009). Semantychna ta slovotvirna struktura ukrainskoi hirnychoi terminolohii. (Thesis abstract). Zaporizhzhia National University, Zaporizhzhia (in Ukrai nian). Sobol, V. (2016). Lviv, 26.03.2016: 27th naukova sesiia naukovoho tovarystva im. Shevchenka v Ukraini. Studia Polsko-Ukrainskie, 3, 193-200 (in Ukrainian). Sobol, V. (2016). Lviv, 26.03.2016: 27th naukova sesiia naukovoho tovarystva im. Shevchenka v Ukraini. Studia Polsko-Ukrainskie, 3, 193-200 (in Ukrainian). 38 УДК 030(477) АКАДЕМІЧНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КОНТЕКСТІ РОЗВИТКУ СУЧАСНОГО ІНФОРМАЦІЙНОГО ПРОСТОРУ Микола Железняк, Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна У статті схарактеризовано явище електронної енциклопедистики й досліджено електронні енциклопедії, створені установами Національної академії наук Украї ни, що функціонують в інтернет-середовищі. Виявлено, що електронні енци- клопедії можна поділити на кілька різновидів, основний із яких – онлайн-енци- клопедії, що найбільшою мірою вирізняються на тлі класичних друкованих ви- дань характеристиками, спричиненими розвитком інформаційних технологій. Відповідно до цього поділу окреслено вітчизняні академічні енциклопедичні ресурси. Висунуто ідею щодо актуальності створення онлайн-енциклопедії про Національну академію наук України, а також констатовано, що наявні онлайн- енциклопедії не забезпечені повною мірою великим спектром технологічних можливостей їх використання. Ключові слова: онлайн-енциклопедистика, електронні енциклопедії, Націо нальна академія наук України, інформаційний простір, інтернет-технології, «Енциклопедія Сучасної України», «Вікіпедія». Найприкметніша особливість світової енциклопедистики початку ХХІ століття – розвиток електронних енциклопедій, зумовлений прогресом інформаційних технологій. А. Альшева справедливо зазначає, що онлайн- енциклопедії є породженням епохи інформаційного суспільства [1, с. 43]. Нині часто зауважують, що електронна енциклопедистика певною мірою витісняє класичну з її традицією укладання багатотомних книжкових видань та лако нічним стилем представлення матеріалів (скорочення слів, дрібний шрифт, обмежений в кількості і розмірі іконографічний матеріал тощо), позаяк низка відомих у світі видавництв відмовилася від друкованих видань й оприлюднює 39 Микола Железняк, Олександр Іщенко їх лише в електронному форматі. До того ж з’явилося багато нових енцикло педичних проектів, що існують винятково в інформаційному просторі, тобто лише в мережі Інтернет. Таку тенденцію спричинено прикметними рисами електронної енциклопедистики, що на тлі класичної енциклопедичної справи виглядають перевагами. Серед них, наприклад, – зручність у створенні енци клопедій (видавці оптимізують свою роботу й економлять кошти на друкові видань) та їх використанні (читачі використовують всі технічні можливості для пошуку, ознайомлення й засвоєння інформації, які забезпечує сучасний стан розвитку інтернет-технологій). Докладніше про електронні енциклопедії див.: [4]. Із появою й розвитком електронної енциклопедистики загальний ін терес до енциклопедично-довідкової інформації у суспільстві не зменшився, а, вірогідно, і збільшився. Підтвердженням цьому може бути той факт, що сайт найпопулярнішої у світі онлайн-енциклопедії «Вікіпедія» в багатьох країнах світу входить до рейтингової десятки найбільш відвідуваних, що засвідчує не абияку зацікавленість суспільства в енциклопедичних знаннях. Згідно зі Статутом Національної академії наук України, серед завдань її діяльності є здійснення пропаганди досягнень науки і техніки, сприяння по ширенню наукових знань, систематичне висвітлення результатів досліджень у засобах масової інформації та інших джерелах1. Уважаємо, що наукові ре зультати кожної установи Національної академії наук України презентувати суспільству доречно у вигляді енциклопедичних видань, адже, як зазначають В. Смолій, Г. 1 Статут Національної академії наук України, зареєстрований Міністерством юсти ції України 27.07.2016 р. С. 7. АКАДЕМІЧНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КОНТЕКСТІ РОЗВИТКУ СУЧАСНОГО ІНФОРМАЦІЙНОГО ПРОСТОРУ Микола Железняк, Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна Боряк, «енциклопедичні студії посідають особливе місце в євро пейській та світовій науці, оскільки не лише узагальнюють і систематизують наші знання, а й представляють їх суспільству» [5, с. 44]. Саме енциклопедич ний формат систематизації знань істотно сприяє їхньому поширенню й спо живанню серед широких верств населення. Водночас, як стверджує Н. Гаври лишина, видання електронних енциклопедій становить посилений інтерес, а також свідчить про престиж та освіченість країни. Загальнодоступна онлайн- енциклопедія «Вікіпедія» показала суспільству чого саме не вистачає сучас ній українській науці, зокрема популяризації маловідомих подій та фактів і спрощеного доступу до інформації [2]. Онлайн-енциклопедії з погляду доступності, ймовірно, мають переваги перед книжковими виданнями, так як нині Інтернет є найбільшим джерелом інформації, яким користується більшість людей. Для ознайомлення з онлайн- енциклопедією необхідно лише скористатися пошуком в Інтернеті, а озна йомлення з книжковою енциклопедією вимагає її попереднє придбання, що потребує часу й коштів. Більше того, саме з точки зору представлення наукової інформації, що актуально для установ Національної академії наук України, електронним виданням властиві й інші переваги на зразок можливості засто сування новітніх інформаційно-комунікативних технологій для зручного по дання наукової інформації (тексту, таблиць, графіків, анімації тощо). 40 Академічні онлайн-енциклопедії ... В теоретико-термінологічному плані електронну енциклопедистику про тиставляють класичній. У свою чергу, термін «електронна енциклопедисти ка» дещо застарілий, і сьогодні більш точно говорити про онлайн-енцикло педистику (або веб-енциклопедистику, чи інтернет-енциклопедистику), яка спочатку була «еволюційною» гілкою в електронній енциклопедистиці, а нині спостерігаємо тенденцію до термінологічного заступлення цього поняття. За визначенням фахівців, електронна енциклопедія – це джерело інформації в електронній формі, яке охоплює найповніший та достовірний довідковий об’єктивний, науково-вивірений матеріал з однієї або декількох галузей знань у вигляді статей, аудіо- та відеоматеріалів для задоволення інформаційних потреб людини з доступом через локальну (CD, DVD диски) та глобальну (Інтернет) мережі [3, с. 400]. Однак нині локальні носії інформації (CD, DVD диски) фактично не використовуються – їх замінив Інтернет. В українському інформаційному просторі нині можна виділити два типи електронних енциклопедій (це стосується як академічних онлайн- енциклопедій, так і будь-яких інших): • цифрові версії друкованих енциклопедій; • власне онлайн-енциклопедії. У свою чергу, серед оцифрованих енциклопедій виокремлюємо кілька різновидів залежно від форми представлення в мережі Інтернет: і і і У свою чергу, серед оцифрованих енциклопедій виокремлюємо кілька різновидів залежно від форми представлення в мережі Інтернет: • ті, що представлені у вигляді електронних книг; • ті, що представлені у вигляді електронних книг; • ті, що представлені у вигляді сайту. • ті, що представлені у вигляді сайту. Рис. 1. Зображення веб-інтерфейсу «Енциклопедії історії України» Рис. АКАДЕМІЧНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КОНТЕКСТІ РОЗВИТКУ СУЧАСНОГО ІНФОРМАЦІЙНОГО ПРОСТОРУ Микола Железняк, Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна 1. Зображення веб-інтерфейсу «Енциклопедії історії України» Цифрові електронні енциклопедії, представлені для читача у традицій ному для електронних книг PDF-форматі (рідше DjVu-форматі), становлять собою копію паперових аналогів, зберігаючи відповідну сторінкову структу 41 Микола Железняк, Олександр Іщенко ру, розмітку тощо. Сюди можна віднести всі паперові енциклопедії, створені в Національній академії наук України, адже ті, що не мають цифрових ко пій, рано чи пізно будуть оцифровані й розміщені в мережі Інтернет як PDF- файли (або DjVu-файли) – на сайті відповідної установи або в електронних бібліотеках, репозитаріях, архівах тощо). Чимало академічних енциклопедій вже мають цифрові копії (створені одночасно з друкованими або оцифровані з друкованих оригінальних видань), серед них, зокрема, «Енциклопедія іс торії України» (розміщено на сайті Інституту історії України НАН України PDF-файлами), «Українська мова: енциклопедія» (розміщено DjVu-файлом на різних сайтах), «Мала гірнича енциклопедія» (розміщено PDF-файлами на різних сайтах) тощо. Та чи можна називати онлайн-енциклопедіями такі видання? Гадаємо, ні, адже вони – друковані енциклопедії, а наявність у них електронних копій не робить їх онлайн-енциклопедіями. Інакше кажучи, їм не властиве те, що передбачають такі поняття, як «онлайн», «інтернет», «веб- технології» тощо. ру, розмітку тощо. Сюди можна віднести всі паперові енциклопедії, створені в Національній академії наук України, адже ті, що не мають цифрових ко пій, рано чи пізно будуть оцифровані й розміщені в мережі Інтернет як PDF- файли (або DjVu-файли) – на сайті відповідної установи або в електронних бібліотеках, репозитаріях, архівах тощо). Чимало академічних енциклопедій вже мають цифрові копії (створені одночасно з друкованими або оцифровані з друкованих оригінальних видань), серед них, зокрема, «Енциклопедія іс торії України» (розміщено на сайті Інституту історії України НАН України PDF-файлами), «Українська мова: енциклопедія» (розміщено DjVu-файлом на різних сайтах), «Мала гірнича енциклопедія» (розміщено PDF-файлами на різних сайтах) тощо. Та чи можна називати онлайн-енциклопедіями такі видання? Гадаємо, ні, адже вони – друковані енциклопедії, а наявність у них електронних копій не робить їх онлайн-енциклопедіями. Інакше кажучи, їм не властиве те, що передбачають такі поняття, як «онлайн», «інтернет», «веб- технології» тощо. Рис. 2. Зображення веб-інтерфейсу «Юридичної енциклопедії» Рис. 2. Зображення веб-інтерфейсу «Юридичної енциклопедії» Цифрові енциклопедії, представлені для читача у веб-форматі, позбав лені прив’язки до друкованого оригіналу з його посторінковою розміткою тексту, до того ж у них забезпечено можливість шукати довідковий матеріал завдяки тій чи іншій навігації сайту. Серед таких енциклопедій назвемо зга дану вище «Енциклопедію історії України» (рис. 2 Режим доступу: http://resource.history.org.ua/cgi-bin/eiu/history.exe?C21COM=F &I21DBN=EIU&P21DBN=EIU 4 Режим доступу: http://astro.lnu.edu.ua/astro 3 Режим доступу: http://leksika.com.ua/legal 3 Режим доступу: http://leksika.com.ua/legal 4 Режим доступу: http://astro.lnu.edu.ua/astro АКАДЕМІЧНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КОНТЕКСТІ РОЗВИТКУ СУЧАСНОГО ІНФОРМАЦІЙНОГО ПРОСТОРУ Микола Железняк, Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна 1), адже на веб-сторінці Ін ституту історії України НАН України можна знайти не лише цифрову копію всіх томів, а й електронну версію енциклопедії у веб-форматі2 – на головній сторінці енциклопедії є можливість здійснювати пошук за гаслами, кожне 2 Режим доступу: http://resource.history.org.ua/cgi-bin/eiu/history.exe?C21COM=F &I21DBN=EIU&P21DBN=EIU 42 Академічні онлайн-енциклопедії ... гасло має окрему сторінку, у статтях перехресні посилання функціонують як гіперпосилання. Інший приклад – «Юридична енциклопедія» (рис. 2) Інсти туту держави і права імені Володимира Корецького НАН України (її розмі щено на сайті «Лексика» в окремому розділі3 поруч з іншими довідковими виданнями, де вона має структуру, безвідносну до друкованих томів). Особливість таких енциклопедій полягає в тому, що, незважаючи на від мінність форми, за змістом вони лишаються копією паперових видань. Рис. 3. Зображення веб-інтерфейсу «Астрономічного енциклопедичного словника» Рис. 3. Зображення веб-інтерфейсу «Астрономічного енциклопедичного словника» Подібні видання коректно йменувати онлайн-енциклопедіями переду сім формально, оскільки з погляду технічного оформлення вони набува ють ознак, що відсутні в друкованому оригіналі та найголовніше – функ ціонують лише при підключенні до Інтернету, мають власну веб-адресу в межах того чи іншого сайту (але не мають власного сайту), тож не можуть «мігрувати» в Інтернеті по різних сайтах, як PDF-файли. «Астрономічний енциклопедичний словник»4 (рис. 3), підготовлений науковцями Головної астрономічної обсерваторії НАН України та Львів ського національного університету імені Івана Франка, поєднує в собі два описані вище різновиди цифрових видань, оскільки цей онлайн-ресурс має власну головну сторінку на сайті згаданого Львівського університету, на якій пропонується навігація матеріалу за літерами, однак кожну літеру представляє окремий PDF-файл. Другий тип електронних енциклопедичних видань – власне онлайн- енциклопедії як окремі енциклопедичні ресурси, що вирізняються власним 3 Режим доступу: http://leksika.com.ua/legal 43 Микола Железняк, Олександр Іщенко сайтом, створеним спеціально для енциклопедії (технічні можливості сайту забезпечують швидкість віднайдення енциклопедії в мережі, зручність по шуку інформації в ній, її читання, засвоєння, опрацювання тощо). Інтернет- ресурси такого типу є онлайн-енциклопедіями і за формою, і за змістом. Деякі зарубіжні енциклопедисти на основі аналізу трьох поважних універсальних видань – «Britannica»5, «Brockhaus»6 і «NE»7 – виокремили 15 релевантних характеристик, що визначають власне онлайн-енциклопедії (табл. 1). Табл. 1. Визначальні особливості онлайн-енциклопедій [6, с. 5 Режим доступу: http://eb.com 6 Режим доступу: https://brockhaus.de 7 Режим доступу: http://ne.se 5 Режим доступу: http://eb.com 6 Режим доступу: https://brockhaus.de 7 Режим доступу: http://ne.se 8 Режим доступу: http://esu.com.ua 9 Режим доступу: http://kobzar.ua 10 Режим доступу: http://mineralopediaukraine.com АКАДЕМІЧНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КОНТЕКСТІ РОЗВИТКУ СУЧАСНОГО ІНФОРМАЦІЙНОГО ПРОСТОРУ Микола Железняк, Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна 22] Характеристики Britannica NE Broсkhaus Доступ із різних ґаджетів + + + Регулярне оновлення застарілих даних + + + Об’єктивність інформації (перевірена експертом) + + + Багатовимірність контенту (медіаресурсний супровід) + + + Багаторівневість контенту + + + Інтегрованість із системою освіти + + + Багатофункціональність веб-сайту енциклопедії + + + Інтеграція з соціальними мережами + + + Інтерактивність + + + Багатомовність + + - Опрацювання інформації (копіювання, зберігання) + + + Наявність підписки + + + Особистий кабінет + + + Моніторинг користувацьких запитів + + + Табл. 1. Визначальні особливості й ій [6 22] Табл. 1. Визначальні особливості онлайн-енциклопедій [6, с. 22] Отже, онлайн-енциклопедії – це цілком відмінне явище від класичних енциклопедичних видань у зв’язку з наявністю в них низки функціональних компонентів, властивих лише інтернет-ресурсам. Одразу зауважимо, що, на жаль, жодна з українських електронних енциклопедій не може похвалитися наявністю всіх із можливих характеристик, які наведено в таблиці. Загалом академічних онлайн-ецниклопедій поки що обмаль. У свою чергу, онлайн-енциклопедії можна поділити на: • ті, що мають паперові аналоги; • ті, що не мають паперових аналогів. Перший різновид онлайн-енциклопедій забезпечує передусім популя ризацію й поширення в інформаційному просторі знань, опублікованих у 5 Режим доступу: http://eb.com 6 Режим доступу: https://brockhaus.de 44 Академічні онлайн-енциклопедії ... друкованих виданнях. Основний масив контенту в таких онлайн-енцикло педіях є адаптацією текстів із друкованих томів. Проте текстовий контент може бути доповнено звуковим, фото- чи відеорядом, що, ясна річ, відсут ній у паперових оригіналах. Чільне місце серед них займає «Енциклопедія Сучасної України» Інституту енциклопедичних досліджень НАН України (рис. 4), яку створюють на основі відповідного багатотомного видання. Сайт енциклопедії8 має власний мультимедійний ресурс, що вирізняється, окрім енциклопедичних статей (кожна з яких має власну сторінку), наявністю фотогалерей, актуальною щоденною інформацією-календарем про відомих осіб, стрічкою енциклопедичних новин, інтеграцією із соціальними мережа ми та обговоренням статей (впроваджено можливість лишати коментарі до статей). Рис. 4. Зображення веб-інтерфейсу «Енциклопедії Сучасної України» Рис. 4. Зображення веб-інтерфейсу «Енциклопедії Сучасної України» Ще один приклад – онлайнова «Шевченківська енциклопедія»9, в осно ві якої 6-томне універсальне довідкове видання з такою ж назвою про жит тєвий і творчий шлях видатного українського поета, яке підготував Інститут літератури імені Тараса Шевченка НАН України за участю шевченкознавців України та діаспори (рис. 5). Онлайн-енциклопедія становить собою портал із багатьма тематичними розділами про видатного українця, а також мульти медійні блоки (музеї та 3-d тури, пам’ятники, місця перебування, аудіокниги, телепередачі тощо – тобто все те, що не можливо реалізувати в рамках книж кових видань). 11 Режим доступу: http://hrushevsky.nbuv.gov.ua 12 Режим доступу: http://eu.univ.kiev.ua АКАДЕМІЧНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КОНТЕКСТІ РОЗВИТКУ СУЧАСНОГО ІНФОРМАЦІЙНОГО ПРОСТОРУ Микола Железняк, Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна 45 Микола Железняк, Олександр Іщенко Рис. 5. Зображення веб-інтерфейсу «Шевченківської енциклопедії» Рис. 5. Зображення веб-інтерфейсу «Шевченківської енциклопедії» Рис. 6. Зображення веб-інтерфейсу «Української мінералопедії» Рис. 6. Зображення веб-інтерфейсу «Української мінералопедії» Другий різновид власне онлайн-енциклопедій – ті, що не мають друко ваних аналогів. Для прикладу назвемо «Українську мінералопедію» – міне ралогічну онлайн-енциклопедію України»10 Інституту геохімії, мінералогії та рудоутворення імені Миколи Семененка НАН України (рис. 6). Щоправда, варто зауважити, що основний масив інформації складають матеріали з ци клу друкованих праць, об’єднаних назвою «Гірнича енциклопедія». Та все ж окремої паперової «Української мінералопедії» не існує. 46 Академічні онлайн-енциклопедії ... До цього різновиду онлайн-енциклопедій можна віднести «Електронний архів Михайла Грушевського»11 Національної бібліотеки України імені Во лодимира Вернадського НАН України (рис. 7). Автори не називають цей проект енциклопедією, оскільки він не складається з енциклопедичних ста тей, а є фундаментальною інформаційною базою про видатного українського діяча, де акумульовано його твори, біографічні документи, листування, праці про нього, іконографічні матеріали (фотодокументи, ілюстрації з видань), ві деоматеріали (документальні та документально-постановочні фільми) тощо. Однак для поширення знань у суспільстві цей інтернет-ресурс має таке ж зна чення, яке б мала персональна енциклопедія про М. Грушевського. Прикмет но, що інформаційний ресурс багатомовний (понад 40 мов світу), отже, є до ступний для мільйонів зацікавлених людей зі всього світу. Рис. 7. Зображення веб-інтерфейсу «е-Архіву Михайла Грушевського» Рис. 7. Зображення веб-інтерфейсу «е-Архіву Михайла Грушевського» В Україні серед онлайн-енциклопедій вирізняються такі, що презентують навчальні заклади (типологічно такі енциклопедії, на думку дослідників, на лежать до регіональних). Йдеться, наприклад, про «Енциклопедію Київського національного університету імені Тараса Шевченка»12, що всебічно охоплює та інформаційно представляє за персоналійним і тематичним принципами історію університету від часу заснування до сьогодні. Прикметно, що ця енци клопедія планувалася від початку як двотомне друковане видання, втім реалі зувалася як онлайн-енциклопедія. Подібні енциклопедії (електронні або дру ковані) мають й інші заклади вищої освіти України (Львівський національ ний університет імені Івана Франка, Національний університет «Острозька академія»). Уважаємо цілком доречним і необхідним створення онлайн- 47 Микола Железняк, Олександр Іщенко енциклопедії про Національну академію наук України – її історію, структу ру, видатних науковців, наукові досягнення світового й національного рівнів тощо, позаяк 100-річна діяльність Академії уповноважує на її експлікацію в енциклопедичному форматі. Між іншим, варто згадати й про Наукове това риство імені Шевченка, яке разом з Інститутом енциклопедичних досліджень НАН України започаткувало в 2012 році відповідну енциклопедію – «Науко ве товариство імені Шевченка» (щоправда, її оприлюднюють як паперове ба гатотомне видання). АКАДЕМІЧНІ ОНЛАЙН-ЕНЦИКЛОПЕДІЇ В КОНТЕКСТІ РОЗВИТКУ СУЧАСНОГО ІНФОРМАЦІЙНОГО ПРОСТОРУ Микола Железняк, Олександр Іщенко Інститут енциклопедичних досліджень НАН України, Київ, Україна Загалом же, зважаючи на нинішній високий рівень розвитку інформацій них технологій, академічних онлайн-енциклопедій, підготовлених установа ми Національної академії наук України, поки що мало, а ті, що є, не викорис товують повний спектр технічних і змістових можливостей при їх створенні. Це актуалізує проблеми української електронної енциклопедистики та необ хідність подальших досліджень і технічних розробок у цьому напрямі. Mykola Zhelezniak, Oleksandr Ishchenko Institute of encyclopedic research, Kyiv, Ukraine Mykola Zhelezniak, Oleksandr Ishchenko Institute of encyclopedic research, Kyiv, Ukraine The article describes the phenomenon of the electronic encyclopedia activity and investigates the electronic Internet encyclopedias created by the institutions of the National Academy of Sciences of Ukraine. It has been discovered that electronic en cyclopedias can be divided into several varieties, the main of which is online ency clopedias, which are highly distinguished in comparison with classical printed pub lications by the characteristics caused by the development of information technolo gy. In accordance with this division, the national academic encyclopedic resources are characterized in the article. The idea of the importance of creating an online encyclopedia about the National Academy of Sciences of Ukraine is presented; it also argued that current online encyclopedias are not fully equipped with a wide range of technological opportunities for their use. Keywords: online encyclopedia activity, electronic encyclopedias, National Academy of Sciences of Ukraine, information space, Internet technologies, Ency clopedia of modern Ukraine, Wikipedia. ЛІТЕРАТУРА 1. Альшева А. Переклад німецької Вікіпедії як шлях до розширення Uanet // Мовні і концептуальні картини світу. 2013. Вип. 46 (1). С. 42–48. 1. Альшева А. Переклад німецької Вікіпедії як шлях до розширення Uanet // Мовні і концептуальні картини світу. 2013. Вип. 46 (1). С. 42–48. 2. Гаврилишина Н. Регіональна енциклопедистика Полтавщини // Уманська старовина. 2016. № 1. 2. Гаврилишина Н. Регіональна енциклопедистика Полтавщини // Уманська старовина. 2016. № 1. 3. Жежнич П., Гірняк М. Особливості формування енциклопедії в сучасних умовах розвитку інформаційних технологій // Вісник Національного уні верситету «Львівська політехніка». 2012. № 732: Комп’ютерні науки та ін формаційні технології. С. 400–405. 4. Железняк М. Українська електронна енциклопедистика: тенденції розвитку та місце в інформаційному просторі держави // Енциклопедичний вісник України. 2017. Ч. 8–9. С. 7–21. 4. Железняк М. Українська електронна енциклопедистика: тенденції розвитку та місце в інформаційному просторі держави // Енциклопедичний вісник України. 2017. Ч. 8–9. С. 7–21. 5. Смолій В., Боряк Г. Сучасна історична енциклопедистика: здобутки і пер спективи розвитку // Вісник НАН України. 2015. № 3. С. 44–60. 5. Смолій В., Боряк Г. Сучасна історична енциклопедистика: здобутки і пер спективи розвитку // Вісник НАН України. 2015. № 3. С. 44–60. 6. Ялафов Ф., Гилязов И., Хабибуллин М. Методологические основы онлайн- энциклопедии // Международный журнал прикладных и фундаментальных исследований. 2017. № 10. С. 20–24. 48 Академічні онлайн-енциклопедії ... REFERENCES Alsheva, A. (2013). Pereklad nimetskoi Vikipedii yak shliakh do rozshyrennia Uanet. Movni i kontseptualni kartyny svitu, 46(1), 42-48 (in Ukrainian). Alsheva, A. (2013). Pereklad nimetskoi Vikipedii yak shliakh do rozshyrennia Uanet. Movni i kontseptualni kartyny svitu, 46(1), 42-48 (in Ukrainian). Havrylyshyna, N. (2016). Rehionalna entsyklopedystyka Poltavshchyny. Umanska starovyna, 1 (in Ukrainian). Havrylyshyna, N. (2016). Rehionalna entsyklopedystyka Poltavshchyny. Umanska starovyna, 1 (in Ukrainian). Smolii, V., Boriak, H. (2015). Suchasna istorychna entsyklopedystyka: zdobutky i perspektyvy rozvytku. Visnyk NAN Ukrainy, 3, 44-60 (in Ukrainian). Smolii, V., Boriak, H. (2015). Suchasna istorychna entsyklopedystyka: zdobutky i perspektyvy rozvytku. Visnyk NAN Ukrainy, 3, 44-60 (in Ukrainian). Yalafov, F., Giliazov, I., Khabibullin, M. (2017). Metodologicheskie osnovy onlain- entciklopedii. Mezhdunarodnyi zhurnal prikladnykh i fundamentalnykh issledovanii, 10, 20-24 (in Russian). Zhelezniak, M. (2017). Ukrainska elektronna entsyklopedystyka: tendentsii rozvytku ta mistse v informatsiinomu prostori derzhavy. Entsyklopedychnyi vis nyk Ukrainy, 8-9, 7-21 (in Ukrainian). Zhezhnych, P., Hirniak, M. (2012). Osoblyvosti formuvannia entsyklopedii v suchasnykh umovakh rozvytku informatsiinykh tekhnolohii. Visnyk Natsionalnoho universytetu «Lvivska politekhnika», 732 (Kompiuterni nauky ta informatsiini tekhno lohii), 400-405 (in Ukrainian). 49 Енциклопедичний вісник України. 2018. Число 10 ÁВТОР також: Енциклопедія електронна), оскільки в них це легко реалізува ти. В енциклопедіях, які виходять друком, А. і. забезпечують публіку ванням окремих додаткових томів. Для багатотомних видань такими публікаціями традиційно є щоріч ники (див.: Том додатковий). У них додають не лише актуалізовані стат ті, а й цілком нові (за актуалізова ним реєстром гасел), що позначають нові актуалізовані поняття, терміни, явища. Незалежно від форми пред ставлення довідкової інформації А. і. в енциклопедіях має здійснюватися регулярно з визначеною періодич ністю. (англ. autor) – особа, яка створила будь-яку наукову працю, літератур ний чи мистецький твір тощо. В ен циклопедичній справі А. – той, хто написав енциклопедичну статтю чи енциклопедію. Може бути од нією особою або колективом (див.: Авторський колектив). Авторство (ініціали і прізвище або повне імя і прізвище) зазначають зазвичай під статею. Не існує стандарту регла ментації порядку подання автора (ініціали та прізвище або повне ім’я та прізвище). В окремих енциклопе дичних статтях автора не зазначають (див.: Стаття редакційна). ÁВТОРСЬКИЙ КОЛЕКТИ́В (англ. questionnaire) – один із методів збирання достовірної інформації для створення енциклопедичних ста тей. Належить до вторинних методів збирання інформації для енцикло педичних видань. А. використовують переважно для написання статей редакційних, рідше – для підготовки статей авторських. В енциклопе дичній справі методика А. принципо во не відрізняється від застосування в інших наукових галузях. (англ. composite authors) – група осіб, які об’єдналися з метою спіль ного написання енциклопедичної статті чи енциклопедії за узгодже ним задумом (див.: Енциклопедії концепція) і планом. MATERIALS TO GLOSSARY FOR ENCYCLOPEDIA SCIENCE Енциклопедичний вісник України. 2018. Число 10 Materials to Glossary for Encyclopedia Science АКТУАЛІЗÁЦІЯ ІНФОРМÁЦІЇ (англ. content updating) – сукупність дій, спрямованих на оновлення в енциклопедичній статті застарі лих даних із метою надання їм від повідності нині актуальній (досто вірній) інформації. Актуалізуватися можуть не лише статті, а й слóвник енциколопедії. У статтях інформацію актуалізують за допомогою редукції чи заміни застарілих даних новими або лише доповненням (розширен ням за рахунок нових важливих ві домостей). Нині А. і. найхарактерні ша для онлайн-енциклопедій (див. БІБЛІОГРÁФІЯ (англ. bibliography) – перелік фахо вих джерел (книг, журналів, статей), що стосуються описуваного матері алу. Бібліографія може бути струк турною частиною енциклопедичної статті або енциклопедії. Див. та кож: Огляд та Опис бібліографічний. ВИДÁННЯ ДОВІДКÓВЕ (англ. reference book) – видання, основним завданням і призначен ням якого є подання матеріалу в зручній формі для швидкого отри мання інформації різного характеру. Розраховано переважно на вибірко вого читача. Уміщує короткі наукові або прикладні (навчальні) відомості. Серед різновидів В. д. – енциклопе дія, енциклопедичний словник, енци клопедичний довідник, енциклопе дичний словник-довідник, довідник, словник, посібник, путівник. АНОТÁЦІЯ (англ. annotation note) – структур на одиниця слóвника енциклопе дичного видання, що є стислим тлу маченням реєстрового слова (див. також: Гасло). Може містити до того ж коротку інформацію про читацьке призначення, основні питання, не обхідні для висвітлення, обсяг май бутньої статті тощо. 51 Матеріали до словника енциклопедичних термінів АПАРÁТ ВИДÁННЯ но до встановлених правил. Найвідо мішим прикладом вікі-енциклопедії є «Вікіпедія». В.-е. зручні з погляду по шуку інформації, зокрема дозволяють швидко знаходити матеріали за клю човим словом. Прикметною особли вістю В.-е. є актуалізація інформації (застарілі дані постійно оновлюють на відміну від друкованих видань, де ак туалізація інформації забезпечується завдяки друку томів додаткових). Се ред вихідних даних В.-е. – веб-адреса і роки існування її у веб-середовищі. (англ. equipment of edition) – додатко ві (довідкові, наукові чи пояснюваль ні) до основного тексту енциклопе дичного видання допоміжні матері али, що призначені для покращення умов користування ним. Структура А. в. залежить від типу енциклопедії. БÁЗА ДÁНИХ (англ. database) – сукупність упоряд кованої інформації в електронному та паперовому вигляді (див. також: Оригінал), що використовується для створення статей в енциклопедич них виданнях. ВКАЗÍВКА (англ. web-encyclopedia) – енцикло педія в цифровому вигляді, що існує в мережі Інтернет. В.-е. створюють або на базі паперового енциклопе дичного видання (шляхом адаптації матеріалу під веб-середовище), або як окремий веб-ресурс, що функціонує лише в мережі Інтернет і не має дру кованих аналогів. Серед останніх ви окремлюють вікі-енциклопедії – сай ти довідкового характеру, створювані спільними зусиллями необмеженої кількості зацікавлених осіб відповід (англ. specification) – інформаційно- методичний документ, що регулює до тримання визначеної концепції енци клопедії та рекомендованої орієнтовної схеми-анкети в підготовці й написанні статей до енциклопедій різного типу. ДÉРЕВО ПОНЯ́ТЬ (англ. conception tree) – сукупність взаємопов’язаних понять із чіткою іє рархічною структурою. В енциклопе дистиці подекуди вживають і термін «дерево гасел». За деревовидною схе мою подають статті в енциклопеді ях систематичних (див. також: Ен циклопедія тематична). В енцикло педіях алфавітних взаємопов’язані поняття відшуковують за посилань системою (див. також: Посилання пе рехресне). Д. п. допомагає авторсько му колективу та редакторам орієн туватися щодо обсягу статей. Термін має таке найменування, оскільки його графічне зображення нагадує пере вернуте дерево – корінь, або корене вий (перший, стартовий) вузол (голов- ний понятійний елемент, від якого відгалужуються решта) розташову ють зверху. Від головного та проміж них елементів можливе відгалуження одного чи більшої кількості елементів. Проміжні понятійні елементи також називають гіллям, або внутрішніми вузлами; понятійні елементи, які не мають дочірніх чкладників, – листка ми, або листковими (термінальними, кінцевими) вузлами. ДÓВІДКА ЕТИМОЛОГÍЧНА (Етимологія) (англ. etymological information) – лінгвістична інформація, що пояс нює походження слова та його спо ріднені зв’язки з іншими словами тієї самої або інших споріднених мов. В енциклопедіях і словниках Д. е. подають найчастіше в дужках до іншомовних слів-заголовків. ВКЛÉЙКА (англ. insert) – окремий відбиток ілюстративного матеріалу чи тексту, приклеєний до корінцевого поля од 52 Materials to Glossary for Encyclopedia Science ГÁСЛО (англ. catchword) – реєстрове слово в слóвнику енцилопедичного видан ня (самостійно або разом з анота цією). У ширшому значенні Г. на зивають заголовок енциклопедичної статті (самостійно чи разом із на повненням статті). ДЕФІНÍЦІЯ нієї із внутрішніх сторінок енцикло педичного видання. (англ. definition) – коротке логічне та науково точне визначення (тлу мачення) поняття, у якому відбито найістотніші його риси. Д. вибудову ють за можливістю однотипними та водночас повноцінними словнико вими довідками. Вони також мають тлумачити сучасний зміст поняття без звернення до історії трансфор мації його розуміння суспільством. В енциклопедіях Д. є найвагомішою інформаційною одиницею. Користу вач може обмежитися лише дефініт ною інформацією, не читаючи всієї статті, і цього буде достатньо для найзагальнішого уявлення про по няття. ЕНЦИКЛОПЕДИ́ СТИКА (Енциклопедознавство) (англ. encyclopedia science) – галузь наукових знань, у якій об’єктом до сліджень є енциклопедичні видання Е. – наука про енциклопедії з охоп ленням усіх проблем їхнього створен ня, зокрема методології та методики укладання, класифікації, визначен ЕНЦИКЛОПЕДИ́ЧНА КУЛЬТУ́РА (англ. encyclopedic culture) – сукуп- ність цінностей, пов’язаних зі ство ренням енциклопедій. Е. к. асоцію ють із розвитком традицій енцикло педичної справи – з її минулим та майбутнім, яке вимагає консолідації й докладання зусиль багатьох на уковців, інституцій тощо, а також ре тельного вивчення досвіду енцикло педичного книговидання. Значного розвитку Е. к. отримала в епоху Ре несансу. ЕНЦИКЛОПЕДИ́ СТ ня типових ознак тощо. Е. як окре мий науковий напрям формується на основі поєднання різних наук, та найбільше – історичних (Е. відно сять до спеціальних історичних дис циплін). Серед наукових проблем Е. вирізняють питання історії енцикло педичного книговидання, форм і кла сифікації енциклопедій (зокрема роз різнення друкованих і електронних; універсальних, спеціалізованих, ре гіональних тощо); методичних засад їхнього укладання, що пов’язані з від бором матеріалів до енциклопедій, мовними особливостями написання статей, поданням інформації (фак тичних даних), актуалізацією інфор мації в них, залученням авторів; ролі та значення енциклопедичних ви дань розвою суспільства тощо (див. також: Енциклопедична справа). (англ. encyclopedist) – 1) укладач енциклопедій (автор статей чи ре дактор видання) або науковець, який досліджує теоретичні й прак тичні проблеми їх укладання (фахі вець з енциклопедистики). Термін «Е.» вживають від 18 ст., коли група французьких мислителів, науковців, письменників та митців почала пра цювати над виданням «Енциклопе дія, або Тлумачний словник науки, мистецтва й ремесел» під редагуван ням Д. Дідро та Ж.-Л. д’Аламбера. 2) Ерудована особа з глибокими уні версальними знаннями в певній чи багатьох галузях науки й життя. При кметні риси Е. – вченість, обізнаність (високий інтелект), начитаність. Е. вирізняється особливим мисленням, що формується на основі поєднання наукових знань (різних наук), мисте цтва, філософії. Історія знає чимало науковців, художників, письменни ків, талант яких виявився і в науці, і в мистецтві. Е. часто називають Арис тотеля, який підсумував здобутки давньогрец. філософів. Окрім знань, Е. має системне логічне мислення, аналітичні здібності та вміння роби ти критичні висновки. ЕКСПÉРТНА ОЦÍНКА СТАТТÍ (англ. expert opinion on an article) – вид редакторської роботи, метою якої є дослідження, перевірка, ана ліз й оцінка наукового та іншого рівнів статті, а також підготовка об ґрунтованих висновків щодо неї. До Е. о. с., зокрема й енциклопедичної, залучають експертів-фахівців відпо відної галузі (див.: Наукове редагу вання). 53 Матеріали до словника енциклопедичних термінів ЕНЦИКЛОПЕДИ́ЧНА ЛІТЕРАТУ́РА (англ. encyclopedic historical literatu re) – книги довідкового характеру (див.: Видання довідкові), що охо плюють як енциклопедії, енцикло 54 Materials to Glossary for Encyclopedia Science ЕНЦИКЛОПЕДИ́ЧНИЙ ЖАНР (англ. encyclopedic genre) – жанро вий різновид видань довідкових, що характеризується власними засадни чими рисами створення, структурою подання інформації, мовною стиліс тикою тощо. Е. ж. властивий пере дусім енциклопедіям та іншій енци клопедичній літературі. Водночас будь-який інший твір (напр., науко ва стаття) може бути написаний за принципами Е. ж. ЕНЦИКЛОПЕДИ́ЧНИЙ СЛОВНИ́К (англ. encyclopedic dictionary) – ви дання довідкове, що містить упоряд кований за алфавітним або тематич ним принципами перелік певних понять (слів, словосполучень), тер мінів тощо із поясненням їхнього значення, але без широкого потрак тування суті явища. На відміну від енциклопедій, в Е. с. основне смисло ве навантаження покладено на дефі ніцію. Статті вирізняються лако нічністю, більшість із них має лише тлумачний характер. Історично тер ЕНЦИКЛОПЕДИ́ЧНИЙ ДОВІДНИ́ К педичні словники, енциклопедичні довідники й енциклопедичні словни ки-довідники, так і літературу, що історично передувала їм (словники, лексикони, компендіуми, зерцала тощо). До найвідомішої в світі Е. л. належать: трактат «Дисципліни» Терренція Варрона (116–27 рр. до н. е.) й «Історія природи» у 37-ми книгах Плінія Старшого (23–79 рр.), словник «Лексикон Свіди» (10 ст.), «Енциклопедія, або Тлумачний слов- ник науки, мистецтва й ремесел» Д. Дідро та Ж.-Л. д’Аламбера, «Бри танська енциклопедія» тощо. В Україні до найвідоміших зразків Е. л. належать такі видання: «Ізборнік Святослава» (1073), «Лексикон…» Памви Беринди (1627), «Українська загальна енцикльопедія» за ред. І. Раковського (1930–1933), «Ен циклопедія українознавства» за ред. В. Кубійовича (1949–1995), «Укра їнська радянська енциклопедія» за ред. М. Бажана (1959–1985) тощо. Серед Е. л. найбільшою за кількістю томів нині є «Енциклопедія Сучас ної України» за ред. А. Жуковського, М. Дзюби, М. Железняка та ін. (ста ном на 2018 рік – 20 томів; готують ся нові). (англ. encyclopedic reference book) – видання довідкове, що містить корот кі відомості наукового, науково-пу бліцистичного або прикладного ха рактеру про певні поняття, терміни тощо. Матеріал в Е. д. розміщують зазвичай за тематичним принци пом, обсяг обмежують 1–3 томами. Більшість із них орієнтовано на ши роке коло читачів. ЕНЦИКЛОПÉДІЇ КОНЦÉПЦІЯ міном «Е. с.» позначали всі видання енциклопедичного характеру, три валий час його вживали як синонім поняття «енциклопедія». (англ. concept of encyclopedia) – ідей ний задум енциклопедії. Е. к. є су купністю взаємопов’язаних і взає мозумовлених ознак видання, ви значених його метою, змістом, фор мою представлення. На основі Е. к. планують характер енциклопедії, її формат, обсяг, залучення авторів, загальні вимоги до змісту й напо внення статей тощо. Е. к. часто ко ригують задля удосконалення в про цесі роботи над енциклопедією. До обговорення концепції фундамен тальних видань, зокрема енциклопе дії національної, залучають широкі кола громадськості та засоби масової інформації. ЕНЦИКЛОПЕДИ́ЧНА СПРÁВА (англ. encyclopedia publishing) – га лузь знань, що охоплює енцикло педистику, передусім її прикладну ділянку, пов’язану з підготовкою, укладанням та виданням енциклопе дій (пошук авторів, написання ста тей, їх редагування, а також друку вання видань та їх розповсюдження тощо). (англ. encyclopedia publishing) – га лузь знань, що охоплює енцикло педистику, передусім її прикладну ділянку, пов’язану з підготовкою, укладанням та виданням енциклопе дій (пошук авторів, написання ста тей, їх редагування, а також друку вання видань та їх розповсюдження тощо). 55 Матеріали до словника енциклопедичних термінів ЕНЦИКЛОПЕДИ́ЧНИЙ СЛОВНИ́К-ДОВІДНИ́К (англ. encyclopedic dictionary) – ви дання довідкове, що містить упоряд кований за алфавітним або тематич ним принципами перелік певних по нять (слів, словосполучень), термінів тощо із поясненням їхнього значення та короткими додатковими відомос тями про них наукового, науково-пу бліцистичного або прикладного ха рактеру. ЕНЦИКЛОПÉДІЯ БІОГРАФÍЧНА (англ. biographical encyclopedia) – енциклопедія, зміст якої формують довідки про життя й діяльність ви датних осіб. Укладають переважно за алфавітним принципом (іноді – за тематичним або хронологічним). За характером інформації можуть бути загальні (про видатних діячів різних родів діяльності, напр., «Ве ликі українці»), та часткові, зокре ма регіональні (про діячів окремого регіону країни, населеного пункту, навчального закладу, товариства, за воду тощо, напр., «Енциклопедія Му качева в іменах») та галузеві (про ді ячів спільного роду діяльності, напр., «Видатні вітчизняні ботаніки»). Ти пологічно Е. б. відносять зазвичай до енциклопедій спеціалізованих, зокрема енциклопедій галузевих або енциклопедій регіональних. ЕНЦИКЛОПÉДІЯ АЛФАВÍТНА допомогою загальновизнаних тер мінів і понять; уникнення сумнів них гіпотез, авторських неологізмів, прогнозів; відсутність як негатив них, так і позитивних емоційних особистісних оцінок); наявність де фініцій; об’єктивність, фактологічна точність, уніфікація, схематичність оформлення статей; використання посилань системи, приміток, кур сивів, довідкового апарату, карт, таблиць, ілюстрацій тощо (див. також: Енциклопедичний жанр). За характером інформації виділяють енциклопедії універсальні, енцикло педії національні, енциклопедії дер жави, енциклопедії спеціалізовані, енциклопедії галузеві, енциклопедії тематичні, енциклопедії проблемні (вузькогалузеві), енциклопедії регіо нальні, енциклопедії біографічні, ен циклопедії персональні. За системою розташування статей – енциклопедії алфавітні, енциклопедії система тичні, енциклопедії хронологічні; за обсягом (реєстрових слів) – одно томні й багатотомні, енциклопедії короткі, малі й великі. За цільовим призначенням – популярні (для як найширшого кола читачів), фахові (наукові, прикладні), енциклопедії дитячі. За формою подання мате ріалу – паперові, енциклопедії елек тронні, веб-ециклопедії (онлайн-ен циклопедії), енциклопедії класичні. Е. створюють переважно на основі її чіткої концепції (див.: Енциклопедії концепція), а також передбачають комплекс підготовчих робіт, серед яких – створення слóвника (при цьому окремі енциклопедії базо вим підґрунтям мали не слóвники, а повноцінні довідкові видання). (англ. encyclopedia in alphabetical or der) – енциклопедія, статті в якій роз міщено за алфавітним принципом. Е. а. зручна в користуванні, забезпе чує читача легким пошуком необхід ної інформації. На відміну від енци клопедії систематичної, не вимагає суцільного перечитування тексту. (англ. encyclopedia in alphabetical or der) – енциклопедія, статті в якій роз міщено за алфавітним принципом. Е. а. зручна в користуванні, забезпе чує читача легким пошуком необхід ної інформації. На відміну від енци клопедії систематичної, не вимагає суцільного перечитування тексту. ЕНЦИКЛОПÉДІЯ (англ. encyclopedic layout) – різновид формату видання, що визначає шири ну та довжину сторінки (у міліметрах) або ширину чи довжину друкованого аркуша (у сантиметрах) енциклопе дії із зазначенням частки, яку займає сторінка. Зазвичай у випускних даних вказують формат друкарського паперу та частку аркуша, при цьому потен ційні відхилення в накладі не повинні перевищувати 1 мм за шириною і ви сотою. Під час вибору Е. ф. враховують цільове призначення енциклопедії (універсальна, дитяча, галузева, спе ціалізована тощо), особливості тех нічного та художнього оформлення видання, фінансові можливості видав ця (зокрема, високий коефіцієнт ви користання площі сторінки дозволяє економити папір, однак ускладнює сприйняття інформації окремими гру пами читачів). (англ. encyclopedia) – видання до відкове, що містить упорядкований за алфавітним або тематичним принципами перелік певних по нять (слів, словосполучень), тер мінів тощо з поясненням їхнього значення. Е. – основний вид ен циклопедичної літератури, подає найповніші порівняно з іншими довідковими виданнями відомос ті зі всіх чи окремих галузей знань про людину чи світ. За повнотою довідкової інформації розрізняють власне Е., енциклопедичні словни ки, енциклопедичні довідники, ен циклопедичні словники-довідники. Основні ознаки Е.: повнота, сис темність і водночас лапідарність викладення інформації; науковість (висвітлення лише достовірних або максимально ймовірних фактів за 56 Materials to Glossary for Encyclopedia Science ЕНЦИКЛОПÉДІЯ ДИТЯ́ЧА (англ. subject encyclopedia) – енци клопедія, що містить систематизова ну інформацію з певної галузі нау ки чи виду практичної діяльності (напр., «Енциклопедія кібернетики», «Фольклористична енциклопедія», «Географічна енциклопедія Украї ни», «Українська літературна енци клопедія», «Мала гірнича енцикло педія», «Геодезичний енциклопе дичний словник»). В Е. г. відсутні терміни загального значення. Серед таких енциклопедій можуть бути вузькоспеціалізовані (напр., «Вироб нича енциклопедія бджільництва»), за обсягом реєстрових слів – повни ми («Повна енциклопедія етикету») або короткими («Коротка банківська й комерційна енциклопедія»). Типо логічно Е. г. зазвичай відносять до енциклопедій спеціалізованих. (англ. children’s encyclopedia) – ен циклопедія, призначена для дітей. Стиль статей в Е. д. науково-художній або науково-популярний. Фактич ний матеріал, мова статей і посилань система узгоджені з особливостями вікової групи, якій призначене ви дання. Чільне місце в Е. д. займають ілюстрації, які часто-густо є основним носієм змісту (зокрема у виданнях для дошкільнят). ЕНЦИКЛОПÉДІЯ ЕЛЕКТРÓННА (англ. digital encyclopedia) – ен циклопедія, яку представлено в цифровому форматі й опубліко вано на електронних носіях (CD- дисках) або в веб-мережі (Інтер неті). Е. е. з’явилися наприкінці 20 ст. (уперше Е. е. на дискетах було випущено в Японії). Нині Е. е. поділяють на цифрові аналоги па перових надрукованих енцикло педій та веб-енциклопедії (онлайн- енциклопедії). Перші можуть бути CD-додатком до таких видань або їх окремою редакцією (напр., з від редагованим, оновленим і допо вненим новим матеріалом). Особ ливість других – перманентна ак туалізація інформації. Крім цього, цифрові відповідники паперових видань можуть виглядати як елек тронна копія з повним збереженням не лише змісту видання, а й фор ми (напр., комп’ютерний оригінал- ЕНЦИКЛОПÉДІЯ ВЕЛИ́КА (англ. great encyclopedia) – енцикло педія, у якій укладачі ставлять за мету охопити якнайбільше реєстро вих слів, щоб якнайповніше подати відомості з тих питань, яким її при свячено. Е. в. належить до найповні ших джерел довідкової інформації. 57 Матеріали до словника енциклопедичних термінів Е. в. є виданнями в багатьох томах. Е. в. найчастіше бувають енциклопе діями універсальними. Е. в. є виданнями в багатьох томах. Е. в. найчастіше бувають енциклопе діями універсальними. «Енциклопедія професійно-техніч ної освіти України», «Енциклопе дія історії України», енциклопедія «Мистецтво України»). ЕНЦИКЛОПÉДІЯ КЛАСИ́ЧНА макет у форматах PDF, DjVu, FB2 тощо). Аналогами паперових ви дань можуть бути й такі Е. е., які ма ють свою форму розміщення й по дання довідкового матеріалу, адап тованого під особливості цифрового формату – із власним інтерфейсом, архітектонікою, навігацією, пошуко вою системою тощо. Е. е. такого типу вирізняються широкими мультиме дійними особливостями: вони міс тять «живі» ілюстрації – матеріали відео-, аудіо- і кінозйомок, що від творюють об’єкти в повноті кольо рів, звуків, динаміки. Ще однією прикметною рисою Е. е. є окремий спосіб подання інформації – гіпер текст, який характеризується нелі нійною архітектурою викладу мате ріалу й забезпечує миттєвий перехід від однієї статті до пов’язаної з нею іншої. Цифровий формат подання енциклопедичної інформації від крив нові перспективи в розвитку видань цього жанру. Зокрема, в мережі Інтернет з’явилися різно манітні онлайн-енциклопедії, най важливішою особливістю яких є мо більність щодо оновлення застарілої інформації. Будь-яку енциклопедію, що публікують на CD-носієві, можна адаптувати під веб-енциклопедію, і навпаки. Вихідні дані Е. е. на CD- носієві охоплюють дату опубліку вання, видавця тощо, натомість веб- енциклопедії, які не мають паперо вих оригіналів, – лише веб-адресу і роки існування в мережі. ЕНЦИКЛОПÉДІЯ ЗАГÁЛЬНА макет у форматах PDF, DjVu, FB2 тощо). Аналогами паперових ви дань можуть бути й такі Е. е., які ма ють свою форму розміщення й по дання довідкового матеріалу, адап тованого під особливості цифрового формату – із власним інтерфейсом, архітектонікою, навігацією, пошуко вою системою тощо. Е. е. такого типу вирізняються широкими мультиме дійними особливостями: вони міс тять «живі» ілюстрації – матеріали відео-, аудіо- і кінозйомок, що від творюють об’єкти в повноті кольо рів, звуків, динаміки. Ще однією прикметною рисою Е. е. є окремий спосіб подання інформації – гіпер текст, який характеризується нелі нійною архітектурою викладу мате ріалу й забезпечує миттєвий перехід від однієї статті до пов’язаної з нею іншої. Цифровий формат подання енциклопедичної інформації від крив нові перспективи в розвитку видань цього жанру. Зокрема, в мережі Інтернет з’явилися різно манітні онлайн-енциклопедії, най важливішою особливістю яких є мо більність щодо оновлення застарілої інформації. Будь-яку енциклопедію, що публікують на CD-носієві, можна адаптувати під веб-енциклопедію, і навпаки. Вихідні дані Е. е. на CD- носієві охоплюють дату опубліку вання, видавця тощо, натомість веб- енциклопедії, які не мають паперо вих оригіналів, – лише веб-адресу і роки існування в мережі. (англ. classic encyclopedia) – енци клопедія, яка стала зразком для на- слідування у створенні енциклопе дичних видань. Найчастіше Е. к. ЕНЦИКЛОПÉДІЯ КЛАСИ́ЧНА – це всесвітньовідомі енциклопедії минулих епох, що мали суттєвий вплив на розвиток енциклопедич ної справи. Напр., такою є 35-том на «Енциклопедія, або Тлумач ний словник науки, мистецтва й ремесел» (1751–1780), яку було створено за систематичним прин ципом подання наукових відомос тей в епоху Просвітництва у Па рижі під керівництвом Д. Дідро та Ж.‑Л. д’Аламбера. У тритомній К. е. «Британська енциклопедія» (1768– 1771), виданій в Единбурзі, вперше застосовано алфавітне розміщення статей як більш зручне для орі єнтування в системі знань. В епоху Просвітництва енциклопедії набу ли вдосконалення; саме вони сфор мували основні риси, принципи та моделі сучасних енциклопедичних видань. Найвизначніші Е. к. цього періоду – «Енциклопедія Брокгауз» (1796–1808) та «Великий універ сальний словник XIX ст.» П. Ла русса (1866–1874). У ХХ ст. низка Е. к. минулих епох перевидавалася, розширювалася, вдосконалювалася, видозмінювалася в інші видання й форми. Наприклад, «Велику енци клопедію Ларусса» (1960–1964), що продовжила традицію згадано го вище універсального словника, створили в елітній, повній і базовій версіях. ХХІ ст. відзначається по явою енциклопедій електронних, у зв’язку з чим поняття «Е к » набу ЕНЦИКЛОПÉДІЯ ДЕРЖÁВИ (англ. encyclopedia of a state) – ен циклопедія, яку присвячено окремій державі (див. також: Енциклопедія національна). Залежно від характе ру інформації Е. д. може належати до енциклопедій загальних (уза гальнює різнорідні дані про народ, його територію, історію, діяльність, культуру, самобутність, здобутки, представників тощо, напр., «Енци клопедія Сучасної України») або енциклопедій спеціалізованих (ви світлює державу в контексті певної теми, проблеми, галузі знань, напр., 58 Materials to Glossary for Encyclopedia Science ЕНЦИКЛОПÉДІЯ КОРÓТКА (англ. concise encyclopedia) – ен циклопедія, що подає лише най основніші терміни, поняття, явища з тих питань, висвітленню яких її при свячено. Енциклопедія може бути короткою не лише через оптимізо ваний перелік гасел, а й за рахунок того, що статтям у ній властиві стислість і лаконічність. Е. к. часто є скороченою версією більш повно го видання, зокрема багатотомно го. Проте Е. к. може бути й першим етапом створення більш широкого видання. Напр., «Шевченківський словник» у 2-х томах став основою під час створення фундаментальної 6-томної «Шевченківської енцикло педії». Е. к. найчастіше належить до енциклопедій галузевих. ЕНЦИКЛОПÉДІЯ ЗАГÁЛЬНА (англ. general encyclopedia) – те саме, що енциклопедія універсальна. (англ. general encyclopedia) – те саме, що енциклопедія універсальна. (англ. general encyclopedia) – те саме, що енциклопедія універсальна. 59 Матеріали до словника енциклопедичних термінів тощо), філософії та релігії, охорони здоров’я, міжнародних зв’язків дер жави. Е. н. має важливе для держави значення, зокрема в контексті утвер дження національної самосвідомості її громадян, патріотизму. Належить здебільшого до багатотомних видань (напр., «Енциклопедія Сучасної України») і є своєрідною «візитів кою» держави. Нині Е. н. створюють переважно в паперовому (книжко вому) і водночас електронному фор матах (див.: веб-енциклопедія). ло додаткового відтінку значення, асоційованого з друкованими енци клопедичними виданнями, в яких дотримано традиційних жанрових ознак. Методологічні підходи й тех нології підготовки матеріалів Е. к. найчастіше застосовують в укладан ні енциклопедій універсальних. Див. також: Енциклопедії концепція. ЕНЦИКЛОПÉДІЯ ПЕРСОНÁЛЬНА (англ. encyclopedia of a person) – ен циклопедія, яку присвячено одній особі – письменнику, науковцю, митцеві, спортсменові тощо. Здебіль шого Е. п. є завершальною формою ґрунтовного вивчення діяльності визначної особи. Типова Е. п. поєд нує біографічну інформацію з нау ковими розвідками про особу (до слідження її місця й ролі в тій галузі знань, до якої належить особа). Е. п. часто складається з передмови, роз логого нарису про особу, словнико вої частини (гасла відповідають на звам творів, оточенню, персонажам тощо), літопису життя й творчості особи, бібліографії, іменного покаж чика, списків основних скорочень та ілюстрацій. Напр., Е. п. у галузі літе ратури особливої цінності набувають у поєднанні з повним виданням тво рів особи. Прикладами Е. п. є «Шев ченківська енциклопедія» та «Фран ківська енциклопедія». Типологічно Е. п. зазвичай відносять до енцикло педій спеціалізованих. ЕНЦИКЛОПÉДІЯ НАЦІОНÁЛЬНА (англ. encyclopedia of a nation) – різновид енциклопедії держави. Ви значальною рисою Е. н. є тематика, пов’язана з відображенням історії та сучасного стану народу, природ ного багатства, економіки, держав ного та суспільного устрою, культу ри (освіти, науки, мистецтва, ЗМІ, книговидання, фізичного виховання 60 Materials to Glossary for Encyclopedia Science ЕНЦИКЛОПÉДІЯ ПРОБЛÉМНА (Енциклопедія вузькогалузева) про регіон, відображають його коло рит, виразні особливості, напр., ен циклопедичні довідники «Полтав щина», «Чернігівщина»), вузько- галузевими (ті, що висвітлюють лише певну галузь чи практичну ді яльність регіону, напр., «Морская энциклопедия Одессы», «Право славная энциклопедия Харьков щины») та біографічними (ті, що інформують про видатних уроджен ців, відомих людей, які жили та пра цювали в регіоні, напр., «Сумщина в іменах»; див. також: Енциклопедія біографічна). Формально до Е. р. можна віднести енциклопедії, при свячені тій чи іншій країні, кільком країнам, які межують між собою і мають багато спільного, або окре мим частинам світу. Див. також: Ен циклопедія держави, Енциклопедія національна. (англ. subject encyclopedia) – різно вид енциклопедії спеціоналізованої, підвид енциклопедії галузевої або енциклопедії регіональної, у якій зі брано матеріали з конкретної про блеми, що становить предмет роз гляду певної частини окремої галузі (напр., «Козацька старшина Геть манщини», «Енциклопедія аграр них фінансів», «Визначні пам’ятки Києва»). Е. п. містить релевантну термінологічну базу з окресленої проблеми. ЕНЦИКЛОПÉДІЯ РЕГІОНÁЛЬНА (англ. regional encyclopedia) – ен циклопедія, що містить відомості про всі чи окремі визначені аспекти певного регіону. В типологічному плані Е. р. відносять до енцикло педій спеціалізованих. Найчастіше Е. р. присвячують окремому регі ону країни – відповідно до адмі ністративно-територіального (на селений пункт, район, область) або історико-географічного (напр., Слобожанщина, Галичина, Волинь, Кривбас, Донбас) поділу. До Е. р. належать також видання, присвяче ні навчальним закладам, установам, організаціям, товариствам, музеям, спілкам, місцевим пам’яткам при роди тощо (напр., «Енциклопедія НТШ», «Енциклопедія Львівського національного університету імені Івана Франка». За інформативністю Е. р. можуть бути загальними (ті, що охоплюють різноманітні знання ЕНЦИКЛОПÉДІЯ СИСТЕМАТИ́ЧНА (англ. conceptual encyclopedia) – ен циклопедія, статті якої укладено за логіко-тематичним (ієрархічним) принципом. На відміну від енци клопедії алфавітної, у якій статті розташовують за алфавітом, та ен циклопедії хронологічної, де стат ті корелюють із датою, у Е. с. статті групують відповідно до деревовид ної структури (див.: Дерево понять). Енциклопедії з таким способом ор ганізації не містять інверсій у назвах статей, їхнім обов’язковим еле ментом є зміст, подекуди додають різноманітні алфавітні покажчики. Кожний том Е. с. присвячено одній або кільком темам, що в сукупності 61 Матеріали до словника енциклопедичних термінів висвітлюють закладену в концеп ції енциклопедії тематику. Статті в Е. с. можуть бути досить розлогими, сягаючи розміру окремого розділу книги. За ієрархічним принципом створюють переважно енциклопедії тематичні (вживають також як то тожне поняття до Е. с.). Іноді в такий спосіб формують один із томів ал фавітних енциклопедій (напр., 17-й том «Української радянської енци клопедії») або укладають частину томів за ієрархічним принципом, а іншу – за алфавітом (напр., «Ен циклопедія українознавства» поді ляється на тематичну та словникову частини). Статті веб-енциклопедій та енциклопедій електронних за бажанням користувача можна від бирати за тематиками або за алфа вітом, а подекуди – й за хронологі єю. У Е. с. переважно не вміщують статей-персоналій або подають їх у незначній кількості. Серед ін. назв – енциклопедія з деревовидним роз ташуванням статей, ієрархічна ен- циклопедія, логічно-тематична ен циклопедія. галузева, енциклопедія регіональна та енциклопедія персональна. У свою чергу, серед галузевих чи регіональ них енциклопедій виділяють енци клопедію проблемну (вузькогалузе ву) та енциклопедію біографічну. ЕНЦИКЛОПÉДІЯ ТЕМАТИ́ЧНА (англ. thematic encyclopedia) – енци клопедія, яку присвячено одній темі (всім її аспектам). В окремий тип Е. т. виділяють через тип розміщен ня статей за деревовидною схемою, тобто за ієрархічним принципом (див.: Дерево понять). Тому доволі часто поняття «Е. т.» вживають як синонім до «енциклопедія система тична». Тематика, яку висвітлюють Е. т., може охоплювати науковий, лі тературний, мистецький та інші на прями, історичні події чи історичні феномени, вулиці, місцевості, мікро райони населеного пункту, пам’ятки чи групи споріднених пам’яток ар хітектури, історії, монументального мистецтва, географічного об’єкту, культурного, релігійного, спортив ного та іншого закладу, етикет в гро мадських місцях, за столом, здоров’я сім’ї, жінки, чоловіка, дитини, краси жінки, дівчинки тощо. Е. т. можуть містити велику кількість ілюстрацій на відповідну тему. За характером інформації Е. т. найчастіше є енци клопедією галузевою або її підви дом – енциклопедією проблемною (вузькогалузевою), що складається з кількох томів. Причому кожен том присвячено окремій темі, хоча всі вони об’єднані спільною тематикою (напр., енциклопедія «Країни світу і Україна»). СПЕЦІАЛІЗÓВАНА (англ. subject encyclopedia) – енци клопедія, яку присвячено окремій частині знань про людину чи світ. Е. с. протиставляють енциклопедії універсальній. Якщо універсальні ен циклопедії лише згадують або озна йомлюють читача з тією чи іншою тематикою, то Е. с. ґрунтовно пода ють весь спектр відповідної інформа ції про неї. Серед Е. с. виокремлюють три основні різновиди: енциклопедія 62 Materials to Glossary for Encyclopedia Science ЕНЦИКЛОПÉДІЯ УНІВЕРСÁЛЬНА ють переважно енциклопедії, при свячені історії певного регіону (див. також: Енциклопедія регіональна) або історії науки, наукового напря му. Хронологічне розташування ма теріалу дає можливість огляду фак тичної послідовності у становленні державності та формуванні нації, розвитку історичних подій на визна ченій території, окремих наукових ідей, тих чи інших світоглядних кон цепцій, соціальних рухів тощо. Серед прикладів перших праць української енциклопедичної літератури хро нологічного характеру – літописи. Див. також: Енциклопедія алфавіт на та Енциклопедія систематична. (англ. universal encyclopedia) – енци клопедія, що охоплює все коло най важливіших знань про людину і світ. Укладають переважно за алфавітом (винятком можуть бути енциклопе дії дитячі; див. також: Енциклопедія алфавітна), оскільки містить значну кількість непов’язаних між собою ста тей. Інколи за деревовидною схемою створюють окремі томи, присвячені конкретній темі (див.: Дерево понять, Енциклопедія систематична). На відміну від інших видів енциклопедій в Е. у. обов’язковим є подання статей- персоналій. Е. у. переважно є енци клопедіями великими. Вони призна чені як для фахівців різних галузей, так і для широкого загалу, оскільки подають наукову інформацію в до ступній формі. Е. у. може висвітлюва ти всі сфери людської діяльності гло бально або на рівні держави (див.: Ен циклопедія національна), як від най давніших часів і до сьогодення (напр., «Велика українська енциклопедія»), так і за певний проміжок часу (напр., «Енциклопедія Сучасної України», яка висвітлює 20 – поч. 21 ст.). У ти пологічному плані Е. у. протиставля ють енциклопедії спеціалізованій. ЗÁГОЛÓВОК (англ. title) – 1) назва статті в ен циклопедичному виданні. Формаль но є одним словом чи словосполу ченням; висвітлює сутність теми, ідеї, предмет, об’єкт, якому присвя чено статтю. З. прийнято виділяти видозміненим шрифтом (напр., на півжирним) та наголошувати (якщо З. складається з кількох слів, то на голос ставлять на кожному з них, окрім односкладових слів). Багато томні енциклопедії, довідники, слов ники та інші видання, побудовані за абеткою, окрім основної назви, містять заголовкові літери. Основні функції З.: структурування значного за обсягом матеріалу, забезпечення зручності в пошуку вибіркової ін формації. Додатковий З., який пояс нює та доповнює основний, назива ють підзаголовком. Найчастіше його подають у круглих дужках або розмі щують окремим рядком, надрукова ХРОНОЛОГÍЧНА (англ. encyclopedia in chronological order) – енциклопедія, статті в якій укладено за хронологічним прин ципом. Заголовками таких енцикло педій є дати – до них прив’язана вся інформація. У вигляді Е. х. створю 63 Матеріали до словника енциклопедичних термінів живописі, скульптурі, графіці, фото графії тощо. живописі, скульптурі, графіці, фото графії тощо. ним відносно основного З. шрифтом меншого кегля. Якщо З. складається з кількох слів, то часто вживають ін версію задля того, щоб першим було ключове слово для того чи іншого поняття. 2) Сукупність різних видів зобра жень певної особи, події, місцевості в живописі, скульптурі, графіці, фо тографії тощо. Залежно від концеп ції та типу енциклопедичних видань їхні укладачі поєднують текстовий контент із певною кількістю іконо графічного матеріалу. Див.: Ілю страція, Фотографія. 2) Назва тематичного розділу в ен циклопедичних виданнях із логічно- тематичним поданням статей (див.: Енциклопедія систематична, Енци клопедія тематична). ЗМІСТ (англ. image) – наочне зображення, що супроводжує текст статті з метою візуалізації змісту. І. є одним із видів друкованої графіки, частиною ху дожнього оформлення видання з об кладинкою, заставками, кінцівками, орнаментальним декором і шрифто вим набором. До І. належать: малюн ки, фотографії, репродукції, карти, креслення, схеми, діаграми. І. не лише унаочнюють зміст статті, а й доповнюють її виклад, поглиблюють інформативність поданого матеріа лу. Часто І. дає можливість суттєво скоротити енциклопедичну статтю. Її розміщують переважно на тій са мій сторінці, що й текст із посилан ням на неї, або на розвороті з таким текстом. Усі І. оформлюють із підпи сом, узгодженим як із зображенням, так і з текстовим матеріалом. Ступінь насичення видання І. вимірюють у відсотках. Середній ступінь насичен ня І. в енциклопедіях –15 %. (англ. contents) – 1) сутність викладе ного в енциклопедичній статті. Осно вна структурна частина статті. (англ. contents) – 1) сутність викладе ного в енциклопедичній статті. Осно вна структурна частина статті. 2) Перелік складових частин енци клопедії – заголовків всіх або най значніших частин видання. З. розмі щують на початку або в кінці. У З. за значають також початкові сторінки, на яких уміщені відповідні частини. Він виконує довідково-пошукову й інформаційно-пояснювальну функ ції. З. є обов’язковим елементом до відково-енциклопедичних видань з логічно-тематичним розташуванням статей (див.: Енциклопедія система тична, Енциклопедія тематична). В енциклопедіях алфавітних інколи друкують перелік заголовків голо вних (найбільших) статей із зазна ченням авторів і сторінок, карт й ілю страцій, рідше – перелік заголовків усіх статей. 2) Перелік складових частин енци клопедії – заголовків всіх або най значніших частин видання. З. розмі щують на початку або в кінці. У З. за значають також початкові сторінки, на яких уміщені відповідні частини. Він виконує довідково-пошукову й інформаційно-пояснювальну функ ції. З. є обов’язковим елементом до відково-енциклопедичних видань з логічно-тематичним розташуванням статей (див.: Енциклопедія система тична, Енциклопедія тематична). В енциклопедіях алфавітних інколи друкують перелік заголовків голо вних (найбільших) статей із зазна ченням авторів і сторінок, карт й ілю страцій, рідше – перелік заголовків усіх статей. ІНІЦІÁЛ (англ. initial cap) – 1) заголовна літе ра книги, нового розділу, частини або абзацу, зазвичай виділена шрифтом більшого розміру. Інша назва – бук виця. І. може бути набірним (пере важно крупнокегельна літера тієї са мої гарнітури шрифту, якою надруко вано основний текст), рисованим або гравійованим; іноді композиційно декорованим, орнаментованим і під кріпленим сюжетною ілюстрацією. Використання буквиць на початку статей на одну літеру набуло поши рення в енциклопедичній літерату рі. У наступному томі багатотомної енциклопедії, який продовжує цикл статей на ту саму літеру, буквиці не розміщують. КÁРТА (англ. map) – зменшене та генера лізоване загальне зображення тих чи інших територій земної поверх ні. К. відображають розміщення та взаємозв’язки об’єктів і явищ у при йнятій системі умовних знаків. Для них також характерний математич ний закон побудови, тобто вико ристання картографічної проекції, що дає змогу перейти від сферичної поверхні Землі до площини. К. кла сифікують за масштабом (плани, ве лико-, середньо- і дрібномасштабні), територіальним охопленням (напр., зоряного неба, природного об’єкта, населеного пункту) і змістом (за гально-географічні: топографічні й оглядово-топографічні; тематичні: природничі, суспільних явищ, сус пільно-природничі; вузькоспеціалі зовані, напр., навігаційні, кадастрові, технічні, проектні). В енциклопедіях їх використовують як ілюстратив ний матеріал (див.: Ілюстрація), подають переважно до статей про країни, області, райони, з природни чо-географічної, демографічної істо ричної та інших тематик. ІНВÉРСІЯ В НÁЗВАХ СТАТÉЙ (англ. iconography) – 1) наукове ви вчення й опис різних видів зобра жень певної особи, події, місцевості в (англ. inversion in article titles) – змі на звичайного порядку слів у назві (англ. inversion in article titles) – змі на звичайного порядку слів у назві 64 Materials to Glossary for Encyclopedia Science статей (див. також: Заголовок). Здійснюють з метою висунення на перше місце слова, що має в назві суттєвіший зміст, та алфавітного групування статей за однокореневи ми словами, що мають змістову спо рідненість (напр., «Геологів України спілка», «Геологічних наук Інсти тут НАНУ», «Геологія інженерна»). Інверсовані назви полегшують по шук відповідної інформації, рубри ки, статті тощо, сприяють процесу встановлення систематизації знань. Іноді для зручності пошуку подають статті відсильні, напр., за таким зразком: «Медицина космічна –див. Космічна медицина». статей (див. також: Заголовок). Здійснюють з метою висунення на перше місце слова, що має в назві суттєвіший зміст, та алфавітного групування статей за однокореневи ми словами, що мають змістову спо рідненість (напр., «Геологів України спілка», «Геологічних наук Інсти тут НАНУ», «Геологія інженерна»). Інверсовані назви полегшують по шук відповідної інформації, рубри ки, статті тощо, сприяють процесу встановлення систематизації знань. Іноді для зручності пошуку подають статті відсильні, напр., за таким зразком: «Медицина космічна –див. Космічна медицина». статті, зазначаючи тих чи інших осіб, роблячи підписи до іконографії або створюючи перелік бібліографічних джерел. КОМПІЛЯ́ЦІЯ (англ. compilation) – один із методів написання статті, який ґрунтуєть ся на запозиченні матеріалів, думок, досліджень з опублікованих джерел різних авторів. Метод К. вимагає по силання на першоджерело, з якого запозичено інформацію. КРИТÉРІЇ ВІДБÓРУ необхідні статті, а також частково їх опрацювати безпосередньо перед редагуванням, верстанням і друком. К. г. формується із групи автори тетних людей – провідних фахівців окремої галузі. Їхні прізвища зазна чають у вихідних даних видання, завдяки цій інформації читач може складати перше враження про його якість. Редколегією керує редактор головний, або голова, іноді – кіль ка співголовів, до її складу входять відповідальний секретар, або відпо відальний редактор, наукові редак тори. В енциклопедичних виданнях К. г. розробляє концепцію, визначає орієнтовні обсяги матеріалу в зна ках, затверджує перелік гасел (див.: словник), рекомендує авторів для написання статей, здійснює оціню вання матеріалів тощо. (англ. selection requirements) – озна- ки, за якими відбирають, визна чають, оцінюють персоналії, події, явища, об’єкти, інституції тощо. К. в. статей ретельно прописують в енци клопедії концепції. К. в. інформації – представлення фактів, подій, поста тей правдиво, повно, об’єктивно, ви важено, збалансовано, незаангажо вано, без ідеологізованих оцінок, з адекватними характеристиками. Ви значаючи масштаб енциклопедич ного видання (див.: Обсяг енцикло педії), зважають на ступінь удоклад нення матеріалу. Напр., для загаль них енциклопедій К. в. інформації зі спеціальних галузей знань є такі орієнтири, як «загальне–конкрет не», «важливіше–менш важливе». В енциклопедіях явища, осіб мину лого й сучасного намагаються пред ставляти симетрично та відповідно до історичних наслідків, ролі в істо рії, віддаючи перевагу особистостям із позитивним внеском у різні сфери діяльності. Відбираючи персоналії, зважають насамперед на формальні ознаки (особи, які займали найвищі посади у важливих установах; науко ве чи мистецьке звання; членство у професійних спілках; наявність дер жавних нагород), які не варто ігно рувати, проте вони не можуть бути абсолютними нормами. На основі експертних оцінок консультантів наукових, членів редколегії, через громадське обговорення слóвників здійснюють альтернативний прин цип підходу до відбору персоналій, наголошуючи на їхньому внеску в розвиток науки, техніки, мистецтва, КОЛÉГІЯ РЕДАКЦÍЙНА (англ. editorial board) – дорадчий і керівний орган, який розробляє ен циклопедії концепцію та визначає формат енциклопедичного видання, допомагає видавництву або друко ваному органу оцінити та вибрати 2) Прописна перша літера імені, по батькові та іноді прізвища. В енци клопедичній справі вживають під час зазначення автора статті, а також у 65 Матеріали до словника енциклопедичних термінів НÁКЛАД (англ. run) – загальна кількість при мірників друкованого видання, ви готовлених поліграфічним підпри ємством. Зазвичай Н. зазначають у вихідних даних видання; визнача ють видавці чи замовники. Види Н.: пробний (первісний, для вивчення попиту на видання), разовий (на клад одного випуску, номера, тому періодичного видання), річний (су купність накладів видавництва, кра їни за рік; загальна кількість усіх ви пусків періодичного видання за рік), додатковий, загальний (сукупність накладів видань, випущених видав ництвом за певний період), середній (частка від ділення суми накладів усіх видань на їхню кількість). Роз різняють Н. малі (до 15 тис. примір ників), середні (до 100 тис.) та масові (понад 100 тис.; у зв’язку зі значни ми обсягами поліграфічних робіт друкують частинами). Напр., одна з найперших енциклопедій у світі «Ен НАУКÓВО-РЕДАКЦÍЙНА РÁДА (англ. scientific editorial board) – ко легіальний орган, що формує мето дичну групу для розроблення енци клопедії концепції та визначення орі єнтовних обсягів статей для кожної галузі знань з урахуванням досвіду підготовки енциклопедій, особли востей національного розвитку та потреб сучасного суспільства, до сягнень науки, появи нових галузей знань тощо. НАПÓВНЕННЯ СТАТТÍ (англ. content) – кількість і характер інформації у статті; внутрішньоста тейна інформація готується автором чи редактором відповідно до вимог видання (див.: Енциклопедії концеп ція). Н. с. залежить від обсягів ста тей, запланованих у слóвнику. Див. також: Оригінал авторський. КОНСУЛЬТÁНТ НАУКÓВИЙ (англ. scientific adviser) – фахівець, що здійснює наукову експертизу та консультації в підготовці матеріалів для енциклопедичного видання. В енциклопедіях К. н. надає поради з проблемних питань свого фаху укла дачам енциклопедичного видання, які опрацьовують оригінал автор ський. 66 Materials to Glossary for Encyclopedia Science літератури, політики та громадсько го життя, актуальності чи історичній значимості спадщини. Важливим є принцип вибірковості (напр., особи, які народилися, навчалися чи по мерли в Україні за певний період; репресовані; дослідники української тематики, популяризатори україн ської культури в діаспорі). К. в. є та кож відомість, резонанс, визнання, цитованість, думка критиків. Осо бливу увагу приділяють явищам, процесам, поняттям, що характери зують добу сучасності (хоча сучас ний матеріал створює певні трудно щі під час добору). циклопедія, або Тлумачний словник науки, мистецтва й ремесел» мала наклад 4250 примірників, натомість одна з найбільших енциклопедій «Британіка» у 32-томному виданні 1988 року мала наклад 200 тис. при мірників. «Українська радянська ен циклопедія» – 80 тис. (1-ше видання) і 50 тис. (2-ге видання) примірників; «Енциклопедія Сучасної України» – 10 тис. примірників. ОБРÓБКА ІЛЮСТРÁЦІЙ (англ. image editing) – перетворення початкового зображення за допомо гою спеціальних комп’ютерних про грам із метою підвищення якості ілю страцій та забезпечення їх придат ності для оптимального розміщення в тексті статті. О. і. передбачає ра стрування, кольороподіл, градаційну й колірну корекцію тощо. ОБКЛÁДИНКА (англ. cover) – обов’язковий кон структивний елемент (з одного ці лісного аркуша цупкого паперу з твердих чи м’яких палітурних ма теріалів) зовнішньої форми енци 67 Матеріали до словника енциклопедичних термінів клопедичного видання, яким по кривають книжковий блок; чотири зовнішні сторінки, де розміщено основні інформативні дані для чи тача (назву, відомості про серію, ви хідні дані тощо). На О. з рекламною метою можуть виносити суттєву для продажу книги інформацію, здатну привернути увагу покупця, – заго ловки чи марки популярних серій, місце випуску, назву видавництва, основні заголовки, цитати. О. бу вають простими (з однієї деталі), складеними (з двох і більше), ілю строваними, декоративними, шриф товими (мальованими, набірними), гнучкими. Книги в О. розраховані на незначний термін експлуатації при інтенсивному використанні чи на се редній при невеликій інтенсивності користування (довідкові книги з тов щиною блоку переважно до 40 мм). Для підвищення захисту, міцності та покращення зовнішнього вигляду книги О. лакують або ламінують. кількістю авторських аркушів, дру кованих аркушів або обліково-ви давничих аркушів. Змістовно О. е. оцінюють за кількістю гасел. Напр., 17-томна «Українська радянська ен циклопедія» містить 45 000 гасел, 12-томна «Українська радянська ен циклопедія» (2-ге видання) – 50 000 гасел, 30-томна «Большая советская энциклопедия» (3-тє видання) – 95 270 гасел, 32-томна «Британіка» (перевидання 2007 року) – 65 000 гасел. ÓБСЯГ СТАТТÍ (англ. article size) – орієнтовна кіль кість знаків тексту чи сторінок визна ченого шрифту, що залежить від сту пеня удокладнення понять, розділів, явищ, персоналій, наявності бібліо графії та ілюстративних матеріалів. На О. с. орієнтують автора під час замовлення її підготовки. Відповід но до обсягу статті умовно поді ляють на великі (розлогі), середні та малі (короткі). В основі загального орієнтира О. с. – важливість явищ, понять, персоналій у відповідному тематичному блоці. О. с. визначають під час випрацювання енциклопедич них концепцій, розділяючи статті на кілька категорій, кількість яких може варіюватися. Перша категорія (об сяг до 100 тис. знаків) – комплексні та найбільші самостійні науки (істо рія, фізика, біологія і т. п.), основні види мистецтва (література, живопис тощо), найважливіші історичні епо хи, країни; 2‑а (30–50 тис.) – науки, значні наукові та науково-технічні проблеми, жанри й види літератури і мистецтва, столиці; 3‑я (10–30 тис.) – ÓБСЯГ ЕНЦИКЛОПÉДІЇ (англ. size of encyclopedia) – кількіс на характеристика енциклопедич ного видання. Технічно О. е., як і будь-якої іншої книги, вимірюють 68 Materials to Glossary for Encyclopedia Science найбільші одиниці в певній науці чи галузі господарства, найважливіші види корисних копалин, найбільші елементи рельєфу, види військ, най більші міста світу, найбільші етно си, мови і писемності, археологічні культури, корифеї науки, мистецтв; 4‑а (3–10 тис.) – наукові дисципліни, важливі наукові поняття, терміни, ва гомі природні об’єкти, основні види тварин і рослин, визначні історичні події, видатні діячі науки та мисте цтва, основні види спорту, машини, транспортні засоби, великі міста; 5‑а (1–3 тис.) – найбільша за кількістю статей категорія, охоплює наукові поняття, відомих діячів науки й мис тецтва, найважливіші пам’ятники культури і мистецтва, окремі природ ні об’єкти, тварин, рослини, мінера ли, гірські породи, хімічні елементи, прилади, пристрої, підприємства, театри, ВНЗи тощо; 6‑а (до 1 тис.) – спеціальні наукові та науково-техніч ні терміни, терміни в галузі фінансів і бізнесу, правові терміни, деталі ма шин, механізмів, мінерали і дрібні природні об’єкти, наукові і культур но-освітні організації, виконавські колективи, побутова техніка, види тканин, одяг, біографічні довідки; 7‑а (50–100 знаків) – статті з посиланя ми. О. с. про персоналії залежить від внеску діяча, значення особистості, її ролі в історії чи галузі діяльності. 1‑а категорія (10–30 тис. знаків) – найвидатніші історичні постаті, дія чі науки, культури всіх часів і на родів, які отримали загальносвітове визнання; 2‑а (3–10 тис.) – видатні політичні й культурні діячі, науков ці; 3‑я (1–3 тис.) – відомі діячі, які зробили значний внесок у розви ток науки, мистецтва, політики; 4‑а (до 1‑ї тис.) – статті словникового типу, до яких належить значна кіль кість біографій. Розміри статей-пер соналій не повинні перевищувати 5‑ти сторінок; винятки бувають, коли йдеться про найбільші постаті історії, науки, літератури, мистецтва. О. с. про фундаментальні поняття, розді ли науки, галузі діяльності, природ ні, історичні, суспільні, політичні та інші явища – до 5–6‑ти сторінок; про конкретніші, спеціальні терміни, по дії – до 1‑ї. В енциклопедіях від О. с. залежить ступінь удокладнення, від бір матеріалів, наявність другоряд них фактів і творчих характеристик. Співвідношення масштабу оповіді про схожих за рівнем величі діячів, які належать до різних галузей, – обов’язкова умова під час роботи над біографічним розділом. Якщо про видатних діячів збереглося мало ві домостей, то О. с. буде меншим за бажаний. На О. с. біографічної статті впливає також наявність у слóвнику окремих статей про вчення, теорії, відкриття, закони, формули і т. д., створених тим чи іншим діячем. ÓБСЯГ ЕНЦИКЛОПÉДІЇ Збільшення кількості статей зумов лює зменшення їхнього обсягу, що спонукає шукати прийнятну пропо рцію, де вирішальним має бути прак тичний підхід, пов’язаний із майбут нім попитом на видання якомога ширшого кола читачів. Збільшення списку статей загрожує тим, що дуже стисла стаття зі спеціальної галузі знань (як дуже скорочений варіант статті зі спеціальної енциклопедії) не задовольнить спеціаліста, а статисти ка звернень до такої статті з боку не спеціаліста буде занизькою. ток науки, мистецтва, політики; 4‑а (до 1‑ї тис.) – статті словникового типу, до яких належить значна кіль кість біографій. Розміри статей-пер соналій не повинні перевищувати 5‑ти сторінок; винятки бувають, коли йдеться про найбільші постаті історії, науки, літератури, мистецтва. О. с. про фундаментальні поняття, розді ли науки, галузі діяльності, природ ні, історичні, суспільні, політичні та інші явища – до 5–6‑ти сторінок; про конкретніші, спеціальні терміни, по дії – до 1‑ї. В енциклопедіях від О. с. залежить ступінь удокладнення, від бір матеріалів, наявність другоряд них фактів і творчих характеристик. Співвідношення масштабу оповіді про схожих за рівнем величі діячів, які належать до різних галузей, – обов’язкова умова під час роботи над біографічним розділом. Якщо про видатних діячів збереглося мало ві домостей, то О. с. буде меншим за бажаний. На О. с. біографічної статті впливає також наявність у слóвнику окремих статей про вчення, теорії, відкриття, закони, формули і т. д., створених тим чи іншим діячем. Збільшення кількості статей зумов лює зменшення їхнього обсягу, що спонукає шукати прийнятну пропо рцію, де вирішальним має бути прак тичний підхід, пов’язаний із майбут нім попитом на видання якомога ширшого кола читачів. Збільшення списку статей загрожує тим, що дуже стисла стаття зі спеціальної галузі знань (як дуже скорочений варіант статті зі спеціальної енциклопедії) не задовольнить спеціаліста, а статисти ка звернень до такої статті з боку не спеціаліста буде занизькою. ÓБСЯГ ЕНЦИКЛОПÉДІЇ найбільші одиниці в певній науці чи галузі господарства, найважливіші види корисних копалин, найбільші елементи рельєфу, види військ, най більші міста світу, найбільші етно си, мови і писемності, археологічні культури, корифеї науки, мистецтв; 4‑а (3–10 тис.) – наукові дисципліни, важливі наукові поняття, терміни, ва гомі природні об’єкти, основні види тварин і рослин, визначні історичні події, видатні діячі науки та мисте цтва, основні види спорту, машини, транспортні засоби, великі міста; 5‑а (1–3 тис.) – найбільша за кількістю статей категорія, охоплює наукові поняття, відомих діячів науки й мис тецтва, найважливіші пам’ятники культури і мистецтва, окремі природ ні об’єкти, тварин, рослини, мінера ли, гірські породи, хімічні елементи, прилади, пристрої, підприємства, театри, ВНЗи тощо; 6‑а (до 1 тис.) – спеціальні наукові та науково-техніч ні терміни, терміни в галузі фінансів і бізнесу, правові терміни, деталі ма шин, механізмів, мінерали і дрібні природні об’єкти, наукові і культур но-освітні організації, виконавські колективи, побутова техніка, види тканин, одяг, біографічні довідки; 7‑а (50–100 знаків) – статті з посиланя ми. О. с. про персоналії залежить від внеску діяча, значення особистості, її ролі в історії чи галузі діяльності. 1‑а категорія (10–30 тис. знаків) – найвидатніші історичні постаті, дія чі науки, культури всіх часів і на родів, які отримали загальносвітове визнання; 2‑а (3–10 тис.) – видатні політичні й культурні діячі, науков ці; 3‑я (1–3 тис.) – відомі діячі, які зробили значний внесок у розви 69 Матеріали до словника енциклопедичних термінів ÓПИС БІБЛІОГРÁФІЧНИЙ (англ. bibliographic description) – су купність головних уніфікованих за складом і послідовністю ідентифі каційних відомостей (прізвище та ім’я автора, назва твору, місце і рік видання, кількість сторінок чи кон кретна сторінка, на яку зроблено посилання в тексті) про друкований твір, його частини, групу творів або невиданий документ, необхідних для розпізнавання й пошуку, поданий у єдиній системі умовних знаків. О. б. може бути коротким, повним, роз ширеним, міжнародним. Послідов ність і систему умовних знаків, набір обов’язкових елементів О. б. в Україні визначено у ДСТУ ГОСТ. Втім кож на редакція може створювати власні правила для О. б., напр., з метою по легшення сприйняття читачами бі бліографічної інформації. ОНЛÁЙН-ЕНЦИКЛОПÉДІЯ (англ. online encyclopedia) – те саме, що веб-енциклопедія (див. також: Ен циклопедія електронна). (англ. online encyclopedia) – те саме, що веб-енциклопедія (див. також: Ен циклопедія електронна). ОРИГІНÁЛ ÁВТОРСЬКИЙ (англ. author’s original) – 1) автор ський текст статті (машинописний чи рукописний), написаний відповід но до встановлених редакцією вимог, чи окремі його матеріали (напр., фо тографії, ілюстрації) про персона лію, об’єкт, поняття, явище, напрям тощо. О. а. є вихідним джерелом для редагування енциклопедичної статті, він зберігається в архіві редакції. Див. також: Оригінал видавничий. (англ. bibliographic book) – 1) один із видів довідкової літератури, що стано вить собою вечерпний перелік науко вих публікацій та документів із певної галузі знань. О. б. призначені переваж но для вчених, фахівців, керівників. 2) Інформаційне видання у вигляді зв’язних характеристик про друкова ну продукцію, об’єднаних за типом і видами цієї продукції, персоналія ми, тематикою тощо. 2) Заповнена анкета зі збирання первинної інформації для підготов ки статті (див.: Анкетування). ОРИГІНÁЛ ВИДАВНИ́ЧИЙ (англ. publisher’s original) – проана лізований та опрацьований редак тором оригінал авторський, допов нений зовнішнім оформленням та розмічений для поліграфічного ви конання. О. в. – повний проект ви дання, основа для виготовлення ори гіналу-макету видання. Див. також: Оригінал авторський. ОФÓРМЛЕННЯ ВИДÁННЯ (англ. symbols) – розроблені скоро чення для записів описів бібліогра фічних, позначки для заміни сло весного напису під ілюстрації. Для спрощення сприйняття картинки розшифрування П. у. подають у під рисунковому підписі (експлікації) чи в основному тексті. (англ. book design) – подання зміс ту енциклопедії як єдиної цілісної композиції видання за допомогою художніх та поліграфічних засобів. Зовнішнє О. в. – виконання зовніш ніх (палітурки, суперобкладинки, форзаца, футляра), внутрішнє – оформлення внутрішніх (титула, по- чаткової, проміжної та кінцевої сто- рінок, ілюстрацій) елементів видан ня. За характером використаних за собів виділяють художнє (охоплює зовнішнє оформлення, ілюстрації, систему книжкових прикрас) і тех- нічне (типові набірні засоби) оформ- лення. ПЕРЕДМÓВА (англ. foreword) – розміщена пе ред основним текстом допоміжна, службова частина апарату видан ня, де описово пояснено читацьке й цільове призначення, особливості видання (структуру, склад і харак тер матеріалу, концепцію, часові межі, критерії відбору, відомості про авторський колектив тощо). У П. висвітлено значення теми енци клопедії, особливості змісту й фор ми книги, джерела написання тощо. П. готує читача до сприйняття ви дання, формує уявлення про нього. П. в енциклопедіях можуть подава ти різними мовами. 2) Елемент покажчика видання (зміс ту, списку літератури та ін.), який по значає місце у тексті видання (сторін ку, розділ, таблицю та ін.), де наведе но відомості, пояснені докладно. 3) Запис, який переадресовує читача до іншого видання, фрагмента тек сту, ілюстрації, таблиці, формули, додатку, параграфа тощо. ПОСИЛÁННЯ (відсилання) (англ. reference) – 1) cловесна або цифрова вказівка на джерело, що роз’яснює, уточнює або доповнює відомості, подані в основному тексті видання. Назву документа, на який дають П., друкують курсивом або по значають його за допомогою цифри чи символа (*), цей документ часто вміщують у примітці. ОРИГІНÁЛ-МАКЕТ ВИДÁННЯ (англ. book layout) – підписаний до тиражування, роздрукований як зра зок і підготований для перенесення тексту в друковану форму оригінал видавничий, сторінки якого збіга ються зі сторінками майбутньої кни ги. О.-м. в. складають за допомогою комп’ютерної видавничої системи. Переданий у друкарню О.-м. в. уне можливлює здійснення авторських і видавничих правок під час виготов лення друкарської форми. 70 Materials to Glossary for Encyclopedia Science ПОСИЛÁННЯ ПЕРЕХРÉСНЕ (англ. cross reference) – вказівка на взаємозв’язок частин (фрагментів) тексту, які пояснюють і доповнюють одна одну (в прямій та зворотній по слідовності). Застосовують П. п. на заголовки, виноски, закладки, на зви, пронумеровані абзаци тощо, але лише в межах однієї статті. ПÍДПИС (англ. image tittle) – текст під ілю страцією, який пов’язує її з осно вним текстом статті, визначає те матичний зміст. 71 Матеріали до словника енциклопедичних термінів ПОШУКÓВА СИСТÉМА (англ. search engine) – програмно- апаратний комплекс, створений для віднайдення інформації у веб- енциклопедіях, а також електронних базах даних, створених зокрема й для підготовки енциклопедій. ПОСИЛÁНЬ СИСТÉМА (відсилань система) За ступенем і характером змін роз різняють: вичитку – виправлення остаточного (погодженого з авто ром) варіанту тексту або технічних похибок при підготовці перевидань без змін тексту; скорочення – ви правлення з метою зменшити текст до потрібного обсягу; оброблення – ідейно-смислові, фактичні, компо зиційні, логічні, стилістичні виправ лення без докорінного перетворен ня тексту; перероблення – глибокі зміни авторського матеріалу, який слугує лише основою для остаточно го тексту видання. Наступний після П. р. етап роботи над текстом – ко ректура. (англ. system of references) – органі зована певним чином сукупність різ номанітних посилань, покликаних забезпечити раціональне та продук тивне опрацювання читачем тексту з метою виокремлення і засвоєння необхідної інформації. РЕДАГУВÁННЯ ЛІТЕРАТУ́РНЕ (англ. copy editing) – перевірка, оці нювання та вдосконалення разом із автором літературної форми тво ру. При написанні енциклопедій Р. л. застосовують у випадку розподі лу праці, коли редактор науковий аналізує та оцінює зміст і наповне ння тексту, а редактор літератур ний – його правописання та мовну стилістику. Часто Р. л. ототожню ють лише із вдосконаленням мов ного оформлення, хоча цей процес є лише складовою частиною редак торської праці. Основні етапи Р. л.: аналіз тематичної організації ен циклопедичної статті (визначення головної і побічних тем, робота над тематичними відхиленнями); ана ліз композиційної структури, оцін ка її логічності; робота з фактоло гічним матеріалом, перевірка його точності та достовірності; виявлен ня й виправлення порушень логіч ПРÁВКА РЕДÁКТОРСЬКА (англ. text editing) – опрацювання редактором оригіналу авторського з метою покращення його змісту та форми. Р. здійснюють відповідно до прийнятих норм і вимог редагування енциклопедичних статей. Головний об’єкт Р. – оригінал авторський (може містити текстову, ілюстрацій ну, а також аудіо- та відеочастини), другорядні – оригінал видавничий, конструкція та проект видання (пе ревірка дотримання поліграфічних норм, технічних регламентів тощо). Основні етапи Р. енциклопедичних статей: первинне ознайомлення з авторським оригіналом (на цьому етапі здебільшого не вносять прав ки, але роблять помітки на полях або фіксують побіжні зауваження на окремому аркуші паперу); перевірка фактичного матеріалу (оцінка щодо коректності та достовірності поданих відомостей, змістовного наповнення (англ. editor’s alteration) – зміна тексту на різних етапах підготовки до видання з метою вдосконалення його змісту, літературної, редак ційно-технічної та графічної форм. Внесенню П. р. передує редактор ський аналіз: вивчення оригіналу авторського, оцінювання його при датності до друку, за необхідності – допомога автору в процесі вдоско налення змісту статті. Основними завданнями П. р. є усунення поми лок, пропусків, огріхів, виявлених під час редакторського аналізу і не виправлених у ході авторського доо працювання; досягнення виразності та ясності формулювань; перевірка фактичного матеріалу; покращен ня структури, мови і стилю. Осно вні принципи виправлення тексту: доречність, послідовність (узгодже ність), зрозумілість, компактність. 72 Materials to Glossary for Encyclopedia Science цифрової інформації, переміщення по надрукованому тексту, вилучен ня і вставка символів і рядків. Серед функцій текстових процесорів – форматування символів (викорис тання різноманітних шрифтів та накреслень) і абзаців (вирівнюван ня ширини сторінки й автоматич не перенесення слів), оформлення сторінок (автоматична нумерація, введення колонтитулів і зносок), оформлення документа (автома тична побудова заголовків і показ ників), перевірка правопису. Сучас ні Р. к. з. дозволяють додавати до тексту графічні об’єкти (ілюстрації, діаграми, фотографії). статті); власне Р. матеріалу (виправ ляють орфографічні, пунктуаційні та стилістичні помилки). РЕДАГУВÁННЯ ЗАГÁЛЬНЕ (англ. general text editing) – різновид редагування, мета якого полягає в редакторській допомозі автору або авторському колективу досягнути єдності змісту та форми й уникнути неточностей і розбіжностей. Осно вними етапами Р. з. є виправлення логічних (порушення порядку ви кладу, неправильне обґрунтування мотивації дії, наявність неузгодже ності думок, понять тощо) і факто логічних (історичних, географічних, друкарських, цифрових та ін.) по милок. Узгодження матеріалу ре дагованої статті з іншими статтями, представленими в цьому виданні. Зазвичай Р. з. здійснює редактор головний. РЕДАГУВÁННЯ НАУКÓВЕ (англ. scientific editing) – аналіз і оцінювання оригіналу авторсько го з наукового погляду. До Р. н. ен циклопедій залучають провідних фахівців (див.: Редактор науко вий) у тій галузі науки, якій при свячено видання, або з якими вони пов’язані. Якщо у процесі підготов ки енциклопедії галузевої Р. н. здій снює зазвичай один вузькопрофіль ний фахівець (або кілька), то до ви дання енциклопедії загальної чи ен циклопедії національної залучають десятки фахівців у різних галузях науки, економіки, культури, літера тури й мистецтв, спорту тощо. РЕДАГУВÁННЯ ТЕХНÍЧНЕ (англ. layout editing) – перевірка матеріального втілення художньо го та графічного задуму видання, а також дотримання технічних пра вил складання оригінал-макету видання і верстки. Основні етапи Р. т. енциклопедичних статей: унормування технічних параметрів складання макету й верстки, контр оль шрифтової палітри, контроль за відступами, величиною шриф тів, форматуванням тексту тощо. Р. т. здійснює редактор технічний. РЕДАГУВÁННЯ КОМП’Ю́ТЕРНІ ЗÁСОБИ (англ. text editing tools) – комп’ю терні програми, які дають можли вість друкувати, зберігати, редагу вати або читати будь-які текстові документи. Їх поділяють на дві основні групи: текстові редактори (програмне забезпечення, за до помогою якого створюють текстові файли без елементів форматуван ня) і текстові процесори (програмне забезпечення, за допомогою якого форматують текст, вставляють у до кументи графіку та інші не текстові об’єкти тощо). Основні операції ре дагування за допомогою текстових редакторів: введення алфавітно- 73 Матеріали до словника енциклопедичних термінів РЕДАГУВÁННЯ СПЕЦІÁЛЬНЕ них зв’язків у тексті; мовна правка; редакційно-технічне опрацювання (робота з текстом оригінал-макету). У процесі Р. л. енциклопедичного контенту редактор літературний повинен намагатися зберегти як найбільше лексико-морфологічних, синтаксичних особливостей автор ського написання, не втрачаючи при цьому риси енциклопедичного жанру. (англ. expert editing) – аналіз та оцінювання оригіналу авторсько го фахівцем (спеціалістом) в тій чи іншій галузі знань. Р. с. застосо вують у випадку, коли для редагу вання текту недостатньо загальних знань редактора, або воно потре бує залучення різнопланових фа хівців. Приклад Р. с. – підготовка до друку енциклопедичних ста тей про країни світу, в якій беруть участь фахівці з історії, географії, економіки, політики тощо, при цьому кожен редактор акцентує увагу на тому, у чому є компетент ний. Різновиди Р. с.: редагування літературне, редагування науко ве, редагування технічне, редагу вання художнє. РЕДАГУВÁННЯ ПЕРВИ́ННЕ (англ. first editing) – первісне озна йомлення редактора з оригіналом авторським. У процесі Р. п. енци клопедичної статті основну увагу звертають на її змістове наповнення та відповідність зазначеній темати ці. При цьому за необхідності редак тор складає план подальшої спільної роботи разом з автором задля вдо сконалення статті згідно з редакцій ними вимогами. РЕДÁКТОР ХУДÓЖНІЙ (англ. creative designer) – редактор, який здійснює редагування художнє енциклопедичних статей та енци клопедії загалом. Р. х. може також виконувати функції дизайнера ви дання, ілюстратора, оформлювача тощо. РЕДÁКТОР НАУКÓВИЙ (англ. scientific editor) – редактор, який здійснює редагування наукове енциклопедичних статей. (англ. scientific editor) – редактор, який здійснює редагування наукове енциклопедичних статей. РЕДАГУВÁННЯ ХУДÓЖНЄ (англ. creative designing) – створення художнього оформлення видання, аналіз та оцінювання ескізів, маке тів й оригіналів цього оформлення, 74 Materials to Glossary for Encyclopedia Science а також пробних відбитків ілюстра цій та елеметів оформлення. Основні етапи Р. х. енциклопедій: створення проектів художнього та технічного оформлення видання; перегляд ав торських оригіналів ілюстрацій і їх оцінювання з погляду можливості технологічного переформатування для поліграфічного відтворення; пе ревірка використаних шрифтів, сим волів, ілюстрацій, композиції кожної сторінки; підготовка до друку обкла динки. Р. х. здійснює редактор ху дожній. 2) Посадова особа, яка керує редак ційною роботою у видавництві, що за ймається енциклопедичною справою. 2) Посадова особа, яка керує редак ційною роботою у видавництві, що за ймається енциклопедичною справою. 2) Посадова особа, яка керує редак ційною роботою у видавництві, що за ймається енциклопедичною справою. РЕДÁКТОР (англ. editor) – фахівець, який опра цьовує, виправляє та готує до опу блікування оригінал авторський. Основні функціональні обов’язки Р. енциклопедичних видань: ґрунтовне ознайомлення з авторським оригі налом і підготовка аргументованого редакторського висновку; формулю вання письмових вимог до автора та співпраця з ним; редагування за твердженого редактором головним авторського оригіналу; узгодження остаточного тексту статті з автором; перевірка та вичитка верстки (з ілю стративним матеріалом). Див. та кож: Редактор головний, Редактор літературний, Редактор науковий, Редактор технічний, Редактор ху дожній. РЕДÁКТОР ЛІТЕРАТУ́РНИЙ (англ. copy editor) – редактор, який здійснює редагування літератур не енциклопедичних статей. Іноді Р. л. залучають і до остаточної ви читки тексту енциклопедії. РЕДÁТОР ТЕХНÍЧНИЙ (англ. layout editor) – редактор, який здійснює редагування техніч не енциклопедичних статей та ен циклопедії загалом. РЕДÁКЦІЯ (англ. editorial staff; redaction) – 1) колектив, який готує будь-яке видан ня до друку. (англ. editorial staff; redaction) – 1) колектив, який готує будь-яке видан ня до друку. 2) Структурна одиниця видавниц тва, виокремлена для роботи в пев ній галузі чи з певними видами пу блікацій (див.: Редакція літератур но-контрольна, Редакція науково- галузева). 3) Опрацювання редактором оригі налу авторського (те саме, що й ре дагування). 4) Варіант того самого літературного чи музичного твору або його окремої частини. 5) Приміщення, у якому працюють редактори. РЕДÁКТОР ГОЛОВНИ́Й (англ. adition and publishing activity) – сукупність послідовних дій праців ників видавництва, спрямованих на підготовку й випуск будь-якого енциклопедичного видання. Основ ні етапи Р.-в. п. у створенні енцикло (англ. chief editor) – 1) фахівець, який очолює роботу редакції або ко легії редакційної енциклопедичного видання. 75 Матеріали до словника енциклопедичних термінів педій: підготовчий (складання і за твердження слóвника алфавітного редактором головним або головною колегією редакційною із залученням редакторів наукових, пошук авто рів для написання статей, збір ори гіналів авторських та ілюстрацій), редакційний (редакторське опрацю вання отриманих оригіналів, погод ження вичитки з авторами), вироб ничий (перетворення оригінал-маке ту видання у друковану продукцію), маркетинговий (реклама та інші за ходи, пов’язані з продажем видання). рюють у процесі підготовки до ви дання енциклопедій універсальних із метою уніфікації форми подання текстів, опрацьованих у галузевих редакціях (див.: Редакція науково- галузева), контролю за дотриман ням норм сучасної української літе ратурної мови та вимог енциклопе дичного стилю. РЕДÁКЦІЯ НАУКÓВО-ГАЛУЗÉВА (англ. scientific editorial staff) – структурна одиниця видавництва. Р. н.-г. створюють у процесі підго товки до видання енциклопедій уні версальних з метою оптимізувати роботу над статтями, що нале жать до тієї самої або споріднених галузей людської діяльності (на уки, освіти, культури, спорту тощо). Основні етапи діяльності Р. н.-г.: пошук авторів з урахуванням ре комендацій редакторів наукових; замовлення тематичних словників, їх рецензування та редагування (вилучення реєстрових слів поді бної тематики, які дублюють одне одного або висвітлюють однакову проблему з різних позицій); редагу вання, рецензування, загальне об говорення та внутрішньотематичне узгодження отриманих від авторів окремих статей або їхніх циклів; кореляція текстів з ілюстраціями та картами. Після опрацювання у Р. н.-г. матеріал передають до ви пускової редакції. РЕЦЕНЗÉНТ енциклопедичних статей оціню ють насамперед повноту висвітлен ня теми з дотриманням загальних вимог енциклопедичного стилю та редакційних стандартів; авторську неупередженість в оцінках і висно вках; використання системи логіч них обґрунтувань, доказів, посилань; застосування наукового апарату ви дання. (англ. reviewer) – особа, яка здійснює рецензування енциклопедичного ви дання (зовнішнє рецензування) чи окремої статті до неї на етапі її за твердження (внутрішнє рецензуван ня). У процесі підготовки енциклопе дії Р. статей може виступати видав ничий редактор, редактор науковий і редактор головний, а також, за не обхідності, позаштатний висококва ліфікований фахівець у певній галузі знань. РОЗРЯ́ДКА (англ. letter spacing) – маркування слова чи групи слів у тексті шляхом збільшення проміжків між буквами та словами. В енциклопедіях Р. вико ристовують із метою виокремлення підстатті у складі загальної статті або окремих елементів складного по няття чи терміна. 2) Документ, у якому зазначено оцінку енциклопедичного видання, підготовленого до друку, та реко мендаційний висновок. Метою Р. є встановлення ступеня готовності видання як завершеної праці. Ви різняють також внутрішню Р., яка стосується окремої енциклопедичної статті. РИСУ́НОК (англ. figure) – графічне зображення будь-якого об’єкта на сторінках ен циклопедії. Посилання на Р. у тексті та в підписі під ілюстрацією скоро чено позначають словом «Рис.», ін коли його курсуючи. РЕЦÉНЗІЯ (англ. review) – 1) публікація, що міс тить розгорнутий критичний ана ліз надрукованого видання (опри люднюють у фахових виданнях). Є жанром наукової статті. РЕЄСТРÓВЕ СЛÓВО (англ. catchword) – те саме, що заго ловок (див. також: Гасло). (англ. catchword) – те саме, що заго ловок (див. також: Гасло). (англ. copy editorial staff) – структур на одиниця видавництва, яку ство 76 Materials to Glossary for Encyclopedia Science РЕЦЕНЗУВÁННЯ (англ. article rubrication) – поділ тек сту на пов’язані сурядним чи під рядним зв’язком складові частини (рубрики). Р. с. в енциклопедіях здій снюють за допомогою заголовків, розрядки, нумерації, шрифтів тощо. При цьому поділ тексту має бути по слідовним і сумірним, рубрики ма ють бути співвіднесені як частини одного цілого. Рубрики, у свою чер гу, поділяють на абзаци. (англ. peer-review) – аналіз та оці нювання рецензентом оригіналь ного енциклопедичного видання або його частини (окремої статті чи їх низки). Застосування Р. в енцикло педичній справі допомагає підібрати відповідний матеріал для опубліку вання та сприяє загальному підви щенню якості видання. У процесі Р. 77 Матеріали до словника енциклопедичних термінів СЛÓВНИК (англ. thesaurus) – повний реєстр гасел, на основі яких формується корпус енциклопедії. Гасла подають здебільшого з короткою анотацією та із зазначенням обсягу майбутніх статей. З погляду добору реєстру гасел С. утворює зміст енциклопе дичного видання, його ідейно-на укову спрямованість. С. забезпечує також створення системи подання СЛÓВНИК АЛФАВÍТНИЙ (англ. thesaurus in alphabetical order) – словник, укладений за алфавітним принципом. У процесі підготовки енциклопедичних видань спочат ку укладають С. а. окремих великих розділів науки (на основі словників тематичних), а потім загальний С. а. видання. СКОРÓЧЕННЯ статей у масштабі всієї енциклопедії, виокремлення циклів статей, випра цювання посилань системи. З під готовкою С. пов’язане планування структури статті, структури сторін ки, розташування бібліографічного, ілюстративного й картографічного матеріалу. Під час створення енци клопедичного видання його С. може поповнюватися новими одиницями. (англ. abbreviation) – подання слів і словосполучень у скороченому ви гляді. Застосовують задля оптимі зації обсягу видання (кількості сто рінок). Види С.: графічні, ініціальні абревіатури, складноскорочені сло ва, змішані. Ці види, у свою чергу, поділяють на загальноприйняті, спе ціальні (характерні для окремих ви дань або застосовувані у спеціальних текстах, напр., у бібліографічному описі, і зрозумілі без розшифруван ня) та індивідуальні (запроваджені лише в тексті певного видання і по требують розшифрування, яке по дають у списку С.). Вимоги до С.: прийнятність у тексті (що ширшому колу читачів його адресовано, то об меженіша кількість С.), зрозумілість для читача без розшифрування або з розшифруванням у списку С. (за умо ви, що таке розшифрування легко запам’ятовується), відсутність омо- німії, послідовність і узгодженість у межах усього видання, а не лише од нієї статті. С. слів застосовують у бі бліографії (крім прізвища автора та основного заголовка). СЛÓВНИК ТЕМАТИ́ЧНИЙ (англ. thematic thesaurus) – словник, укладений за тематичним принци пом. Підготовка енциклопедичного видання зазвичай розпочинається з укладання С. т. для кожної галузі знань, які відображають підпорядку вання термінології (від загальних до спеціальних термінів). На основі них формують словники алфавітні. СТАТТЯ́ ÁВТОРСЬКА (англ. author’s article) – стаття, на писана автором, який є фахівцем у тій галузі знань, до якої належить ре єстрове слово енциклопедії (див. та кож: Гасло). С. а. готується на основі авторитетних достовірних джерел, завдяки яким виклад інформації на буває об’єктивності, точності, всебіч ності та неупередженості. С. а. пода ють зазвичай із зазначенням автора. СТАТТЯ́ ВІДСИ́ЛЬНА (англ. related article) – стаття, що скеровує читача до іншого реєстро вого слова енциклопедії. Разом із по кликанням на іншу статтю може міс тити елементарне тлумачення. СТАТТЯ́ БІОГРАФÍЧНА (англ. biographical article) – стат тя, що містить визначальні відомос ті про особу (науковця, культурного діяча, митця, спортсмена, політика тощо). Складається з біографічної довідки й представлення фактичних досягнень особи, часто містить ко ротку характеристику творчого чи наукового доробку. (англ. biographical article) – стат тя, що містить визначальні відомос ті про особу (науковця, культурного діяча, митця, спортсмена, політика тощо). Складається з біографічної довідки й представлення фактичних досягнень особи, часто містить ко ротку характеристику творчого чи наукового доробку. СТАТТЯ́ (англ. article) – основна структурна одиниця енциклопедії, що тлумачить реєстрове слово (див. також: Гас ло). С. подає найвагоміші відомості про реалію (явище, процес, об’єкт, особу тощо), презентованою реє стровим словом. Характеризується об’єктивністю (науковістю), точніс тю, авторською неупередженістю, всебічним викладом, простотою по дання матеріалу. Структура і обсяг С. визначається редакторською енци клопедії концепцією та особливостя ми власне реалії. Сукупність С. утво рює корпус будь-якої енциклопедії. Становить окремий жанр наукової чи науково-популярної довідкової літератури. Тематично енциклопе дичні С. поділяють на блоки: 1) при родні утворення, відомості з геології та фізичної географії, рослинний і тваринний світ, демографія, питан ня екології; 2) геополітичні регіони, держави, землі, адміністративно-те риторіальні одиниці; 3) нації, етно си, етнічні групи, національні мен шини, їхні культури і мови; 4) націо нальні та міжнародні установи – по літико-адміністративні, економічні, правові, громадські, освітні, наукові, мистецькі тощо; 5) галузі діяльнос ті – економіка, політика, наука, ме дицина, виховання, мистецтво та ін.; 6) історичні утворення (цивілізації, держави), історичні періоди (епохи), історичні події; 7) сфери свідомості: міфи, релігії, ідеології; 8) пізнаваль СПІВÁВТОРСТВО (англ. coauthoring) – співпраця двох чи більше осіб у написанні спільної статті чи всієї енциклопедії, відно сини між якими визначено певною угодою, напр., видавничим догово ром; С. визначає належність двом чи кільком особам авторського права на статтю чи енциклопедичне видання. 78 Materials to Glossary for Encyclopedia Science Не є С. надання будь-якої технічної допомоги, а також участі у створенні ідеї чи концепції твору. Не є С. надання будь-якої технічної допомоги, а також участі у створенні ідеї чи концепції твору. Не є С. надання будь-якої технічної допомоги, а також участі у створенні ідеї чи концепції твору. на діяльність: науки природничі й гуманітарні, їхні найважливіші роз діли, філософія та її основні розділи; 9) найважливіші поняття – повсяк денні, наукові, філософські; 10) ви датні особистості. СТРУКТУ́РА СТАТТÍ ма фахівцями, кожен із яких ви світлює зміст реєстрового слова з того аспекту, на якому професійно знається. Зазвичай С. к. представ ляє складне (багатоаспектне) явище, яке в різних галузях знань має влас не значення. Напр., реєстрове сло во «людина» містить біологічний, психологічний, філософський, соці альний та ін. аспекти, а отже, стаття про людину вимагає кількох авто рів. У написанні статті про державу можуть бути окреслені питання гео графії, історії, правознавства, соціо логії, демографії, етнографії, куль турології, мовознавства тощо. С. к. може бути і стаття біографічна, коли видатна особа досягла успіхів у різних сферах життя, не пов’язаних між собою. С. к. подають із зазна ченням авторів кожного розділу чи галузі, іноді в дужках вказуючи, яко го саме. (англ. article design) – організація текстового й графічного матеріалу статті, завдяки чому наповнення статті набуває ознак логічної по слідовності й цілісності. У С. с. тра диційно виділяють такі одиниці (складові частини): заголовок (назва статті), дефініція (тлумачення реє стрового слова), текст (фактичний матеріал), бібліографія, ілюстрація, автор. Проте С. с. може мати іншу організацію залежно від мети, кон цепції енциклопедичного видання, редакційної політики тощо. СТАТТЯ́ ОГЛЯДÓВА (англ. review article) – стаття, що подає огляд основних проблем, за гальних рис, тенденцій, властивих тому поняттю, якому присвячене реєстрове слово енциклопедичного видання. С. о. є одним із піджанрів енциклопедичної статті. СТРУКТУ́РА СТОРÍНКИ (англ. page design) – організація сто рінкового матеріалу, полоси й по лів. Полоса складається з тексту, ілюстрацій, колонцифри, колонти тулу, сигнатури й ін. елементів. Роз міри полоси залежать від формату видання і визначаються одиницями типографічної системи вимірювань. Здебільшого С. с. має вигляд прямо кутника, оточеного полями різних розмірів. Полосу, з якої починається книга чи її розділ, називають почат ковою (може бути зі спуском – не задрукованим проміжком у верхній частині; спуск іноді заповнюють за головком або орнаментально-деко ративними елементами); полосу, яка завершує видання чи його частину, – кінцевою; полосу, яка містить тільки ілюстративний матеріал, – ілюстра ційною. Поля книги – незадрукова ні ділянки сторінки навколо полос. Розрізняють корінцеве (внутрішнє), верхнє, зовнішнє і нижнє поля. Роз СТАТТЯ́ КÓМПЛЕКСНА (англ. overview article) – стаття в енциклопедії, підготовлена кілько 79 Матеріали до словника енциклопедичних термінів СТАТТЯ́ РЕДАКЦÍЙНА (англ. editorial article) – стаття, на писана редакторами видання, а не фахівцями з тієї галузі знань, до якої належить реєстрове слово енцикло педичного видання. С. р. подають без авторства або з ремаркою, що вказує на таку особливість статті. 80 Materials to Glossary for Encyclopedia Science міри поля залежать від типу видан ня, площі сторінки і полоси. лишаються пустими і доповнюються необхідними даними). За розміром і характером верстання – на клоч кові (займають лише частину поло си), поперечні, поздовжні, двійчасті, дво- і багатополосні. За характером оформлення лінійками – закриті (глухі), півзакриті, відкриті. Розгор нена Т. – Т., розміщена на обох сто рінках розвороту. ТОМ (англ. volume) – 1) самостійно оформ лена друкована структурна одини ця енциклопедичного видання. Має свої титул, назву та пагінацію. 2) Збірка декількох окремих випусків багатотомного або продовжуваного видання. Об’єднують випуски в один Т. з метою забезпечити єдність зміс ту й тематики. Кожен випуск одного Т. має свою внутрішню нумерацію, яку супроводжує наскрізна нумера ція всіх випусків. ТÉРМІН (англ. term) – слово або словосполу чення, що позначає чітко окреслене поняття певної галузі науки, техніки, суспільного життя тощо. Основними ознаками Т. є системність, наявність дефініції, тенденція до однозначнос ті у межах термінології певної галузі, стилістична нейтральність, точність семантики, інформативність. ТАБЛИ́ЦЯ (англ. table) – особлива форма пе редавання змісту, що відрізняється від суцільного тексту організацією слів і чисел у колонки (графи) й го ризонтальні рядки. Між заголовка ми колонки, рядка та їхнім спільним елементом встановлюють безсловес ний, графічний змістовий зв’язок, зрозумілий читачам без переведен ня у словесну форму. Осн. елемен тами Т. є нумераційний і тематич ний заголовки, головка (заголовок і підзаголовок колонки), прографка, боковик (заголовок рядків), хвіст, колонки і рядки. Осн. вимоги до Т.: логічність і економність побудови, зручність читання. За призначен ням їх класифікують на аналітичні (зокрема статистичні; застосовують для висвітлення статистичного ма теріалу), проформи (оформляють тільки головку; є зразком для звіт них даних), пустографки (графи за СХÉМА СТАТТÍ (англ. article format) – послідовність викладу матеріалу, що забезпечує логічний взаємозв’язок та вмотиво вану співмірність усіх частин стат ті. Відображає змістове наповнення статті. Існують спільні для всіх енци клопедичних видань С. с., напр., про науковий напрям, наукове поняття, персоналію. Крім цього, кожна ре дакція залежно від тематики видан ня напрацьовує власні С. с., напр., про річку, рослину, архітектурний об’єкт тощо. ЦИТУВÁННЯ (англ. citation) – дослівне викорис тання фрагментів (уривків) будь- якого тексту – цитат. Застосовують на підтвердження думки шляхом по силання на цитоване джерело. Осн. вимогами до цитат є доцільність їх використання і точність відтворен ня. Цитату беруть у лапки (рідше виділяють видозміною шрифту) та забезпечують посилання на джере ло інформації. Іноді при Ц. пропус кають слова (пропуск позначають трьома крапками), якщо це не спо творює зміст оригіналу. В енцикло педичних виданнях має мінімальне застосування. 2) Пронумерований том, виданий як доповнення до попередньо ви значеної кількості томів (напр., 11-й додатковий том до словникової час тини «Енциклопедії українознав ства»). Декілька Т. д. до одного ви дання можуть мати свою нумерацію. 2) Пронумерований том, виданий як доповнення до попередньо ви значеної кількості томів (напр., 11-й додатковий том до словникової час тини «Енциклопедії українознав ства»). Декілька Т. д. до одного ви дання можуть мати свою нумерацію. 2) Пронумерований том, виданий як доповнення до попередньо ви значеної кількості томів (напр., 11-й додатковий том до словникової час тини «Енциклопедії українознав ства»). Декілька Т. д. до одного ви дання можуть мати свою нумерацію. ТРАНСКРИ́ПЦІЯ (англ. transcription) – спеціальна сис тема письма, яку використовують для точного відтворення слів і текстів ін шої мови. Розрізняють три типи Т. Фонетична Т. – спосіб передавання на письмі усної мови з усіма її звуковими особливостями (кожна літера позна чає на письмі той самий звук, а кожен звук передається завжди тією самою літерою). Фонематична Т. передає лише фонемний склад слів і текстів. Практична Т. – запис за допомогою засобів національного алфавіту ін шомовних власних назв, термінів та ін. неперекладних слів. Практичну Т. використовують у спеціальній літера турі, географічних картах тощо. ТОМ ДОДАТКÓВИЙ (англ. additional volume) – 1) ненуме рований том багатотомного видан ня, що містить довідковий апарат 81 Матеріали до словника енциклопедичних термінів видання. Його випускають поза ого лошеною кількістю томів. видання. Його випускають поза ого лошеною кількістю томів. видання. Його випускають поза ого лошеною кількістю томів. ЕНЦИКЛОПЕДИЧНА ХРОНІКА Тетяна Березюк, Оксана Давиденко Інститут енциклопедичних досліджень НАН України, Київ, Україна ФОТОГРÁФІЯ (англ. photo) – зображення будь- чого, отримане внаслідок фотогра фування. В енциклопедичній справі Ф. використовують як ілюстрації до статей, іноді – як видавничий ори гінал. Ф. в енциклопедіях бувають різних типів: монохромні (чорно-бі лі) і кольорові, репортажні (фіксують явища, особи, предмети без втручан ня фотографа) і постановочні (зре жисовані фотографом) тощо. 82 Тернопедія: тернопільська енциклопедія На межі 2017–2018 років з’явилася онлайн-енциклопедія «Тернопільська енциклопедія» (скорочено: «Тернопедія»)1, основною метою якої є розповсю дження відомостей про Тернопільщину та видатних людей цього краю. Онлайн-енциклопедію технічно реалізовано на базі MediaWiki, тому візуаль но вона нагадує «Вікіпедію». Її заснував краєзнавець Микола Василечко. На презентації цього енциклопедичного веб-проекту, що відбулася 2 лютого 2018 року в бібліотеці Тернопільського державного медичного університету імені І. Горбачевського, він зауважив, що «Тернопедія» відрізняється від «Вікіпедії», у якій теж міститься чимало інформації про Тернопільщину, пе редусім за стилем і наповненістю. У «Тернопедії» мають змогу дописувати і редагувати люди під власними іменами (у «Вікіпедії», як відомо, авторство не передбачено); тут також дозволено публікувати оригінальні дослідження, що загалом не властиво енциклопедичним виданням. Нині, станом на кінець 2018 року, енциклопедія містить трохи більше 3,7 тисяч статей. За задумом же автора, її обсяг складатимуть 50 тисяч статей. 1 Режим доступу: http://ternopedia.te.ua. УКРАЇНСЬКА ЕНЦИКЛОПЕДИЧНА СПРАВА В ПОДІЯХ УДК 82-94 2 Режим доступу: http://subcultures.wiki. 3 Луцюк А. Енциклопедія світових субкультур. Навіщо потрібна чергова енцикло педія? // Інформаційне агентство «Центр новин»: електронне видання. 2018. Режим доступу: http://centernews.com.ua/?p=126269. 4 Сокіл В. Почин української фольклористичної енциклопедії // Народознавчі зо шити. 2014. № 3 (117). С. 590–619. Енциклопедія світових субкультур У травні 2018 року на базі MediaWiki було створено онлайнову «Енци 1 Режим доступу: http://ternopedia.te.ua. 1 Режим доступу: http://ternopedia.te.ua. Енциклопедичний вісник України. 2018. Число 10 © Інститут енциклопедичних досліджень НАН України Тетяна Березюк, Оксана Давиденко клопедію світових субкультур»2. Веб-ресурс, що, між іншим, має свідоцтво про державну реєстрацію як засіб масової інформації, підготовлено трьома мова ми – українською, російською, англійською. Загальний опис енциклопедії за пропоновано на її сайті: «Це унікальний науково-популярний проект, який створено в партнерстві з “Великою українською енциклопедією”. Його мета – багатовекторний аналіз світових субкультур. Кожне явище, організація і осо бистість будуть розглянуті з позиції послідовників, учених і засобів масової інформації». На думку професора Національного педагогічного університету імені М. Дра гоманова Ірини Богачевської, члена редакторської групи цього видання, «Енциклопедія світових субкультур» із погляду особливостей подання мате ріалу є цілком інноваційною. Стереотипні енциклопедії не в змозі охопити всю палітру інформації. Наприклад, думки послідовників різних течій і суб культур зазвичай подаються в інтерпретованому варіанті. Традиційно думки академічної науки й засобів масової інформації також знаходяться в різних інформаційних полях. Тут же ми бачимо системний комплексний аналіз, що значно спрощує завдання для дослідників3. 5 Вовк М. Київська академічна фольклористична школа: здобутки і перспективи // Естетика і етика педагогічної дії: Зб. наук. праць. Київ; Полтава, 2011. С. 157. 6 Рішення Івано-Франківської обласної ради № 816-20/2018 від 02.03.2018 р. Про затвердження обласної комплексної програми «Відзначення в області 100-річ чя створення Західноукраїнської Народної Республіки і 100-річчя Акту Злуки Укра їнської Народної Республіки та Західноукраїнської Народної Республіки» на 2018– 2019 роки. Українська фольклористична енциклопедія 21 вересня 2018 року в рамках національного книжкового ярмарку «Форум видавців у Львові» (Book Forum Lviv) в Музеї етнографії та худож нього промислу відбулася презентація «Української фольклористичної ен циклопедії», яку підготував колектив фахівців Інституту народознавства НАН України. «Українська фольклористична енциклопедія» – перша енци клопедія про український фольклор. Вона підсумовує досягнення академіч ної народознавчої науки за 200 років, подаючи інформацію про фольклорис тів, збірники, часописи, наукові установи, жанри фольклору та про самих персонажів, створених народною традицією. Аналогічні фундаментальні праці мають країни-сусіди, наприклад, Польща («Słownik folklore polskiego»), Білорусь (енциклопедія «Беларускі фальклор»). Донині українська фолькло ристика в енциклопедичному форматі частково була представлена в «Українській музичній енциклопедії»; відтепер – у власній галузевій енци клопедії. Керівник цього проекту, науковий редактор і водночас упорядник – док тор філологічних наук, професор Василь Сокіл (завідувач відділу фолькло ристики Інституту народознавства НАН України). В одній зі своїх публікацій4 він зауважував, що ідея створити таку академічну енциклопедію виникла ще 84 Енциклопедична хроніка в 1920-х роках – її ініціювала етнографічна комісія Української академії наук, розпочавши роботу з виписування карток для «Словника українського фольк лору» з метою скласти компендіум – повну базу даних українського фольклору. Проте словник тоді так і не уклали. У 2011 році Мирослава Вовк наголошувала: «Відсутність наукового зводу термінів, понять, які стосуються сфери фольклору й фольклористики, гальмує теоретичні опрацювання багатьох проблем, негативно позначається на під готовці кадрів, видавничій практиці тощо. Об’єктивно назріла потреба ство рення фольклористичного енциклопедичного словника з висвітленням пи тань методології наукової галузі, теорії фольклору, історії фольклористики»5. Відтак маємо сподівання, що поява «Української фольклористичної енцикло педії» сприятиме поступу вітчизняної фольклористичної науки. Коли це число «Енциклопедичного вісника України» було підготовлено до друку, в «Літературній Україні» (№45–46, 6 грудня 2018 р.) натрапили на інтерв’ю з упорядником і науковим редактором «Української фольклористич ної енциклопедії» у 2-х томах Миколою Дмитренком. Її підготовку здійснюють науковці Інституту мистецтвознавства, фольклористики та етнографії імені М. Т. Рильського НАН України. Перший том цієї енциклопедії (А–Л) щойно побачив світ. Отже, відтепер маємо два однакові видання не лише тематично, а й за назвою. 7 Режим доступу: http://knyga1.wixsite.com. Енциклопедія голодомору 12 листопада 2018 року в Національному музеї «Меморіал жертв Голодо мору» в рамках відзначення 85-х роковин Голодомору-геноциду відбулася презентація «Енциклопедії Голодомору 1932–1933 років в Україні», уклада чем якої є доктор історичних наук, професор Василь Марочко. Метою видання укладач назвав необхідність інформувати прийдешні покоління про Голодомор. Енциклопедію видано за сприяння Міністерства культури України. У передмові до видання, що містить понад тисячу тематичних статей, за значено: «Енциклопедія ввібрала основні події і явища, узагальнила історіо графічні та меморіальні практики. Її завдання – інформувати прийдешні по коління про Голодомор в Україні, не переконувати опонентів, відвертих нігі лістів, а правдиво подавати історичні факти, нагадувати про вічні цінності християнського милосердя. Видання не вичерпує тему людського горя, завда ного українському народу, змісту і трагедійності самого історичного явища. Автор сподівається на появу регіональних енциклопедичних видань, які сто сувалися б кожного району і села, що опинилися в 1932–1933 рр. в епіцентрі Голодомору». Енциклопедія ЗУНР У листопаді 2018 року виповнилося 100 років від дня проголошення Західно-Української Народної Республіки, а в січні 2019 р. – 100-річчя від часу підписання Акту Злуки ЗУНР та УНР. Відповідно до комплексної про грами Івано-Франківської області «Відзначення в області 100-річчя створен ня Західноукраїнської Народної Республіки і 100-річчя Акту Злуки Україн ської Народної Республіки та Західноукраїнської Народної Республіки»6, ухвалено опублікувати тритомник «Західноукраїнська Народна Республіка 1918–1923. Енциклопедія». Це видання готують науковці Прикарпатського національного універси тету імені В. Стефаника. Перший том енциклопедії ЗУНР презентував 31 жовтня 2018 року в Івано-Франківську на Міжнародній науковій конферен ції «Західно-Українська Народна Республіка: революція, державність, со борність. До 100-річчя утворення ЗУНР» відповідальний редактор – профе сор Іван Кугутяк. Ректор Прикарпатського національного університету імені 85 Тетяна Березюк, Оксана Давиденко В. Стефаника Ігор Цепенда зауважив, що є сподівання, що інші томи будуть завершені невдовзі, і тритомна праця стане повноцінним фундаментом для подальших активних досліджень ЗУНР. Українська музична енциклопедія У 2018 році вийшов друком 5-й том «Української музичної енциклопедії», що висвітлює гасла на літеру «П». Нагадаємо, що «Українська музична енци клопедія» — це галузева енциклопедія, яку від 2006 року готує й друкує Інститут мистецтвознавства, фольклористики та етнології імені М. Т. Риль ського НАН України. Доречно додати, що цього ж року опубліковано книгу «Музичні адреси Києва» – інформаційно-довідкове видання, присвячене київським музикан там, співакам, акторам, які здійснили внесок у розвиток як української, так і світової музичної культури. Видання подає біографічний матеріал у вигляді путівника по Києву; воно прикметне й тим, що містить численні архівні мате ріали про персоналії, відгуки про них відомих діячів культури тощо. Автори – Вiра Горбатюк і Алла Вишнева – на веб-сторінці7 цього видання зазначають, що задум створити таку працю виник у них ще 1982 року, коли Київ святкував 1500-річний ювілей, відтоді й тривала робота над цим проектом. Велика українська енциклопедія 13 грудня 2018 року відбулася презентація онлайн-версії універсальної «Великої української енциклопедії», яку за участі провідних науковців України готує державна наукова установа «Енциклопедичне видавництво». За задумом творців, електронна ВУЕ відрізняється від того, що пропонується 86 Енциклопедична хроніка на сторінках друкованої ВУЕ. Електронна ВУЕ ― енциклопедичний веб- ресурс із багатим ілюстративним (світлини, карти, схеми, графіки, діаграми, креслення, малюнки, рисунки), аудіо- та відеоматеріалом, що інтегрується з текстом. Веб-сторінка е-ВУЕ: https://vue.gov.ua. на сторінках друкованої ВУЕ. Електронна ВУЕ ― енциклопедичний веб- ресурс із багатим ілюстративним (світлини, карти, схеми, графіки, діаграми, креслення, малюнки, рисунки), аудіо- та відеоматеріалом, що інтегрується з текстом. Веб-сторінка е-ВУЕ: https://vue.gov.ua. Українська Вікіпедія Станом на кінець листопада 2018 року українська «Вікіпедія» містить по над 870 тисяч статей, перебуваючи за цим показником на 16 місці серед «Вікіпедій» іншими мовами світу, на 11 місці серед європейських та на 3-му – серед слов’янських. Для порівняння: в кінці листопада 2017 року було опублі ковано 750 тисяч статей, отже, енциклопедичний онлайн-ресурс за рік збага тився на 120 тисяч статей. Звісно, за обсягом контенту українська «Вікіпедія» перевершує будь-яке багатотомне видання, однак якість статей у ній часто стає об’єктом критики дослідників. Загалом «Вікіпедії» будь-якою мовою властива спільна проблема, яка поки що немає методів усунення (на відміну від низки інших, наприклад, таких, як інформаційний вандалізм, з яким на вчилися боротися) – статті, що мають різні гасла, однак порушують спільні питання, описують те саме явище, реалію, предмет, процес тощо, не узгоджені між собою і за змістом часто суперечать одна одній. Що стосується безпосередньо української «Вікіпедії», то часто зауважу ють, що багато статей у ній за глибиною довідковості, повноцінністю контенту поступаються аналогічним статтям, написаним іншими мовами, зокрема й уміщеним у російській «Вікіпедії». Серед прикметних особливостей української «Вікіпедії» назвемо таку: згідно ж з її даними8, енциклопедичний онлайн-ресурс містить багато статей, які у первісному вигляді було скопійовано з «Енциклопедії українознавства», (понад 6 тисяч), «Української радянської енциклопедії», а також інших енци клопедичних чи довідкових джерел. Можливо, це підвищує якість довідкової інформації у «Вікіпедії», однак суперечить принципам підготовки енцикло педичних видань, науковій етиці тощо, чим «Вікіпедія» вкотре демонструє, що не підходить під визначення терміна «енциклопедія». 8 Режим доступу: https://uk.wikipedia.org/wiki/Українська_Вікіпедія. Оригінал-макет виготовив Р. П. Судик ENCYCLOPEDIC NEWSREEL Tetiana Bereziuk, Oksana Davydenko NASU Institute of Encyclopedic Resarch, Kyiv, Ukraine Tetiana Bereziuk, Oksana Davydenko NASU Institute of Encyclopedic Resarch, Kyiv, Ukraine 8 Режим доступу: https://uk.wikipedia.org/wiki/Українська_Вікіпедія. 87 Енциклопедичний вісник України Наукове видання Число 10 (Спецвипуск до 100-річчя Національної академії наук України) Число 10 (Спецвипуск до 100-річчя Національної академії наук України) Видання містить наукові розвідки з енциклопедистики (серед них – доповіді учас ників круглого столу з нагоди100-річчя НАН України «Енциклопедистика в діяльності Національної академії наук України та Наукового товариства імені Шевченка», що відбувся 26 червня 2018 року), матеріали до словника енцикло педичних термінів, запропоновані фахівцями Інституту енциклопедичних дослід жень НАН України, а також опис подій 2018 року, пов’язаних з енциклопедичною справою. Для викладачів, аспірантів, студентів та усіх, хто цікавиться проблемами енциклопедистики, біографістики, книгознавства та суміжних наукових напрямів. За достовірність інформації, що міститься в опублікованих матеріалах, відповіда ють автори. Редколегія намагається зберегти особливості авторського стилю. Оригінал-макет виготовив Р. П. Судик Рекомендовано до друку вченою радою Інституту енциклопедичних досліджень НАН України (протокол № 10 від 30 жовтня 2018 р.). Свідоцтво про державну реєстрацію друкованого засобу масової інформації від 12 жовтня 2009 р. (серія КВ № 15829 – 4301Р). Свідоцтво про державну реєстрацію друкованого засобу масової інформації від 12 жовтня 2009 р. (серія КВ № 15829 – 4301Р). Свідоцтво про внесення суб’єкта видавничої справи до Державного реєстру видавців, виготівників і розповсюджувачів видавничої продукції ДК № 3484 від 19 травня 2009 р. Свідоцтво про внесення суб’єкта видавничої справи до Державного реєстру видавців, виготівників і розповсюджувачів видавничої продукції ДК № 3484 від 19 травня 2009 р.
https://openalex.org/W2566961083	https://www.scielo.br/j/aabc/a/Ngb6GLtHDzCfvSPVbCgpf9v/?lang=en&format=pdf	English	null	Geostatistical Approach for Spatial Interpolation of Meteorological Data	Anais da Academia Brasileira de Ciências	2,016	cc-by	7,556	ABSTRACT Meteorological data are used in many studies, especially in planning, disaster management, water resources management, hydrology, agriculture and environment. Analyzing changes in meteorological variables is very important to understand a climate system and minimize the adverse effects of the climate changes. One of the main issues in meteorological analysis is the interpolation of spatial data. In recent years, with the developments in Geographical Information System (GIS) technology, the statistical methods have been integrated with GIS and geostatistical methods have constituted a strong alternative to deterministic methods in the interpolation and analysis of the spatial data. In this study; spatial distribution of precipitation and temperature of the Aegean Region in Turkey for years 1975, 1980, 1985, 1990, 1995, 2000, 2005 and 2010 were obtained by the Ordinary Kriging method which is one of the geostatistical interpolation methods, the changes realized in 5-year periods were determined and the results were statistically examined using cell and multivariate statistics. The results of this study show that it is necessary to pay attention to climate change in the precipitation regime of the Aegean Region. This study also demonstrates the usefulness of the geostatistical approach in meteorological studies. Key words: geostatistical interpolation, geographic information system, ordinary kriging, meteorological data. Geostatistical Approach for Spatial Interpolation of Meteorological Data Derya Ozturk1 and Fatmagul Kilic2 1Department of Geomatics Engineering, Ondokuz Mayis University, Kurupelit Campus, 55139, Samsun, Turkey 2Department of Geomatics Engineering, Yildiz Technical University, Davutpasa Campus, 34220, Istanbul, Turkey Manuscript received on February 2, 2015; accepted for publication on March 1, 2016 Manuscript received on February 2, 2015; accepted for publication on March 1, 2016 Correspondence to: Derya Ozturk E-mail: dozturk@omu.edu.tr INTRODUCTION Measurement and evaluation of the spatially distributed meteorological data have become important in connection with climate-change impact studies, determination of water budgets at different temporal and spatial scales, as well as validation of atmospheric and hydrological models. Meteorological data are usually available from a limited number of meteorological stations (Hofierka et al. 2002), mostly because it is not economically and technically possible to obtain meteorological data throughout the entire surface. For this reason, spatial interpolation of the meteorological variables obtained from the certain sample points is performed in order to create a model for the entire surface. Spatial interpolation is the procedure of estimating the value of unsampled points using existing observations (Waters 1997). Methods for spatial interpolation can be classified into two main categories Correspondence to: Derya Ozturk E-mail: dozturk@omu.edu.tr An Acad Bras Cienc (2016) 88 (4) Derya Ozturk and Fatmagul Kilic 2122 as deterministic and geostatistical (Burrough and McDonnell 1998, Matthews 2002). Deterministic interpolation techniques calculate the values of unsampled points and create surfaces from measured points, based on either the extent of similarity or the degree of smoothing (Matthews 2002). Deterministic methods do not use probability theory (Waters 1997). Geostatistical interpolation techniques use the statistical properties of the measured points, quantify the spatial autocorrelation among the measured points and account for the spatial configuration of the sample points around the estimation location (Matthews 2002). Kriging is a geostatistical technique for optimal spatial estimation (Waller and Gotway 2004). Kriging provides a solution to the problem of estimation based on a continuous model of stochastic spatial variation and takes the variogram model (Webster and Oliver 2007). Today, with the developments in computer and Geographical Information System (GIS) technologies, the statistical methods have been integrated with GIS and the geostatistical methods have constituted a strong alternative to deterministic methods in the interpolation of the spatial data. In addition, statistical methods to analyze the interpolated layers have allowed a better understanding of the changes occurred in the specific time period.f Climate change is one of the biggest threats for the entire globe (Kropp 2015). Climate changes affect the natural balance of the earth and ecosystems and whole life is disrupted (National Academy of Sciences 2009) Climate change is most often measured by changes in primary climate variables, such as temperature and precipitation. These variables are the main drivers of climate changes (Sheffield and Wood 2012). INTRODUCTION For this reason, to understand and monitor the changes and their causes and effects accurately, changes should be determined both spatially and quantitatively and the results should be evaluated in detail. In this study it is aimed to investigate the spatial distribution of precipitation and temperature of the Aegean Region in Turkey for years 1975, 1980, 1985, 1990, 1995, 2000, 2005 and 2010 by the Ordinary Kriging method and statistically examine the results using cell statistics and multivariate statistics to understand the changes. This study demonstrates the usefulness of the geostatistical approach for both interpolation of meteorological data and analysis and comparison of the results. MATERIALS AND METHODS The Aegean Region is one of Turkey’s seven geographical regions. It is surrounded by the Aegean Sea on the west and takes its name from the Aegean Sea (Ozcaglar 2014). In this study, the area comprising eight provinces located in the Aegean Region has been analyzed. The total area is approximately 90,000 km2 (Figure 1). The coastal areas of the Aegean Region has a Mediterranean climate. The effects of the Mediterranean climate extend up to 100-150 km inland from the coast. In coastal areas, winters are mild and summers are very hot and dry. The interior side of the region is affected by the continental climate (Sensoy et al. 2008). In the present study, the time series of monthly precipitation and temperature data from 98 meteorological stations for the years 1975, 1980, 1985, 1990, 1995, 2000, 2005 and 2010 were used. Spatial distributions of the stations are shown in Figure 1. The geospatial interpolation of temperature and precipitation data and all statistical analyses of the precipitation and temperature layers were performed using ArcGIS 10.0 software (Esri, Redlands, CA). The method of creating an estimation surface layer with the Ordinary Kriging is explained in Section “Creating An Estimation Surface Layer with the Ordinary Kriging” and statistical analyses of layers is presented in Section “Statistical Analyses of Layers”. An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA 2123 Figure 1 - The location of the study area (The Aegean Region, Turkey) and spatial distributions of the meteorological stations. Figure 1 - The location of the study area (The Aegean Region, Turkey) and spatial distributions of the meteorological stations. CREATING AN ESTIMATION SURFACE LAYER WITH THE ORDINARY KRIGING ariogram (distance h) = 0.5 * average[(value at location i – value at location j)2] (1) CREATING AN ESTIMATION SURFACE LAYER WITH THE ORDINARY KRIGING Estimation with the Kriging interpolation method has a two-step process: (i) fitting a model: creation of the variograms and covariance functions to estimate the statistical dependence (spatial autocorrelation) values that depend on the model of autocorrelation and (ii) making an estimation: estimation of the unknown values (ESRI 2014a). The first step in the Ordinary Kriging is to create a semivariogram from the scatter point set to be interpolated. A semivariogram consists of (i) an empirical semivariogram (experimental variogram) and (ii) a model semivariogram (GMS User Manuel 2012). Semivariogram is a mathematical model of the semivariance as a function of lag and displays the statistical correlation of nearby points (Prasad et al. 2007). Spatial autocorrelation (means feature similarity) is based on both feature locations and feature values simultaneously (not only based on feature locations or attribute values alone). Given a set of features and an associated attribute, it evaluates whether the pattern expressed is clustered, dispersed, or random (Matthews 2002). Empirical semivariogram, computed by (Eq.1) for all pairs of locations separated by distance h (ESRI 2014a): (1) Semivariogram (distance h) = 0.5 * average[(value at location i – value at location j)2] (1) An Acad Bras Cienc (2016) 88 (4) 2124 Derya Ozturk and Fatmagul Kilic The formula involves calculating the difference squared between the values of the paired locations. Figure 2 shows the pairing of one point (the red point) with all other measured locations. This process continues for each measured point (ESRI 2014a). Often, each pair of locations has a unique distance, and there are often many pairs of points. To plot all pairs quickly becomes unmanageable. Instead of plotting each pair, the pairs are grouped into lag bins. The empirical semivariogram is a graph of the averaged semivariogram values on the y-axis and the distance (or lag) on the x-axis (Figure 3) (ESRI 2014a). When two locations are close to each other (far left on the x-axis of the semivariogram cloud), then they are expected to be similar (low on the y-axis of the semivariogram cloud) (ESRI 2014a, Prasad et al. 2007). Cell Statistics In a local function, the value at each location on the output raster is a function of the input values at that location. When computing a local function, input rasters can be combined and a statistic can be calculated. In ArcGIS software, several cell statistics can be calculated for raster layers: (i) MEAN: Calculates the mean (average) of the inputs, (ii) MAXIMUM: Determines the maximum (largest value) of the inputs, (iii) MEDIAN: Calculates the median of the inputs, (iv) MINIMUM: Determines the minimum (smallest value) of the inputs, (v) RANGE: Calculates the range (difference between largest and smallest value) of the inputs, (vi) STD: Calculates the standard deviation of the inputs (ESRI 2014b). CREATING AN ESTIMATION SURFACE LAYER WITH THE ORDINARY KRIGING The basic equation used in the Ordinary Kriging is as (Eq.2) (ESRI 2014a, GMS User Manuel 2012, Borga and Vizzaccaro 1996): ∑ = ∧ = N i i i s Z s Z 1 0 ) ( ) ( λ (2) ∑ = ∧ = N i i i s Z s Z 1 0 ) ( ) ( λ (2) Where; ) ( is Z : the measured value at the ith location ) ( is Z : the measured value at the ith location iλ : an unknown weight for the measured value at the ith location ) ( 0s : the estimation location iλ : an unknown weight for the measured value at the ith location iλ : an unknown weight for the measured value at the ith location iλ : an unknown weight for the measured value at the ith location ) ( 0s : the estimation location N: the number of measured values N: the number of measured values With Kriging method, the value ) ( 0s Z ∧ at the point 0s , where the true unknown value is ) ( 0s Z , is estimated by a linear combination of the values at N surrounding data points (Borga and Vizzaccaro 1996).i With Kriging method, the value ) ( 0s Z ∧ at the point 0s , where the true unknown value is ) ( 0s Z , is estimated by a linear combination of the values at N surrounding data points (Borga and Vizzaccaro 1996).i In the Ordinary Kriging, the weight, iλ , depends on a fitted model to the measured points, the distance to the estimation point, and the spatial relationships among the measured values around the estimation location (ESRI 2014a) and the Kriging weights are calculated by minimizing the variance (Li and Heap 2008). The Ordinary Kriging is the most widely used Kriging method (Wackernagel 2003) and this method assumes that the data set has a stationary variance but also a non-stationary mean value within the search radius. The Ordinary Kriging is highly reliable and recommended for most data sets (Vertical Mapper User Guide 2008). CREATING AN ESTIMATION SURFACE LAYER WITH THE ORDINARY KRIGING “As pairs of locations become farther apart (moving to the right on the x-axis of the semivariogram cloud), they should become more dissimilar and have a higher squared difference (moving up on the y-axis of the semivariogram cloud)” (ESRI 2014a).i Once the empirical variogram is obtained, the next step is to define a model semivariogram (GMS User Manuel 2012). Semivariogram modeling is a main step between spatial description and spatial estimation. The empirical semivariogram provides information on the spatial autocorrelation of datasets, however does not supply information for all possible directions and distances. For this reason, it is necessary to fit Figure 2 - Calculation of the difference squared between the paired locations. An Acad Bras Cienc (2016) 88 (4) Figure 2 - Calculation of the difference squared between the paired locations. Figure 3 - Empirical semivariogram graph example. Figure 2 - Calculation of the difference squared between the paired locations. Figure 2 - Calculation of the difference squared between the paired locations. Figure 3 - Empirical semivariogram graph example. Figure 3 - Empirical semivariogram graph example. An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA 2125 a model (a continuous function or curve) to the empirical semivariogram (ESRI 2014a). There are many semivariogram models. Some of the most common are linear, circular, spherical, exponential, and Gaussian model (Li and Heap 2008). The selected model influences the estimation of the unknown values and each model is designed to fit different types of phenomena more accurately (ESRI 2014a). Once the model variogram is obtained, it is used to calculate the weights used in Kriging (GMS User Manuel 2012). Multivariate Statistics The multivariate statistics allow exploration of relationships between many different data layers or types of attributes. In band collection function, main statistical measures (minimum, maximum, mean and standard deviation) can be calculated for every layer and in addition to these standard statistics, the covariance and correlation matrices can also be determined (ESRI 2014b). An Acad Bras Cienc (2016) 88 (4) 2126 Derya Ozturk and Fatmagul Kilic RESULTS AND DISCUSSION The time series of monthly precipitation and temperature data for the years 1975, 1980, 1985, 1990, 1995, 2000, 2005 and 2010 were used for preparing spatial distribution layers of precipitation and temperature of the Aegean Region, Turkey. The Ordinary Kriging interpolation was applied for each month and a total of 192 interpolations were performed (96 for precipitation and 96 for temperature) and grid layers with 250-meter pixel size were formed. The Ordinary Kriging interpolation results of the precipitation and temperature data for January are shown in Figures 4 and 5, respectively. Based on the multivariate statistics (band collection), spatial analyses were applied for the monthly precipitation and temperature layer series which calculated by the Ordinary Kriging. Table I (for precipitation) and Table II (for temperature) represent the main statistics, including the minimum, maximum, mean and standard deviation values. In addition, the correlation coefficients were calculated with these analyses (Tables III and IV). When examining Table I, it was seen that the highest “average precipitation” and the highest “precipitation” values were in December 1990. Table II shows that both highest “average temperature” and highest “temperature” values were in August 2010.fi According to Table III, correlation coefficients for precipitation are between -0.04847 and 0.92382 for January, 0.18609 and 0.92908 for February, -0.57255 and 0.74793 for March, -0.43776 and 0.76531 for April, -0.62850 and 0.86128 for May, -0.26385 and 0.79177 for June, -0.22994 and 0.74375 for July, 0.01302 and 0.80480 for August, -0.20455 and 0.69922 for September, -0.68396 and 0.74704 for October, Figure 4 - The Ordinary Kriging interpolation results of the precipitation data for the month of January (For the years of 1975, 1980, 1985, 1990, 1995, 2000, 2005, and 2010). Figure 4 - The Ordinary Kriging interpolation results of the precipitation data for the month of January (For the years of 1975, 1980, 1985, 1990, 1995, 2000, 2005, and 2010). An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA 2127 Figure 5 - The Ordinary Kriging interpolation results of the temperature data for the month of January (For the years of 1975, 1980, 1985, 1990, 1995, 2000, 2005, and 2010). Figure 5 - The Ordinary Kriging interpolation results of the temperature data for the month of January (For the years of 1975, 1980, 1985, 1990, 1995, 2000, 2005, and 2010). 0.29150 and 0.85486 for November, 0.22520 and 0.90808 for December. Table I Main statistics for precipitation layers (Minimum, Maximum, Mean, Standard deviation). Main statistics for precipitation layers (Minimum, Maximum, Mean, Standard deviation). RESULTS AND DISCUSSION According to Table IV, correlation coefficients for temperature are between 0.95152 and 0.99524 for January, 0.95804 and 0.99591 for February, 0.91016 and 0.99459 for March, 0.94823 and 0.99361 for April, 0.96744 and 0.99414 for May, 0.94961 and 0.99084 for June, 0.93391 and 0.99041 for July, 0.94175 and 0.99293 for August, 0.96714 and 0.99254 for September, 0.97214 and 0.99542 for October, 0.93409 and 0.99328 for November, 0.96537 and 0.99665 for December. Correlations above 0.80 generally are accepted as high correlations. Correlations between 0.50 and 0.80 are usually considered as medium (moderate) correlations and correlations below 0.50 are typically regarded as low correlations (Wang et al. 1990). Accordingly, very high correlation values were observed between temperature values of years 1975, 1980, 1985, 1990, 1995, 2000, 2005 and 2010 for all months (Table IV). But, the correlations between layers of precipitation were examined, both high and low correlation values were observed. For precipitation layers, the highest correlation was observed between the year of 1975 and 2010 for January; 2005 and 2010 for February; 1985 and 1995 for March; 2005 and 2010 for April; 1990 and 2000 for May; 1990 and 1995 for June; 1975 and 2010 for July; 1975 and 2000 for August; 1980 and 2000 for September; 2000 and 2010 for October; 2005 and 2010 for November; 1980 and 1990 for December. By calculating cell statistics, a statistic for each cell in an output raster can be calculated based on the values of multiple input rasters (ESRI 2014b). In this study; maximum, minimum, mean, median, range and standard deviation layers were produced by using precipitation and temperature layers for years 1975, 1980, 1985, 1990, 1995, 2000, 2005 and 2010 for all months. Totally 144 statistical layers were obtained An Acad Bras Cienc (2016) 88 (4) 2128 Derya Ozturk and Fatmagul Kilic Table II Main statistics for temperature layers (Minimum, Maximum, Mean, Standard deviation). Table II STATISTICS of INDIVIDUAL LAYERS - TEMPERATURE Layer(Year) MIN MAX MEAN STD Layer(Year) MIN MAX MEAN STD January 1975 -1.6080 11.9795 3.7326 3.1861 July 1975 20.8040 28.7429 25.1050 2.1311 1980 -2.6259 11.4972 3.0345 3.4320 1980 21.6467 29.0423 25.5491 1.7273 1985 2.2792 13.7292 7.0922 2.9161 1985 18.8785 28.5773 24.1568 2.6798 1990 -4.3366 11.9610 2.5856 3.4689 1990 20.9858 29.3868 25.8605 2.2016 1995 1.8665 12.9328 6.2080 2.7793 1995 19.5084 29.1838 24.6306 2.9346 2000 -5.0773 9.6267 1.1922 3.7585 2000 22.6168 30.2668 27.1807 2.0244 2005 2.2003 12.6267 6.3250 2.6454 2005 21.7852 29.3529 26.1972 2.1397 2010 1.5386 13.2897 6.6969 2.7832 2010 21.6157 30.2688 26.5241 2.1923 February 1975 -0.4336 11.9043 4.1513 3.0501 August 1975 20.1038 27.2521 23.9362 1.9830 1980 -0.3540 11.8185 4.6182 2.9815 1980 20.3963 28.3603 24.5688 2.0282 1985 -4.1206 10.0607 1.7981 3.7145 1985 21.4755 28.3776 25.3309 1.8210 1990 0.0275 12.6155 5.6012 3.1362 1990 19.6488 27.6955 24.3883 2.2268 1995 3.9147 13.8931 7.8196 2.5229 1995 20.3723 28.2541 24.5760 2.0307 2000 -1.8539 11.9216 4.6025 3.3023 2000 20.0756 28.9870 25.3378 2.4911 2005 1.0839 11.0291 5.1357 2.7220 2005 22.1073 28.9807 26.0388 1.9475 2010 4.8857 14.0768 8.8109 2.4160 2010 25.2166 30.3014 28.1310 1.5564 March 1975 6.6072 15.0204 10.1282 2.2135 September 1975 16.5458 26.1924 20.8361 2.7081 1980 3.7778 13.6001 7.4684 2.5244 1980 14.3647 24.4542 19.1395 2.5351 1985 1.5080 13.6020 7.9737 2.9179 1985 15.2840 25.1916 20.5854 2.4419 1990 5.8160 14.7953 9.8410 2.4662 1990 15.4788 25.2687 19.8176 2.4584 1995 4.9791 14.0615 8.4624 2.4898 1995 17.0612 25.4172 21.0791 2.1735 2000 3.4803 12.7911 7.1545 2.6171 2000 16.3220 25.8142 21.1166 2.4484 2005 5.0723 13.9870 8.8848 2.4185 2005 16.4752 25.7482 20.7710 2.5786 2010 5.8425 16.4715 10.4517 2.3320 2010 18.3427 25.9256 21.7630 2.1259 April 1975 11.4027 17.6734 13.9034 1.7218 October 1975 11.0174 21.0976 14.8899 2.6129 1980 8.4474 15.4275 11.4915 2.0839 1980 12.3536 21.1985 16.2249 2.3637 1985 10.4556 17.2842 13.9654 2.0125 1985 8.7044 19.1918 13.0156 2.5467 1990 9.9327 17.4434 13.1470 2.2247 1990 11.7813 21.7325 16.0564 2.4525 1995 8.2844 15.9269 11.5358 2.2356 1995 9.2743 19.8509 13.8540 2.5798 2000 11.7082 17.5848 14.3388 1.8857 2000 10.5590 20.9450 14.9599 2.6980 2005 9.9162 17.1927 13.1155 2.1300 2005 9.7082 20.0835 13.8896 2.6384 2010 9.9424 18.6159 13.8762 2.4406 2010 10.2576 21.7663 14.9154 2.9379 May 1975 13.7103 20.7852 17.0399 2.3309 November 1975 5.2976 16.6203 9.3378 2.6591 1980 14.1786 20.2122 16.8566 1.7605 1980 7.4070 18.1094 11.4330 2.6440 1985 15.8079 22.5850 19.2854 2.1122 1985 8.4017 18.0259 12.0326 2.3988 1990 13.3384 21.6440 17.3523 2.5074 1990 7.6179 18.0775 12.0642 2.6051 1995 15.4864 21.1762 18.3836 1.6126 1995 1.5022 13.8388 6.7709 3.0380 2000 13.9277 22.0098 18.0709 2.5511 2000 7.1828 18.5339 11.8420 2.5075 2005 14.9138 21.6488 18.4084 2.1777 2005 5.1913 15.7904 9.1067 2.5619 2010 14.5255 22.5254 19.3349 2.2301 2010 9.6838 19.5951 13.9344 2.3800 June 1975 17.7122 24.6564 21.2720 2.2485 December 1975 -3.2787 13.1689 3.8009 3.8370 1980 18.1593 25.9312 21.7894 2.0464 1980 2.1513 13.9154 6.7185 2.9002 1985 18.0790 26.3428 22.1907 2.3044 1985 2.0959 14.5184 6.6195 3.0085 1990 17.3079 25.9777 21.8186 2.6735 1990 2.4093 14.2393 7.3080 3.0477 1995 19.8111 27.7873 24.1458 2.4046 1995 1.0647 14.7638 7.1749 3.1052 2000 18.0133 27.5768 23.1268 2.8309 2000 1.3021 14.4294 6.5028 3.1720 2005 17.8073 25.9265 22.0558 2.4529 2005 3.0349 13.6756 7.2157 2.8025 2010 18.0412 26.3838 22.1602 2.4779 2010 5.2619 15.6738 9.0097 2.6403 Main statistics for temperature layers (Minimum, Maximum, Mean, Standard deviation). An Acad Bras Cienc (2016) 88 (4) Table I STATISTICS of INDIVIDUAL LAYERS - PRECIPITATION Layer(Year) MIN MAX MEAN STD Layer(Year) MIN MAX MEAN STD January 1975 15.0962 290.5551 105.2915 68.9066 July 1975 0.0000 11.4688 3.8647 3.2307 1980 57.4809 301.0310 144.8527 41.7206 1980 0.0000 6.4559 1.5206 0.9994 1985 46.7396 325.7432 160.8042 58.6881 1985 0.0000 7.6697 0.7246 0.7361 1990 0.0000 35.0969 12.1264 5.1998 1990 0.0000 39.4809 5.0383 5.5698 1995 18.9700 272.5263 120.9055 59.4265 1995 0.0000 189.5152 34.9476 30.6981 2000 42.8687 132.2835 69.6099 16.4874 2000 0.0000 23.2685 3.9946 4.4414 2005 14.4151 305.8113 70.4621 46.7614 2005 0.0000 85.0155 24.6473 19.6237 2010 34.0842 249.2391 114.5032 48.2253 2010 0.0173 12.8247 5.7179 3.1505 February 1975 35.9389 164.3231 58.1565 23.4616 August 1975 0.0000 66.0855 12.1734 13.2959 1980 11.4949 100.1111 35.0220 14.2885 1980 0.0000 29.5959 4.3619 5.3946 1985 29.6399 149.1992 73.9867 26.1003 1985 0.0000 41.5747 10.0517 7.4810 1990 7.0911 143.2509 49.7590 25.1979 1990 0.4801 67.0008 10.8518 7.5046 1995 6.6114 117.6134 27.3189 15.0847 1995 0.2385 16.9660 7.1374 3.8129 2000 49.3334 109.7159 86.5541 15.9437 2000 0.0000 26.2174 6.2557 5.7277 2005 18.2838 330.9904 117.0124 68.6871 2005 0.4208 17.9898 8.6525 4.3138 2010 47.3964 343.1137 151.4875 62.9992 2010 0.0000 20.6179 7.7861 5.9152 March 1975 26.2257 107.8506 64.0406 15.5155 September 1975 0.0000 49.7015 10.9055 10.0935 1980 49.5541 159.2850 95.6635 22.8128 1980 0.0000 55.4421 16.4988 14.7347 1985 27.8837 106.6610 59.4646 17.9716 1985 0.0000 6.5261 1.9362 1.3202 1990 6.9016 39.8629 23.6062 5.8286 1990 1.4236 38.1317 20.5715 8.0680 1995 61.6728 222.2994 126.3867 33.2200 1995 0.0000 46.1658 17.5078 11.7651 2000 38.4527 143.0037 88.8729 20.7716 2000 0.0000 22.9868 5.9550 5.8907 2005 33.6981 117.8575 79.2205 15.7518 2005 4.7207 35.6864 15.2393 6.0027 2010 2.0541 66.2231 30.9691 13.4006 2010 22.0556 44.4905 33.0731 4.0500 April 1975 18.2819 98.7922 51.6425 12.2515 October 1975 9.5328 90.6642 33.0401 13.0100 1980 29.4372 69.5153 47.0104 7.3046 1980 4.3897 46.2036 22.3987 9.1381 1985 2.0838 44.1912 19.7078 9.1412 1985 11.1300 151.5788 42.3043 26.9946 1990 19.9863 90.5741 54.8812 12.3692 1990 7.2120 42.6564 24.6231 8.0837 1995 18.1657 89.0869 51.9969 14.4514 1995 6.0607 56.4756 34.5488 14.9560 2000 26.9695 152.4105 85.1713 26.0013 2000 3.6411 98.6216 34.3385 19.8806 2005 15.9945 90.1445 39.7732 13.9404 2005 11.2323 121.7506 33.1998 17.9017 2010 9.5546 83.4186 37.2400 14.8342 2010 49.5620 311.1042 116.5370 49.2287 May 1975 39.9216 119.2508 71.7452 17.9338 November 1975 39.6952 239.7979 112.4898 38.7931 1980 10.6646 77.5801 36.4267 10.0239 1980 31.0222 174.3634 93.1593 26.5030 1985 1.4462 92.5336 33.3793 9.2920 1985 28.2310 180.9006 85.0800 32.2572 1990 0.0000 62.5613 23.3644 13.0269 1990 13.8738 74.1966 32.1743 11.6332 1995 0.0000 76.9429 27.1503 9.6845 1995 31.6046 177.6392 88.1167 27.7271 2000 1.0087 79.3443 31.0651 20.8717 2000 1.8513 201.2959 54.5331 44.2754 2005 6.2719 83.2165 44.0559 17.0640 2005 43.2833 254.8676 114.4541 41.6780 2010 12.1662 46.3693 26.6590 6.6865 2010 8.2926 51.3080 28.8157 9.8915 June 1975 15.4860 100.2484 50.0390 17.3824 December 1975 39.7735 188.7916 88.9672 25.1946 1980 0.0000 80.8975 24.9794 12.4476 1980 28.2021 296.8829 147.5694 61.1975 1985 0.0000 45.2143 16.5010 11.0090 1985 19.9052 135.5423 51.3575 16.5321 1990 0.0000 54.2057 20.5578 14.1597 1990 43.9064 361.6299 165.4436 71.0610 1995 0.0021 47.5019 9.5297 10.6071 1995 24.6129 165.1980 71.9208 30.6091 2000 0.0000 37.4081 11.7682 8.3772 2000 14.4990 329.9915 52.2345 36.1115 2005 0.3222 58.3159 27.5195 12.8268 2005 11.9330 203.2546 65.3737 31.7432 2010 0.7233 141.3197 55.6312 24.9145 2010 59.0785 231.4372 119.4989 34.7257 STATISTICS of INDIVIDUAL LAYERS - PRECIPITATION An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA 2129 Table III Correlation matrix for precipitation layers. Table II An Acad Bras Cienc (2016) 88 (4) 2130 Derya Ozturk and Fatmagul Kilic An Acad Bras Cienc (2016) 88 (4) Table III CORRELATION MATRIX (Precipitation-January) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.78846 0.78386 0.12940 0.87703 0.75682 0.77189 0.92382 1980 0.78846 1.00000 0.86062 0.17125 0.81056 0.69908 0.65659 0.77735 1985 0.78386 0.86062 1.00000 0.16711 0.69942 0.63916 0.60556 0.81675 1990 0.12940 0.17125 0.16711 1.00000 -0.04847 0.39700 0.01441 0.06176 1995 0.87703 0.81056 0.69942 -0.04847 1.00000 0.55791 0.78129 0.86215 2000 0.75682 0.69908 0.63916 0.39700 0.55791 1.00000 0.61256 0.74714 2005 0.77189 0.65659 0.60556 0.01441 0.78129 0.61256 1.00000 0.81718 2010 0.92382 0.77735 0.81675 0.06176 0.86215 0.74714 0.81718 1.00000 CORRELATION MATRIX (Precipitation-February) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.77978 0.71037 0.75465 0.78511 0.32408 0.62331 0.59694 1980 0.77978 1.00000 0.70680 0.65260 0.75107 0.18609 0.51814 0.44678 1985 0.71037 0.70680 1.00000 0.67623 0.59647 0.25324 0.33989 0.25617 1990 0.75465 0.65260 0.67623 1.00000 0.79150 0.64538 0.84264 0.77847 1995 0.78511 0.75107 0.59647 0.79150 1.00000 0.50060 0.71130 0.68328 2000 0.32408 0.18609 0.25324 0.64538 0.50060 1.00000 0.53958 0.66191 2005 0.62331 0.51814 0.33989 0.84264 0.71130 0.53958 1.00000 0.92908 2010 0.59694 0.44678 0.25617 0.77847 0.68328 0.66191 0.92908 1.00000 CORRELATION MATRIX (Precipitation-March) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.43577 0.43600 0.54000 0.42494 0.60580 0.65125 0.24777 1980 0.43577 1.00000 0.48775 0.59721 0.63492 0.69036 0.10031 -0.57255 1985 0.43600 0.48775 1.00000 0.24778 0.74793 0.23000 -0.00553 -0.38343 1990 0.54000 0.59721 0.24778 1.00000 0.34694 0.57528 0.44113 0.00851 1995 0.42494 0.63492 0.74793 0.34694 1.00000 0.51069 -0.00559 -0.43359 2000 0.60580 0.69036 0.23000 0.57528 0.51069 1.00000 0.39025 -0.20299 2005 0.65125 0.10031 -0.00553 0.44113 -0.00559 0.39025 1.00000 0.61318 2010 0.24777 -0.57255 -0.38343 0.00851 -0.43359 -0.20299 0.61318 1.00000 CORRELATION MATRIX (Precipitation-April) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.04649 -0.29210 0.22152 0.08140 -0.09371 -0.43776 -0.29386 1980 0.04649 1.00000 0.20565 0.08084 0.25487 -0.06780 0.46926 0.44613 1985 -0.29210 0.20565 1.00000 0.11292 0.31988 0.64021 0.74893 0.63354 1990 0.22152 0.08084 0.11292 1.00000 0.53602 0.27626 -0.03302 -0.19927 1995 0.08140 0.25487 0.31988 0.53602 1.00000 0.62172 0.26567 -0.06769 2000 -0.09371 -0.06780 0.64021 0.27626 0.62172 1.00000 0.33247 0.06834 2005 -0.43776 0.46926 0.74893 -0.03302 0.26567 0.33247 1.00000 0.76531 2010 -0.29386 0.44613 0.63354 -0.19927 -0.06769 0.06834 0.76531 1.00000 CORRELATION MATRIX (Precipitation-May) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.00014 0.51378 0.40567 0.04118 0.58152 -0.03261 -0.35965 1980 0.00014 1.00000 -0.14016 0.09716 0.11290 0.16423 -0.14988 -0.11414 1985 0.51378 -0.14016 1.00000 -0.22438 -0.12253 -0.13165 -0.22834 0.13157 1990 0.40567 0.09716 -0.22438 1.00000 0.28300 0.86128 0.64427 -0.51083 1995 0.04118 0.11290 -0.12253 0.28300 1.00000 0.19170 0.21629 0.00804 2000 0.58152 0.16423 -0.13165 0.86128 0.19170 1.00000 0.29651 -0.62850 2005 -0.03261 -0.14988 -0.22834 0.64427 0.21629 0.29651 1.00000 0.05753 2010 -0.35965 -0.11414 0.13157 -0.51083 0.00804 -0.62850 0.05753 1.00000 An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA 2131 Table III (continuation) CORRELATION MATRIX (Precipitation-June) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.20265 0.20593 0.28976 0.32791 0.31104 0.10884 -0.22344 1980 0.20265 1.00000 -0.25895 0.25642 -0.01494 -0.26385 0.30535 0.13046 1985 0.20593 -0.25895 1.00000 0.61296 0.63628 0.78552 0.42126 0.32951 1990 0.28976 0.25642 0.61296 1.00000 0.79177 0.60613 0.66090 0.48602 1995 0.32791 -0.01494 0.63628 0.79177 1.00000 0.76352 0.57111 0.30044 2000 0.31104 -0.26385 0.78552 0.60613 0.76352 1.00000 0.27430 0.25948 2005 0.10884 0.30535 0.42126 0.66090 0.57111 0.27430 1.00000 0.48441 2010 -0.22344 0.13046 0.32951 0.48602 0.30044 0.25948 0.48441 1.00000 CORRELATION MATRIX (Precipitation-July) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.22850 -0.22994 0.71580 0.46294 0.37854 0.62662 0.74375 1980 0.22850 1.00000 0.20738 0.21785 0.23528 0.29285 0.10786 0.54332 1985 -0.22994 0.20738 1.00000 0.11996 0.04063 0.20946 0.10074 0.05492 1990 0.71580 0.21785 0.11996 1.00000 0.29884 0.37246 0.57299 0.65655 1995 0.46294 0.23528 0.04063 0.29884 1.00000 0.04565 0.39870 0.56466 2000 0.37854 0.29285 0.20946 0.37246 0.04565 1.00000 0.55639 0.27101 2005 0.62662 0.10786 0.10074 0.57299 0.39870 0.55639 1.00000 0.40386 2010 0.74375 0.54332 0.05492 0.65655 0.56466 0.27101 0.40386 1.00000 CORRELATION MATRIX (Precipitation-August) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.62665 0.45484 0.33267 0.52327 0.80480 0.47424 0.60626 1980 0.62665 1.00000 0.63235 0.23099 0.34192 0.67653 0.49201 0.68952 1985 0.45484 0.63235 1.00000 0.27238 0.49517 0.47897 0.36196 0.71590 1990 0.33267 0.23099 0.27238 1.00000 0.11132 0.34003 0.01302 0.18870 1995 0.52327 0.34192 0.49517 0.11132 1.00000 0.35925 0.67713 0.59460 2000 0.80480 0.67653 0.47897 0.34003 0.35925 1.00000 0.51379 0.66336 2005 0.47424 0.49201 0.36196 0.01302 0.67713 0.51379 1.00000 0.55531 2010 0.60626 0.68952 0.71590 0.18870 0.59460 0.66336 0.55531 1.00000 CORRELATION MATRIX (Precipitation-September) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.37163 0.19678 0.59756 0.53491 0.04033 0.02862 0.39440 1980 0.37163 1.00000 0.57914 0.66755 -0.08048 0.69922 0.38872 0.20890 1985 0.19678 0.57914 1.00000 0.36662 -0.10389 0.58103 0.36137 0.23253 1990 0.59756 0.66755 0.36662 1.00000 0.37907 0.41984 0.03516 0.24139 1995 0.53491 -0.08048 -0.10389 0.37907 1.00000 -0.20455 -0.19451 0.65751 2000 0.04033 0.69922 0.58103 0.41984 -0.20455 1.00000 0.52987 0.17506 2005 0.02862 0.38872 0.36137 0.03516 -0.19451 0.52987 1.00000 0.31895 2010 0.39440 0.20890 0.23253 0.24139 0.65751 0.17506 0.31895 1.00000 CORRELATION MATRIX (Precipitation-October) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.06983 0.66618 -0.34307 -0.21368 0.26494 0.62937 0.25635 1980 0.06983 1.00000 0.49524 0.12709 0.66087 -0.57686 0.44764 -0.60944 1985 0.66618 0.49524 1.00000 -0.08372 0.13923 0.01497 0.61832 -0.06324 1990 -0.34307 0.12709 -0.08372 1.00000 0.50000 -0.16595 -0.20978 -0.04511 1995 -0.21368 0.66087 0.13923 0.50000 1.00000 -0.68396 0.06114 -0.63589 2000 0.26494 -0.57686 0.01497 -0.16595 -0.68396 1.00000 -0.10283 0.74704 2005 0.62937 0.44764 0.61832 -0.20978 0.06114 -0.10283 1.00000 -0.27208 2010 0.25635 -0.60944 -0.06324 -0.04511 -0.63589 0.74704 -0.27208 1.00000 Table III (continuation) An Acad Bras Cienc (2016) 88 (4) Derya Ozturk and Fatmagul Kilic 2132 A A d B Ci (2016) 88 (4) 2132 Derya Ozturk and Fatmagul Kilic Table III (continuation) CORRELATION MATRIX (Precipitation-November) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.79762 0.65602 0.45331 0.41620 0.62822 0.79794 0.72336 1980 0.79762 1.00000 0.77541 0.57684 0.59180 0.52085 0.72144 0.68886 1985 0.65602 0.77541 1.00000 0.63039 0.56208 0.64761 0.72962 0.73200 1990 0.45331 0.57684 0.63039 1.00000 0.34262 0.47725 0.34958 0.29150 1995 0.41620 0.59180 0.56208 0.34262 1.00000 0.45066 0.43236 0.34430 2000 0.62822 0.52085 0.64761 0.47725 0.45066 1.00000 0.64668 0.55346 2005 0.79794 0.72144 0.72962 0.34958 0.43236 0.64668 1.00000 0.85486 2010 0.72336 0.68886 0.73200 0.29150 0.34430 0.55346 0.85486 1.00000 CORRELATION MATRIX (Precipitation-December) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.79379 0.25259 0.81574 0.78905 0.50197 0.86966 0.64845 1980 0.79379 1.00000 0.31704 0.90808 0.90569 0.39818 0.78649 0.80757 1985 0.25259 0.31704 1.00000 0.22520 0.48945 0.76838 0.31490 0.26742 1990 0.81574 0.90808 0.22520 1.00000 0.85097 0.42111 0.74077 0.78907 1995 0.78905 0.90569 0.48945 0.85097 1.00000 0.56697 0.77087 0.77156 2000 0.50197 0.39818 0.76838 0.42111 0.56697 1.00000 0.52801 0.38538 2005 0.86966 0.78649 0.31490 0.74077 0.77087 0.52801 1.00000 0.65563 2010 0.64845 0.80757 0.26742 0.78907 0.77156 0.38538 0.65563 1.00000 Table IV Correlation matrix for temperature layers. GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA CORRELATION MATRIX (Temperature-January) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.99001 0.98543 0.99064 0.98531 0.99384 0.99098 0.97266 1980 0.99001 1.00000 0.99524 0.98171 0.99200 0.99217 0.99463 0.97417 1985 0.98543 0.99524 1.00000 0.97166 0.99482 0.98869 0.99503 0.97539 1990 0.99064 0.98171 0.97166 1.00000 0.97136 0.98582 0.97629 0.95152 1995 0.98531 0.99200 0.99482 0.97136 1.00000 0.98977 0.99353 0.97975 2000 0.99384 0.99217 0.98869 0.98582 0.98977 1.00000 0.99041 0.97014 2005 0.99098 0.99463 0.99503 0.97629 0.99353 0.99041 1.00000 0.98280 2010 0.97266 0.97417 0.97539 0.95152 0.97975 0.97014 0.98280 1.00000 CORRELATION MATRIX (Temperature-February) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.99420 0.99281 0.98748 0.98275 0.96240 0.98388 0.97305 1980 0.99420 1.00000 0.99591 0.99286 0.98342 0.97515 0.98280 0.96708 1985 0.99281 0.99591 1.00000 0.99162 0.98447 0.97700 0.98697 0.97381 1990 0.98748 0.99286 0.99162 1.00000 0.98965 0.98378 0.98806 0.97410 1995 0.98275 0.98342 0.98447 0.98965 1.00000 0.98594 0.98194 0.98254 2000 0.96240 0.97515 0.97700 0.98378 0.98594 1.00000 0.96256 0.95804 2005 0.98388 0.98280 0.98697 0.98806 0.98194 0.96256 1.00000 0.98685 2010 0.97305 0.96708 0.97381 0.97410 0.98254 0.95804 0.98685 1.00000 CORRELATION MATRIX (Temperature-March) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.96776 0.93293 0.96541 0.98026 0.97802 0.96322 0.91016 1980 0.96776 1.00000 0.97682 0.97817 0.98798 0.98687 0.99252 0.96439 1985 0.93293 0.97682 1.00000 0.97683 0.95402 0.95607 0.96738 0.93709 1990 0.96541 0.97817 0.97683 1.00000 0.97132 0.97380 0.97428 0.93204 1995 0.98026 0.98798 0.95402 0.97132 1.00000 0.99459 0.98740 0.93968 2000 0.97802 0.98687 0.95607 0.97380 0.99459 1.00000 0.98191 0.93228 2005 0.96322 0.99252 0.96738 0.97428 0.98740 0.98191 1.00000 0.96909 2010 0.91016 0.96439 0.93709 0.93204 0.93968 0.93228 0.96909 1.00000 Table III (continuation) Table III (continuation) CORRELATION MATRIX (Precipitation-November) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.79762 0.65602 0.45331 0.41620 0.62822 0.79794 0.72336 1980 0.79762 1.00000 0.77541 0.57684 0.59180 0.52085 0.72144 0.68886 1985 0.65602 0.77541 1.00000 0.63039 0.56208 0.64761 0.72962 0.73200 1990 0.45331 0.57684 0.63039 1.00000 0.34262 0.47725 0.34958 0.29150 1995 0.41620 0.59180 0.56208 0.34262 1.00000 0.45066 0.43236 0.34430 2000 0.62822 0.52085 0.64761 0.47725 0.45066 1.00000 0.64668 0.55346 2005 0.79794 0.72144 0.72962 0.34958 0.43236 0.64668 1.00000 0.85486 2010 0.72336 0.68886 0.73200 0.29150 0.34430 0.55346 0.85486 1.00000 CORRELATION MATRIX (Precipitation-December) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.79379 0.25259 0.81574 0.78905 0.50197 0.86966 0.64845 1980 0.79379 1.00000 0.31704 0.90808 0.90569 0.39818 0.78649 0.80757 1985 0.25259 0.31704 1.00000 0.22520 0.48945 0.76838 0.31490 0.26742 1990 0.81574 0.90808 0.22520 1.00000 0.85097 0.42111 0.74077 0.78907 1995 0.78905 0.90569 0.48945 0.85097 1.00000 0.56697 0.77087 0.77156 2000 0.50197 0.39818 0.76838 0.42111 0.56697 1.00000 0.52801 0.38538 2005 0.86966 0.78649 0.31490 0.74077 0.77087 0.52801 1.00000 0.65563 2010 0.64845 0.80757 0.26742 0.78907 0.77156 0.38538 0.65563 1.00000 Table IV Correlation matrix for temperature layers. An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA Correlation matrix for temperature layers. An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA 2133 Table IV (continuation) CORRELATION MATRIX (Temperature-April) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.97750 0.97998 0.97378 0.97661 0.96197 0.96881 0.94823 1980 0.97750 1.00000 0.98631 0.98860 0.99361 0.96976 0.98274 0.97421 1985 0.97998 0.98631 1.00000 0.98679 0.98503 0.97772 0.98419 0.96170 1990 0.97378 0.98860 0.98679 1.00000 0.99253 0.98757 0.99332 0.98398 1995 0.97661 0.99361 0.98503 0.99253 1.00000 0.97755 0.98891 0.97271 2000 0.96197 0.96976 0.97772 0.98757 0.97755 1.00000 0.98612 0.96112 2005 0.96881 0.98274 0.98419 0.99332 0.98891 0.98612 1.00000 0.97864 2010 0.94823 0.97421 0.96170 0.98398 0.97271 0.96112 0.97864 1.00000 CORRELATION MATRIX (Temperature-May) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.98754 0.99057 0.97503 0.96946 0.98393 0.98151 0.97436 1980 0.98754 1.00000 0.99004 0.97957 0.98300 0.98179 0.98569 0.97928 1985 0.99057 0.99004 1.00000 0.97169 0.98487 0.97795 0.97774 0.97943 1990 0.97503 0.97957 0.97169 1.00000 0.97585 0.99414 0.98976 0.96744 1995 0.96946 0.98300 0.98487 0.97585 1.00000 0.97536 0.97691 0.97110 2000 0.98393 0.98179 0.97795 0.99414 0.97536 1.00000 0.98905 0.97046 2005 0.98151 0.98569 0.97774 0.98976 0.97691 0.98905 1.00000 0.96891 2010 0.97436 0.97928 0.97943 0.96744 0.97110 0.97046 0.96891 1.00000 CORRELATION MATRIX (Temperature-June) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.95346 0.98094 0.97460 0.97601 0.97138 0.96753 0.97943 1980 0.95346 1.00000 0.98204 0.98186 0.95395 0.97538 0.98243 0.94961 1985 0.98094 0.98204 1.00000 0.99084 0.97684 0.99073 0.99018 0.97364 1990 0.97460 0.98186 0.99084 1.00000 0.98152 0.98635 0.98838 0.97215 1995 0.97601 0.95395 0.97684 0.98152 1.00000 0.96700 0.96597 0.95207 2000 0.97138 0.97538 0.99073 0.98635 0.96700 1.00000 0.98993 0.96894 2005 0.96753 0.98243 0.99018 0.98838 0.96597 0.98993 1.00000 0.96989 2010 0.97943 0.94961 0.97364 0.97215 0.95207 0.96894 0.96989 1.00000 CORRELATION MATRIX (Temperature-July) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.97647 0.98897 0.99041 0.97678 0.98756 0.98871 0.97928 1980 0.97647 1.00000 0.96587 0.97782 0.93391 0.96840 0.96744 0.95022 1985 0.98897 0.96587 1.00000 0.98248 0.98509 0.97307 0.98407 0.97415 1990 0.99041 0.97782 0.98248 1.00000 0.95961 0.98519 0.98069 0.97109 1995 0.97678 0.93391 0.98509 0.95961 1.00000 0.96299 0.98156 0.96335 2000 0.98756 0.96840 0.97307 0.98519 0.96299 1.00000 0.98577 0.96678 2005 0.98871 0.96744 0.98407 0.98069 0.98156 0.98577 1.00000 0.96754 2010 0.97928 0.95022 0.97415 0.97109 0.96335 0.96678 0.96754 1.00000 CORRELATION MATRIX (Temperature-August) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.98710 0.98710 0.98982 0.98119 0.98817 0.98214 0.96580 1980 0.98710 1.00000 0.98525 0.98710 0.97927 0.98774 0.97624 0.95483 1985 0.98710 0.98525 1.00000 0.98983 0.98460 0.99000 0.98436 0.95388 1990 0.98982 0.98710 0.98983 1.00000 0.98274 0.99293 0.98298 0.96703 1995 0.98119 0.97927 0.98460 0.98274 1.00000 0.98346 0.98776 0.94175 2000 0.98817 0.98774 0.99000 0.99293 0.98346 1.00000 0.99239 0.97248 2005 0.98214 0.97624 0.98436 0.98298 0.98776 0.99239 1.00000 0.96056 2010 0.96580 0.95483 0.95388 0.96703 0.94175 0.97248 0.96056 1.00000 Table IV (continuation) An Acad Bras Cienc (2016) 88 (4) Derya Ozturk and Fatmagul Kilic 2134 (72 for precipitation and 72 for temperature). GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA Figures 6 and 7 show the cell statistics of the precipitation and CORRELATION MATRIX (Temperature-September) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.98432 0.98128 0.98609 0.97975 0.98318 0.98589 0.99125 1980 0.98432 1.00000 0.99155 0.98712 0.98378 0.98371 0.97888 0.97036 1985 0.98128 0.99155 1.00000 0.98127 0.97791 0.98163 0.97309 0.96714 1990 0.98609 0.98712 0.98127 1.00000 0.98041 0.97355 0.97007 0.98024 1995 0.97975 0.98378 0.97791 0.98041 1.00000 0.99027 0.98815 0.96733 2000 0.98318 0.98371 0.98163 0.97355 0.99027 1.00000 0.99254 0.97132 2005 0.98589 0.97888 0.97309 0.97007 0.98815 0.99254 1.00000 0.97438 2010 0.99125 0.97036 0.96714 0.98024 0.96733 0.97132 0.97438 1.00000 CORRELATION MATRIX (Temperature-October) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.98886 0.99071 0.98472 0.99004 0.99062 0.99542 0.98544 1980 0.98886 1.00000 0.97972 0.98269 0.98005 0.98243 0.98307 0.97214 1985 0.99071 0.97972 1.00000 0.98881 0.99341 0.99234 0.98544 0.97534 1990 0.98472 0.98269 0.98881 1.00000 0.98608 0.98709 0.97632 0.97474 1995 0.99004 0.98005 0.99341 0.98608 1.00000 0.99196 0.98567 0.97342 2000 0.99062 0.98243 0.99234 0.98709 0.99196 1.00000 0.98698 0.98191 2005 0.99542 0.98307 0.98544 0.97632 0.98567 0.98698 1.00000 0.98408 2010 0.98544 0.97214 0.97534 0.97474 0.97342 0.98191 0.98408 1.00000 CORRELATION MATRIX (Temperature-November) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.98838 0.99165 0.97041 0.98973 0.96630 0.99328 0.94459 1980 0.98838 1.00000 0.98876 0.98825 0.97959 0.97029 0.98830 0.96251 1985 0.99165 0.98876 1.00000 0.97865 0.98934 0.97822 0.98729 0.95602 1990 0.97041 0.98825 0.97865 1.00000 0.95958 0.97309 0.97731 0.97502 1995 0.98973 0.97959 0.98934 0.95958 1.00000 0.96471 0.98812 0.93409 2000 0.96630 0.97029 0.97822 0.97309 0.96471 1.00000 0.96795 0.96403 2005 0.99328 0.98830 0.98729 0.97731 0.98812 0.96795 1.00000 0.95475 2010 0.94459 0.96251 0.95602 0.97502 0.93409 0.96403 0.95475 1.00000 CORRELATION MATRIX (Temperature-December) Layer(Year) 1975 1980 1985 1990 1995 2000 2005 2010 1975 1.00000 0.98203 0.98128 0.98008 0.98588 0.98097 0.96599 0.96537 1980 0.98203 1.00000 0.99205 0.99665 0.98918 0.99443 0.99171 0.97762 1985 0.98128 0.99205 1.00000 0.98926 0.98818 0.99459 0.98883 0.98108 1990 0.98008 0.99665 0.98926 1.00000 0.98819 0.99248 0.99313 0.98019 1995 0.98588 0.98918 0.98818 0.98819 1.00000 0.98769 0.98406 0.98327 2000 0.98097 0.99443 0.99459 0.99248 0.98769 1.00000 0.98867 0.97993 2005 0.96599 0.99171 0.98883 0.99313 0.98406 0.98867 1.00000 0.98158 2010 0.96537 0.97762 0.98108 0.98019 0.98327 0.97993 0.98158 1.00000 Table IV (continuation) Table IV (continuation) Table IV (continuation) (72 for precipitation and 72 for temperature). Figures 6 and 7 show the cell statistics of the precipitation and temperature for the month of January. f Here, range layers give the most important information. GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA Range layers indicate the difference between the largest and the smallest value of the inputs. When examining the range layers, it was understood that the month with largest changes is January for precipitation and November for temperature. An Acad Bras Cienc (2016) 88 (4) GEOSTATISTICAL INTERPOLATION OF METEOROLOGICAL DATA 2135 Figure 6 - Cell statistics of the precipitation for the month of January. Figure 7 - Cell statistics of the temperature for the month of January. Figure 6 - Cell statistics of the precipitation for the month of January. Figure 6 - Cell statistics of the precipitation for the month of January. Figure 6 - Cell statistics of the precipitation for the month of January. Figure 7 - Cell statistics of the temperature for the month of January. Figure 7 - Cell statistics of the temperature for the month of January. CONCLUSIONS Meteorological data are required in many fields such as environment, agriculture and management of natural disasters where spatial data are used. But meteorological data are generally available from a limited number of stations. For this reason, interpolation techniques are used to obtain complete surface information. In recent years, depending on the technological developments in computer and GIS, geostatistical methods are used in order to determine the spatial distribution of meteorological data and the Ordinary Kriging method is nowadays a preferable option in the literature. Unlike the deterministic methods, geostatistical interpolation techniques also utilize the statistical properties of the measured points. In geostatistical techniques the autocorrelation among the measured points is determined and spatial configuration of the sampling points around the estimation point is taken into consideration. In this study, spatial distributions of precipitation and temperature of the Aegean Region in Turkey for years 1975, 1980, 1985, 1990, 1995, 2000, 2005 and 2010 in 5-year periods were determined by the Ordinary An Acad Bras Cienc (2016) 88 (4) Derya Ozturk and Fatmagul Kilic 2136 Kriging method. The time series of monthly precipitation and temperature data from 98 meteorological stations were used for the Ordinary Kriging. To evaluate and interpret the results, multivariate statistics (band collection) and cell statistics were applied for the monthly precipitation and temperature layer series. The results revealed that a significant change in precipitation regime in the Aegean Region was occurred. 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https://openalex.org/W2512095835	https://diglib.uibk.ac.at/ulbtirolfodok/content/titleinfo/4523640/full.pdf	English	null	Implications of atmospheric conditions for analysis of surface temperature variability derived from landscape-scale thermography	International journal of biometeorology	2,016	cc-by	10,206	Implications of atmospheric conditions for analysis of surface temperature variability derived from landscape-scale thermography Albin Hammerle1 & Fred Meier2 & Michael Heinl1 & Angelika Egger1 & Georg Leitinger1 Received: 18 September 2015 /Revised: 8 August 2016 /Accepted: 8 August 2016 /Published online: 25 August 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Thermal infrared (TIR) cameras perfectly bridge the gap between (i) on-site measurements of land surface tempera- ture (LST) providing high temporal resolution at the cost of low spatial coverage and (ii) remotely sensed data from satellites that provide high spatial coverage at relatively low spatio- temporal resolution. While LST data from satellite (LSTsat) and airborne platforms are routinely corrected for atmospheric effects, such corrections are barely applied for LST from ground-based TIR imagery (using TIR cameras; LSTcam). We show the consequences of neglecting atmospheric effects on LSTcam of different vegetated surfaces at landscape scale. We compare LST measured from different platforms, focusing on the comparison of LST data from on-site radiometry (LSTosr) and LSTcam using a commercially available TIR camera in the region of Bozen/Bolzano (Italy). Given a digital elevation mod- el and measured vertical air temperature profiles, we developed a multiple linear regression model to correct LSTcam data for atmospheric influences. We could show the distinct effect of atmospheric conditions and related radiative processes along the measurement path on LSTcam, proving the necessity to cor- rect LSTcam data on landscape scale, despite their relatively low measurement distances compared to remotely sensed data. Corrected LSTcam data revealed the dampening effect of the atmosphere, especially at high temperature differences between the atmosphere and the vegetated surface. Not correcting for these effects leads to erroneous LST estimates, in particular to an underestimation of the heterogeneity in LST, both in time and space. In the most pronounced case, we found a tempera- ture range extension of almost 10 K. Keywords Surface temperature . Thermal infrared camera . Atmospheric correction . Digital elevation model . Alpine environment Int J Biometeorol (2017) 61:575–588 DOI 10.1007/s00484-016-1234-8 Int J Biometeorol (2017) 61:575–588 DOI 10.1007/s00484-016-1234-8 ORIGINAL PAPER * Albin Hammerle albin.hammerle@uibk.ac.at 1 University of Innsbruck, Innsbruck, Austria 2 Departement of Ecology, Technische Universtität Berlin, Berlin, Germany Introduction Land surface temperature (LST) is a key variable for numer- ous environmental functions. It represents the combined result of all energy exchange processes between the atmosphere and the land surface. Thus, LST has become a basic requirement for model validation or model constraining in surface energy and water budget modelling on various scales (Kalma et al. 2008; Kustas and Anderson 2009; and references therein). It serves as a metric for soil moisture and vegetation condition in eco/hydrological modelling and environmental monitoring (Czajkowski et al. 2000; Kustas and Anderson 2009) and has been used in the area of thermal anomalies and high- temperature events detection (Sobrino et al. 2009; Teuling et al. 2010). Further, LST data is widely used in urban climate studies to quantify the surface urban heat island and to explore its relationship with urban surface properties and air tempera- ture variability as well as for surface-atmosphere exchange processes in urban environments (Voogt and Oke 2003; Weng 2009). LST can be retrieved from various platforms and instru- ments, depending on the application requirements regarding spatial and temporal resolution. Remote sensing platforms provide data with global coverage. They can routinely either provide LST at a coarse spatial resolution at relatively high overpass frequencies (e.g., Terra-MODIS, Aqua-MODIS, NOAA-AVHRR) or provide less frequent but moderate * Albin Hammerle albin.hammerle@uibk.ac.at Int J Biometeorol (2017) 61:575–588 576 justified by relatively short atmospheric path lengths. Existing methods for ground-based TIR imagery are either simple, i.e., based on the assumption of a homogenous sensor-target distance and constant atmospheric transmission value (Yang and Li 2009), or more complex by using a radi- ative transfer code, atmospheric data and under consideration of differences in atmospheric path lengths (Meier and Scherer 2012; Meier et al. 2011; Sugawara et al. 2001). resolution LST data (e.g., Terra-ASTER, Landsat). Recent developments in the thermal remote sensing system even show a trend towards coarser spatial resolutions (e.g., Sentinel mission). Airborne systems on the other hand can provide relatively high temporal as well as high spatial reso- lution LST information on a regional scale, with the drawback of high costs. Infrared radiometers mounted on site provide LSTat any temporal resolution integrated over a given field of view on the expense of spatial coverage. This paper compares LST data measured from different platforms. Introduction The main objective is to quantify the differences between LST data from a ground-based TIR imagery (LSTcam) and LST data from on-site radiometry (LSTosr). Subsequently, an empirical model, based on a digital elevation model and measured vertical air temperature profiles, was developed. This model corrects LSTcam for atmospheric influences. Thermal infrared (TIR) cameras have been continuously refined since their broad commercial launch in the early 1990s and have found wide application since the 2000s due to lower costs for uncooled focal plane sensor arrays and their improved spatial and thermal resolution (Schuster and Kolobrodov 2004). The high spatial and temporal resolution, the operational simplicity, and increasing data storage capabil- ities led to an increasing popularity of this system in many ecological research areas (e.g., Hristov et al. 2008; Katra et al. 2007; McCafferty 2007). Furthermore, we discuss the consequences of neglecting atmospheric influences on LST data derived from ground- based TIR imagery at the landscape scale. While thermal remote sensing has already been widely ap- plied in landscape ecology (Quattrochi and Luvall 1999 and references therein), the demand for high-resolution data (both, temporally and spatially) is unabated. Particularly in alpine landscapes that are characterized by high spatial heterogeneity and temporal dynamics (resulting from small-scale variations in slope, aspect, and altitude), highly resolved LST data are needed (Bertoldi et al. 2010; Heinl et al. 2012; Scherrer and Körner 2010; Scherrer et al. 2011). Methods The basis of the study was the comparison of surface temper- atures measured (i) continuously by infrared radiometers mounted above the canopy (on-site radiometry), (ii) frequent- ly by a TIR camera operated at an elevated position within the study region (ground-based TIR imagery), and (iii) by satellite remote sensing (satellite-based TIR imagery). All thermal remote sensing data, independent of the instru- ment used, is influenced by atmospheric conditions and radi- ative processes along the measurement path (Chandrasekhar 1960). Several atmospheric correction approaches have been established depending on sensor characteristics, e.g., the split window technique (SWT) for multi-channel sensors (Becker and Li 1990; Kerr et al. 1992; Price 1984; Sobrino et al. 1991), where Bsplit window^ refers to radiance differences observed by each atmospheric window of the respective TIR channel. There are different SWT algorithms depending upon spectral emissivity, water vapor content, view angle, or purely empir- ical algorithms. Radiative transfer models together with atmo- spheric profile data of pressure, temperature, and humidity are often used to determine SWT algorithms or to perform atmo- spheric corrections of TIR data derived from single-channel sensors (Berk et al. 1998; Richter and Schläpfer 2002; Schmugge et al. 1998). While these methods are commonly applied to data derived from satellite (Dash et al. 2002; Prata et al. 1995) or airborne platforms (Jacob et al. 2003; Lagouarde et al. 2000; Lagouarde et al. 2004), such correc- tions are not routinely applied in ground-based TIR imagery in natural and urban environments at the landscape scale (Heinl et al. 2012; Scherrer and Körner 2010; Scherrer and Körner 2011; Scherrer et al. 2011; Tonolla et al. 2010; Wawrzyniak et al. 2013; Westermann et al. 2011), partially Study region and experimental setup Numbers denote locations of on-site measurements and corresponding numbers refer to site numbers in Tables 1, 2, and 4. Locations of ground-based TIR imagery and of the microwave radiometer are marked with X and O, M003; onset; Bourne, MA, USA) above the canopy, soil tem- perature (Tsoil) at 0.1 and 0.25 m soil depth (PT 100; EMS; Brno, Czech Republic), and soil water content (SWC) in 0.25 m soil depth (EC-10; Decagon Devices; Pullman, WA, USA). Surface temperatures were derived using an infrared radiometer (SI-111; Apogee Instruments; Logan, UT, USA) mounted 1 m above the canopy. This sensor is sensitive in the electromagnetic spectrum from 8 to 14 μm. Given the half- angle field of view of 22° and the different canopy heights, the visible surface areas ranged from 2 to 8 m2. Data were mea- sured every minute and stored as 10 min average values. Land surface temperatures (LST) derived from on-site radiometry are henceforth referred to as LSTosr. ASCII files and further analyzed using MATLAB (R2013b, The MathWorks, Inc., USA). Despite the mean absolute dif- ferences between LSTosr and LSTcam (0.8 K) being lower than the TIR camera accuracy (±1.5 K), the two systems were intercalibrated in an experimental setup. LST measured by the ground-based TIR imagery are referred to as LSTcam. Surface emissivity (ε) was considered equal to 1 for both LSTosr and LSTcam unless specified differently, as pixels of interest were completely covered by vegetation having a high emissivity at all wavelengths. Satellite-based TIR imagery was derived from ASTER Level 2B03 data products with a spatial resolution (pixel size) of 90 m, acquired on demand for seven dates in 2012 (21 and 28 June 2012; 7 July 2012; 8 and 24 August 2012; 11 and 18 October 2012). The images provide kinetic temperatures at about 11:15 CET and represent the single pixel values at the location of each microclimate station. The standard deviation is calculated over this target pixel and the eight neighboring pixels. Data affected by clouds were not considered for the analyses so that the number of remotely sensed data per site ranges between three and seven observations. LST derived from remote sensing are henceforth referred to as LSTsat. For ground-based TIR imagery, an elevated site on top of a cliff edge (1077 m a.s.l.) was chosen as camera position (Table 1). Study region and experimental setup The study was conducted in the region of Bozen/Bolzano in the northernmost part of Italy (Fig. 1). The city of Bozen/ Bolzano is located in a basin at the transition of the central Alps to the southern Alps, surrounded by four mountain ranges. Ten microclimate stations were erected in the vicinity of the city which spanned an elevational range from 239 to 857 m a.s.l. and covered three different land-use types (vine- yard, orchard, and grassland). These three land-use types cover 16, 29, and 4 % of the investigated rural area, respectively (woodland 48 %). Three out of ten microclimate stations were located in vineyards, six in orchards, and one in a managed grassland. While vineyards and orchards are by far the dominating land-use types in this region, grasslands only occurred at higher elevations (Table 1). No site was positioned closer than 20 m to any building. Meteorological measurements included air temperature (Tair) and relative humidity (RH) at 2 m above ground (Hobo Pro v2-U23-002; onset; Bourne, MA, USA), air tem- perature 1 m above the canopy (PT 100; EMS; Brno, Czech Republic), incoming solar radiation (SR) (S-LIB- Int J Biometeorol (2017) 61:575–588 577 Fig. 1 Study area in the basin of Bozen/Bolzano (I). Numbers denote locations of on-site measurements and corresponding numbers refer to site numbers in Tables 1, 2, and 4. Locations of ground-based TIR imagery and of the microwave radiometer are marked with X and O, Fig. 1 Study area in the basin of Bozen/Bolzano (I). Numbers denote locations of on-site measurements and corresponding numbers refer to site numbers in Tables 1, 2, and 4. Locations of ground-based TIR imagery and of the microwave radiometer are marked with X and O, respectively. The tetragon within the figure represents the transformed marked section in Fig. 7 and Fig. 8 (black squares). Inset upper left: schematic overview of the experimental setup. Map data: Google, DigitalGlobe respectively. The tetragon within the figure represents the transformed marked section in Fig. 7 and Fig. 8 (black squares). Inset upper left: schematic overview of the experimental setup. Map data: Google, DigitalGlobe respectively. The tetragon within the figure represents the transformed marked section in Fig. 7 and Fig. 8 (black squares). Inset upper left: schematic overview of the experimental setup. Map data: Google, DigitalGlobe Fig. 1 Study area in the basin of Bozen/Bolzano (I). Processing of ground-based TIR imagery To cover all field sites by ground-based TIR imagery at one time, we had to pan the camera and take five TIR images (scenes). While we always tried to position the camera the same way and choose the same field of view, the different scenes were not perfectly congruent. Thus, we chose one ref- erence thermal image per scene and used the BComputer Vision System Toolbox^ of MATLAB (R2013b, The MathWorks, Inc., USA) to align all TIR images of one scene with each other. More precisely, we (i) used the SURF blob detector (detectSURFFeaturs-function; Bay et al. 2008) to identify matching regions in the two TIR images, (ii) estimat- ed the geometric transformation from matching point pairs (estimateGeometricTransform-function; Torr and Zisserman 2000), and (iii) applied the geometric transformation to the TIR image (imwrap-function). Subsequently transformed TIR images (n = 3169) were filtered based on a three-step quality check. (i) All images obviously not matching the corresponding reference TIR im- age by visual inspection were selected and removed (remain- ing n = 2909; 92 %). (ii) Any TIR image not exceeding a certain R2 value (night 0.6; day 0.8), when compared with the reference scene or with less than five matching points found in the SURF blob detector algorithm described above were removed (remaining n = 2106; 66 %). (iii) For any av- eraging interval with multiple LSTcam measurements, only the one closest in time to the LSTosr measurement was used, fur- ther reducing the number of remaining LSTcam measurements for the ten sites (remaining n = 2011; 63 %). Applying these algorithms and filters resulted in a dataset of TIR image per scene that perfectly matched each other. In order to get the line-of-sight geometry for each TIR image pixel, the proce- dure described in the following section was applied. Study region and experimental setup Measurements were done using the TIR camera BJenoptik VarioCAM high resolution^ (Infratec; Dresden, Germany), which is sensitive in the electromagnetic spectrum from 7.5 to 14 μm. The camera resolution of 768 × 576 pixels in combination with the standard lens (focal length 25 mm) resulted in pixel sizes ranging from 2.2 to 6.3 m depending on the given atmospheric path length (APL) per site (Table 1). TIR images were taken on 13 days throughout the summer and autumn season 2012 from an exposed position ca. 840 m above the valley floor. While data were restricted to daytime measurements on some days, we conducted 24-h measure- ments on others. Measurements were done at least half hourly (higher frequency around sunrise and sunset or at the times of a satellite overpass), resulting in roughly 250 acquisition times where all ten LSTosr sites were covered simultaneously. Image processing was done using IRBIS® software (InfraTec; Dresden, Germany). All TIR images were exported as A vertical air temperature profile was measured at the airport in Bozen/Bolzano (BZO) using a microwave ra- diometer (MTP-5HE; ATTEX Ltd., Moscow, Russia) (Fig. 1). This radiometer measured air temperature pro- files up to 1000 m above surface (50 m vertical resolu- tion; 10 min time resolution) with a temperature accuracy from ±0.3 K (0–500 m) up to ±0.4 K (>500 m). Radiometer data were provided by BAutonome Provinz Bozen Südtirol/Provincia autonoma die Bolzano Alto Adige^ (Landesagentur für Umwelt/Agenzia provinciale Int J Biometeorol (2017) 61:575–588 578 Table 1 Key characteristics of investigated field sites including measures of their positioning relating to the camera position for ground-based TIR imagery (Cam) Number in Fig. 1 site name Management x_GPS y_GPS Altitude (m a. s. Study region and experimental setup l.) Hillslope (°) Aspect (°) Situation: cam position-site Atmospheric path length (APL) (m) Horizontal distance (m) Altitude difference (m) Cutting angle (AC) (°) View zenith angle (AVZ) (°) 1 Schreckbichl Vineyard 11.295882 46.460708 474 11.0 63 4624 4584 603 18.3 82.5 2 Girlan Orchard 11.279210 46.470331 400 3.5 340 5759 5719 678 5.7 83.2 3 Unterrain Orchard 11.279432 46.488236 240 <3 124 6072 6014 837 8.0 82.1 4 Terlan Orchard 11.252084 46.511405 243 <3 243 9038 8999 834 5.3 84.7 5 Kaiserau Orchard 11.313778 46.477665 245 <3 323 3260 3152 833 13.7 75.2 6 Jennerhof Orchard 11.315806 46.486065 245 <3 240 3457 3355 832 13.3 76.1 7 Moritzing Orchard 11.299137 46.498861 239 <3 20 5275 5208 838 9.1 80.9 8 Alte Mendl Str Vineyard 11.329031 46.496685 250 <3 192 3523 3424 827 13.8 76.4 9 Glaninger Weg Vineyard 11.334097 46.509057 430 21.6 164 4535 4488 647 29.7 81.8 10 Wiesmanhof Grassland 11.339721 46.524530 857 9.3 149 6049 6045 221 10.8 87.9 X Kohlern – 11.353672 46.471006 1077 – – – – – – – per l’ambiente; Labor für physikalische Chemie/Laboratorio di chimica fisica). Average path temperatures (Tpath) were cal- culated for each LSTcam measurement as the arithmetic mean over the corresponding temperature profile segment, defined by the site and camera elevation. Results Data were collected continuously at the field sites from 1 May 2012 until 31 October 2012. Tair ranged from −5.2 °C (30 October 2012) to 37.3 °C (20 August 2012), with a mean Tair of 18.8 °C during that period (30-year average for that period, 19.4 °C (Hydrographisches Amt Bozen/Ufficio idrografico Bolzano)). While the coolest site on average was the Wiesmanhof site, representing the highest-located site (Fig. 1, Table 1), the lowest air temperatures were measured at the Terlan site. The highest air temperature was measured at the Alte Mendel Strasse site, a site located in close proximity to Bozen/Bolzano (Fig. 1, Table 2). LSTosr ranged from −5.7 °C (30 October 2012; Terlan) to 49.1 °C (26 July 2012; Wiesmanhof), with an average LSTosr of 18.0 °C during the measurement period (Table 2). Average wind speed ranged from 0.8 m s−1 (Terlan site) up to 1.5 m s−1 (Wiesmanhof), and mean solar radiation (SR) ranged from 196 W m−2 (Girlan) to 226 W m−2 (Glaninger Weg) among the ten field sites (Table 2). Multiple regression model to correct LSTcam Establishing the multiple regression model was done using IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp; Armonk, NY). The LST model was built using ordinary least squares (OLS) regression based on 1839 observations with LSTosr as dependent variable and four independent variables (LSTcam; Tpath; difference of LSTcam and Tpath; APL). (i) All independent variables were tested regarding multi-collineari- ty, (ii) scatter plots of the dependent vs. each independent variable were analyzed to check for non-linearity, (iii) the significant independent variables were selected by forced en- try OLS using ca. 50 % of available data (897 observations; calibration dataset), (iv) a residual analysis was performed for checking OLS assumptions, and finally (v) the LST model was employed to predict the dependent variable for the re- maining validation dataset (942 observations; validation dataset). Derivation of line-of-sight geometry parameters for ground-based TIR imagery The oblique view of the TIR camera and the topography of the observed landscape produce different line-of-sight (LOS) ge- ometry parameters for each TIR image pixel. The LOS is fully described by APL, by the altitude of the observed surface, and by the view zenith angle (AVZ) under which the TIR camera observes the surface. The calculation of spatially distributed LOS values for every TIR image pixel is based on the idea that every TIR image pixel has a corresponding 3D geographic coordinate (x, y, z). In order to find these pixel-specific Int J Biometeorol (2017) 61:575–588 579 coordinates, the perspective projection of the three- dimensional (3-D) landscape onto the two-dimensional (2- D) TIR image plane was modelled using a DEM of the study region with a spatial resolution of 20 m (Autonomous Province of Bolzano, South Tyrol, Italy). Further, we had to know the geographic coordinates of the TIR camera location and the geographic coordinates of the center pixel of the TIR image (exterior orientation) as well as the size of the 2-D image plane (768 × 576 pixels) and the horizontal and vertical field of view (FOV) of the camera lens (interior orientation). The horizontal FOV is 30° and the vertical FOV is 23°. A detailed description of the perspective projection of the 3-D DEM and calculation of FOV parameters are given in Meier et al. (2011). Results Tair air temperature 2 m above ground, LSTosr land surface temperature from on-site radiometry, SR shortwave radiation Numbers (nr.) refer to numbers in Fig. 1 T i air temperature 2 m above ground LST land surface temperature from on-site radiometry SR shortwave radiation Numbers (nr.) refer to numbers in Fig. 1 Numbers (nr.) refer to numbers in Fig. 1 T i t t 2 b d LST l d f t t f it di t SR h t di ti ) g erature 2 m above ground, LSTosr land surface temperature from on-site radiometry, SR shortwave radiation LSTcam vs. LSTosr Radiant LSTcam and LSTosr were well correlated at nine out of our ten sites (Fig. 3). As mentioned in the pre- vious paragraph, the Wiesmanhof field site was exclud- ed from any further analyses. Slope and offset of the regression lines ranged from 0.69 to 0.92 and −0.81 to 5.94 K, respectively (Fig. 3). The coefficient of deter- mination (R2) and the MAE ranged from 0.82 to 0.95 and 1.51 to 3.63 K, respectively, with an average MAE of 2.61 K (Fig. 3). This intercomparison was done using original LSTcam data, not corrected for any atmospheric influences. Generally, a good correlation of LSTosr (kin) and LSTsat (kin) could be found applying these transformations, whereas there is a distinct outlier datum in the Wiesmanhof dataset (Fig. 2, left panel). In contrast, comparing LSTcam (kin) with LSTsat (kin) did not reveal any outliers for the Wiesmanhof data (Fig. 2, right panel). But, as evident from Fig. 2, LSTcam (kin) and LSTsat (kin) estimates are clearly offset and show a higher mean absolute error (MAE) compared to LSTosr (kin) data. As shown in Fig. 3, LSTcam are lower on average in all cases compared to LSTosr, especially at higher temperatures, clearly indicating the necessity to account for atmospheric effects on LST measurements at landscape scales by TIR cameras. The Wiesmanhof site was excluded in any further analysis because of the following: (i) LSTosr (kin) does not always coincide with LSTsat (kin) at the Wiesmanhof site, while this site does not stand out when comparing LSTsat (kin) with LSTcam (kin) data; (ii) in nine out of ten cases LSTosr (kin) and LSTcam (kin) data are well correlated (except for the Wiesmanhof site at high temperatures) (Fig. 3); and (iii) pho- tographs of the measured plot at Wiesmanhof (taken regularly at times of data collection or maintenance work; not shown) showed withered vegetation right below the sensor during periods with high air temperatures (end of July and around At all sites, uncorrected LSTcam is on average be- tween 1.19 and 3.52 K lower than LSTosr. While these average deviations appear to be rather small, the differ- ences between LSTcam and LSTosr show a pronounced diel cycle. The observed differences between these two methods (ΔLST = LSTosr −LSTcam) ranged from −3.9 up to 11.5 K at the maximum. On average, ΔLST was negative during the night time hours, ranging between −3 and −1 K. LSTsat compared to LSTcam and LSTosr During the measurement campaign, LSTsat could be retrieved from seven satellite overpasses. Excluding data with cloud cover, 58 data points could be used from our ten field sites to compare LSTsat with LSTosr and 32 data points to compare LSTsat and LSTcam. LSTsat data are available as kinetic (kin) LST (routinely corrected for atmospheric effects); thus, LSTosr and LSTcam data had to be recalculated from radiant tempera- ture by applying ε = 0.97 (deciduous vegetation and grass; Jensen 2007) and an environmental temperature (Tsky; K) that was modelled as a function of vapor pressure (ea; kPa) and air l conditions at the ten field sites throughout the measurement campaign 1 May 2012 until 31 October 2012 Table 2 Meteorological conditions at the ten field sites throughout the measurement campaign 1 May 2012 until 31 October 2012 Site Tair 200 cm (°C) LSTosr (°C) SR (W m−2) Wind speed (m s−1) Nr. Name Min Max Mean Min Max Mean Mean Mean 1 Schreckbichl −1.5 35.9 18.6 −3.1 38.6 17.9 206 1.4 2 Girlan −2.6 35.9 18.2 −2.6 37.8 17.2 196 1.1 3 Unterrain −3.8 36.9 19.0 −4.4 33.3 17.6 210 1.1 4 Terlan −5.2 36.5 18.2 −5.7 33.6 17.4 201 0.8 5 Kaiserau −2.9 36.7 19.5 −3.3 39.1 18.3 202 1.0 6 Jennerhof −2.4 36.9 19.8 −3.1 36.1 18.7 205 0.8 7 Moritzing −3.7 37.0 19.2 −3.7 33.8 18.0 209 1.1 8 Alte Mendl Str −2.3 37.3 20.2 −3.1 40.2 19.4 196 0.9 9 Glaninger Weg −1.1 36.8 19.3 −2.9 43.0 18.8 226 1.3 10 Wiesmanhof −3.5 32.3 16.3 −5.0 49.1 16.8 213 1.5 Numbers (nr.) refer to numbers in Fig. 1 580 Int J Biometeorol (2017) 61:575–588 temperature (Tair; K) following Campbell and Norman (1998) (rearranged): the 20th of August) while no dryness was observed at the rest of the meadow (plot not representative). Tsky ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1:72⋅ ea Tair 1 7 ⋅T air ð Þ4 4 s LSTcam vs. LSTosr At sunrise, mean ΔLST rose, reached its Fig. 2 Left panel: intercomparison of kinetic land surface temperatures measured on site by radiometry (LSTosr (kin)) and from remote sensing (ASTER Level 2B03) (LSTsat (kin)). Right panel: intercomparison of kinetic land surface temperatures measured by ground-based TIR imagery (LSTcam (kin)) and from remote sensing (ASTER Level 2B03) (LSTsat (kin)). Red line: 1:1 line, black solid line: regression line, black Fig. 2 Left panel: intercomparison of kinetic land surface temperatures measured on site by radiometry (LSTosr (kin)) and from remote sensing (ASTER Level 2B03) (LSTsat (kin)). Right panel: intercomparison of kinetic land surface temperatures measured by ground-based TIR imagery (LSTcam (kin)) and from remote sensing (ASTER Level 2B03) (LSTsat (kin)). Red line: 1:1 line, black solid line: regression line, black dashed line: 95 % prediction interval of regression line, grey dashed line: 95 % prediction interval of observations, grey shaded area: ±1.5 K on 1:1 line marking camera accuracy. Error bars on LSTsat data refer to the standard deviation within a 3 × 3 pixel area centered around LSTosr locations. Error bars on LSTcam (kin) data refer to the camera accuracy of ±1.5 K dashed line: 95 % prediction interval of regression line, grey dashed line: 95 % prediction interval of observations, grey shaded area: ±1.5 K on 1:1 line marking camera accuracy. Error bars on LSTsat data refer to the standard deviation within a 3 × 3 pixel area centered around LSTosr locations. Error bars on LSTcam (kin) data refer to the camera accuracy of ±1.5 K Fig. 2 Left panel: intercomparison of kinetic land surface temperatures measured on site by radiometry (LSTosr (kin)) and from remote sensing (ASTER Level 2B03) (LSTsat (kin)). Right panel: intercomparison of kinetic land surface temperatures measured by ground-based TIR imagery (LSTcam (kin)) and from remote sensing (ASTER Level 2B03) (LSTsat (kin)). Red line: 1:1 line, black solid line: regression line, black dashed line: 95 % prediction interval of regression line, grey dashed line: 95 % prediction interval of observations, grey shaded area: ±1.5 K on 1:1 line marking camera accuracy. Error bars on LSTsat data refer to the standard deviation within a 3 × 3 pixel area centered around LSTosr locations. Error bars on LSTcam (kin) data refer to the camera accuracy of ±1.5 K Int J Biometeorol (2017) 61:575–588 581 Fig. LSTcam vs. LSTosr Big black dots refer to bin averaged data including their error bar (1 stdv) Upper panel: absolute difference; grey horizontal bar refers to ±1.5 K (camera accuracy). Lower panel: relative difference. Big black dots refer to bin averaged data including their error bar (1 stdv) Fig. 5 Correlations of the temperature difference between path temperature (Tpath) and land surface temperatures from on-site radiometry (LSTosr) and the measurement difference between LSTosr and land surface temperatures from ground TIR imagery (LSTcam) (ΔT) (grey dots). Statistical validation of the model was done applying the model to the remaining 50 % of observation data, which re- sulted in an adjusted R2 = 0.93 (LSTosr = 1.00 LSTosr predict- ed −0.19; RMSE = 1.68 K). values ranging from 0.91 to 1.00 and −0.18 to 3.34 K, respectively. R2 improved noticeably and ranged be- tween 0.98 and 0.99, and the MAE was reduced from 2.61 K on average for uncorrected data to a range of 0.49 to 1.15 K (mean 0.74 K) for the nine sites. Based on the available dataset (n = 1839) and the three selected independent variables LSTcam, LSTcam −Tpath, and APL, the LST model was given by: While this finding does show the importance of at- mospheric corrections on the data, this correlation is not relevant for any data correction as this method would require information on actual LST on landscape scale. LSTosr predicted ¼ −3:971 þ 1:086 LSTcam þ 0:767 LSTcam−Tpath þ 0:000469 APL; ð1Þ LSTosr predicted ¼ −3:971 þ 1:086 LSTcam þ 0:767 LSTcam−Tpath LST model calibration and validation þ 0:000469 APL; ð1Þ ð1Þ representing a highly significant model for LST (p < 0.001; adj. R2 = 0.93; RMSE = 1.70 K) (Fig. 6). In order to correct LST on landscape scale, a multiple linear regression model was set up to model LSTosr by the use of four independent variables (LSTcam, LSTcam −Tpath, Tpath, and APL). Given a variance inflation factor (VIF) well above ten indicating multi-collinearity, Tpath was excluded as an inde- pendent variable from further analysis. With VIFs lower than 1.33, none of the remaining three independent variables (LSTcam, LSTcam −Tpath, and APL) gave evidence for further multi-collinearity (Kutner et al. 2003; Pan and Jackson 2008; Rogerson 2001). Furthermore, no scatterplot of dependent vs. independent variables revealed non-linear dependencies. According to the standardized coefficients beta (~β ), LSTcam exerted the highest influence on the LST model, followed by the difference of LSTcam and Tpath (LSTcam −Tpath) and atmospheric path length (APL) (Table 3). LSTcam vs. LSTosr 3 Correlations of land surface temperatures measured by on-site radiometery (LSTosr) and ground-based TIR imagery (LSTcam) per site including correlation statistics. Grey dotted lines: 1:1 line; black bold lines: sls-regression line Fig. 3 Correlations of land surface temperatures measured by on-site radiometery (LSTosr) and ground-based TIR correlation statistics. Grey dotted lines: 1:1 line; black bold lines: sls-regression line Fig. 3 Correlations of land surface temperatures measured by on-site radiometery (LSTosr) and ground-based TIR imagery (LSTcam) per site including correlation statistics. Grey dotted lines: 1:1 line; black bold lines: sls-regression line maximum of 3.9 K around noon, and decreased again from then on (Fig. 4). could be explained to a very large extent. Eighty-one percent of the residual variation is explained by ΔT (n = 1839; p < 0.01) (Fig. 5). Given Tpath from radiometer measurements, we calcu- lated the difference between LSTosr and Tpath (ΔT). Given ΔT, the residuals between LSTcam and LSTosr Correcting LSTcam data according to this correlation of the residuals with ΔT does result in slope and offset Fig. 4 Upper panel: mean diel variations of land surface temperatures measured by on-site radiometry (LSTosr) and by ground-based TIR imagery (LSTcam), as well as path temperature (Tpath). Only data at times with LSTcam data available were used. Lower panel: mean diel variation of the differences between LSTosr and LSTcam as well as the differences between LSTosr and Tpath. Error bars refer to 1 stdv in any case. For reasons of clarity, error bars are shown for LSTcam data only in the upper panel Fig. 4 Upper panel: mean diel variations of land surface temperatures measured by on-site radiometry (LSTosr) and by ground-based TIR imagery (LSTcam), as well as path temperature (Tpath). Only data at times with LSTcam data available were used. Lower panel: mean diel variation of the differences between LSTosr and LSTcam as well as the differences between LSTosr and Tpath. Error bars refer to 1 stdv in any case. For reasons of clarity, error bars are shown for LSTcam data only in the upper panel 582 Int J Biometeorol (2017) 61:575–588 Fig. 5 Correlations of the temperature difference between path temperature (Tpath) and land surface temperatures from on-site radiometry (LSTosr) and the measurement difference between LSTosr and land surface temperatures from ground TIR imagery (LSTcam) (ΔT) (grey dots). Upper panel: absolute difference; grey horizontal bar refers to ±1.5 K (camera accuracy). Lower panel: relative difference. LST model application Average differences of LSTosr and Tpath during all measure- ment campaigns ranged from −6 to 10 K. To demonstrate consequences of these temperature differences, two differ- ent situations for one field of view were selected, including the stations Kaiserau, Jennerhof, Terlan, and Unterrain (scene 2). On 2 August 2012 at 11:30 CET, a mean differ- ence between LSTosr of these sites and Tpath of 6.7 K was observed (example 1), while on 24 August at 03:00 CET, Thethreeindependentvariablesgeneratedahighlysignificant model (p < 0.001) with a determination coefficient of 0.92 (ad- justed R2; root mean squared error (RMSE) = 1.7 K) based onca. 50 % randomly chosen observations (calibration dataset). Residual analysis revealed no noticeable pattern (heteroscedasticity) and no obvious deviation from normal distribution. Int J Biometeorol (2017) 61:575–588 583 Fig. 6 a Correlation of measured land surface temperatures from on-site radiometry (LSTosr) with modelled LST including the regression line. b Standardized residuals vs. unstandardized predicted values. c p-p-plot of observed (grey) vs expected (black) cumulative residual distribution Fig. 6 a Correlation of measured land surface temperatures from on-site radiometry (LSTosr) with modelled LST Standardized residuals vs. unstandardized predicted values. c p-p-plot of observed (grey) vs expected (black) cumulat Fig. 6 a Correlation of measured land surface temperatures from on-site radiometry (LSTosr) with modelled LST including the regression line. b Standardized residuals vs. unstandardized predicted values. c p-p-plot of observed (grey) vs expected (black) cumulative residual distribution these two temperatures differed by −2.5 K on average (ex- ample 2). These values represent rather high and low mea- sured differences for that scene. to areas covered by vegetation, as the model setup was done using data from such areas only. Results covering settlement or industrial areas (right and lower thermal image area, respectively) could thus not be validated. Presented in Table 4 are the meteorological conditions for the times of examples 1 and 2. Data presented in Table 4 represent average conditions for these specific dates of the year and times of the day. 2 August (example 1) was charac- terized by bright sunshine until the time of presented measure- ments, while on 24 August (example 2), it was partly cloudy around midday and clear sky conditions for the rest of the day. LST model application While LSTosr and Tair were relatively similar at the time of example 1, Tpath was several degrees cooler on average, with differences ranging from −6 down to −11 K (Table 4). In contrast, at the time of example 2, the average Tpath was 2.1 K warmer than the average LSTosr, with differences rang- ing from −0.2 up to 3.5 K. This application of the LST model on landscape scale clearly shows that correcting for atmospheric influences (i) amplifies the measured LST spectrum (for the pronounced case in Fig. 7, the LST range was extended by as much as 10 K for the marked section) and (ii) shifts median tempera- tures depending on the difference between Tpath and surface temperature. Discussion Various studies on LST have been conducted using ground- based TIR cameras on landscape scale. These instruments gained popularity in ecosystem research due to their high tem- poral and spatial resolution as well as their operational sim- plicity (Corsi 2010; Pron and Bissieux 2004). In this study, we Consequences of these conditions on LSTcam and ac- cording corrections on these data by the LST model at landscape scale are shown in Fig. 7 and Fig. 8. The marked section in panels a–f was used to restrict data Table 3 Three variables exhibited significance and were used in our final LST model LST model Unstandardized coefficients Standardized coefficients ~β T-value (t) Significance (p value, two sided) VIF βi S.E. (Constant) −3971 0.247 −16,060 0.000 LSTcam 1.086 0.008 0.909 142.350 0.000 1.086 LSTcam −Tpath 0.767 0.029 0.194 26.258 0.000 1.256 APL 0.000469 0.000 0.128 16.697 0.000 1.105 S.E. standard error, VIF variance inflation factor, LSTcam land surface temperatures measured by ground-based TIR imagery, Tpath measurement path temperature, APL atmopsheric path length Table 3 Three variables exhibited significance and were used in our final LST model S.E. standard error, VIF variance inflation factor, LSTcam land surface temperatures measured by ground-based TIR imagery, Tpath measurement path temperature, APL atmopsheric path length Int J Biometeorol (2017) 61:575–588 584 Table 4 Meteorological conditions on reference days 2 August 2012 11:30 (example 1; E1) and 24 August 2012 03:00 (example 2; E2) Site LSTosr (°C) Tair 2 m (°C) Tpath (°C) LSTcam (°C) SR (W m−2) Wind speed (m s−1) RH (%) Nr. Discussion Name E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 1 Schreckbichl 31.8 18.5 27.5 20.1 22.9 20.6 26.0 – 728 0 0.28 1.64 61 76 2 Girlan 30.1 17.7 28.4 20.0 23.1 20.7 25.5 20.6 – 0 1.24 0.68 57 77 3 Unterrain 29.3 19.4 32.1 20.7 23.5 20.9 26.5 21.0 728 0 1.00 0.52 60 80 4 Terlan 29.9 18.1 29.7 19.6 23.5 20.9 25.7 20.6 684 0 0.28 0.52 55 84 5 Kaiserau 29.6 17.4 30.7 18.2 23.5 20.9 27.6 20.5 756 0 0.84 0.28 53 95 6 Jennerhof 32.2 18.7 30.7 20.1 23.5 20.9 28.6 20.8 764 0 0.84 0.84 48 82 7 Moritzing 29.6 18.7 29.3 20.0 23.5 20.9 26.8 – 810 0 1.08 0.68 61 86 8 Alte Mendl Str 34.7 21.1 31.2 22.6 23.5 20.9 30.1 – 686 0 1.16 1.00 48 70 9 Glaninger Weg 32.6 18.7 28.3 20.7 23.0 20.6 28.7 – 828 0 1.48 0.76 49 75 Mean 31.1 18.7 29.8 20.2 23.4 20.8 27.3 20.7 762 0 0.91 0.77 55 81 Italicized sites are covered by scene 2 shown in Fig. 7 and Fig. 8. Numbers (nr.) refer to numbers in Fig. 1 for atmospheric effects, using our multiple linear regression model, LSTsat and LSTcam (kin) agreed reasonably well, re- ducing the MAE from 3.55 to 2.45 K (data not shown). Beside atmospheric effects, the offset could to some degree also be a result of thermal anisotropy, i.e., the LST depends on the viewing direction of the sensor (Christen et al. 2012; Kimes 1980; Lagouarde et al. 2000; Lagouarde et al. 2004; Voogt and Oke 2003). Under cloudless conditions, the satellite observes determined the magnitude of atmospheric effects on ground- based radiant surface temperature by comparison of LSTcam with LSTosr. Furthermore, we established a multiple linear regression model to correct LSTcam data and to show the ef- fects of Tpath and APL on LSTcam data. p While LSTosr and LSTsat data did show a good correlation, LSTcam (kin) data, not corrected for atmospheric effects, were clearly offset compared to LSTsat data (Fig. 2). Once corrected Fig. 7 Example 1 (2 August 2012, 11:30 CET)—a elevation model, as seen by ground-based TIR imagery. b Resulting atmospheric path lengths (APL) for each pixel. c Average path temperatures (Tpath) for the time the infrared images were taken. Discussion c Average path temperatures (Tpath) for the time the infrared thermograms were taken. d Land surface temperatures as measured by ground-based TIR imagery (LSTcam). e Resulting LSTcam from model application (LSTcam corr). f Difference between LSTcam and LSTcam corr. g Temperature ranges of LSTcam and LSTcam corr for the entire scene. h Temperature ranges of LSTcam and LSTcam corr for the marked section in panels a–f. Grey shadings in g and h refer to min–max range, 90 % percentile, 50 % percentile (IQR), and the median (black line), respectively. i Histogram of the differences in panel f for the entire scene predominantly sunlit surfaces because of the nadir view. The oblique view of the TIR camera includes more vertical sur- faces which could be cooler due to shading. analysis (i) choosing the pixel within these regions matching the LSTosr readings worst and (ii) choosing the one matching the LSTosr reading closest. Even between these two extreme scenarios, the mean difference in the RMSE comparing LSTosr with LSTcam was as low as 0.16 K (range 0.0–0.63 K) and differences in R2 were below 0.01 in any case. This low sen- sitivity on the exact pixel location can be attributed to the land cover in that region, which shows low variability at small scales due to the intensive pomiculture. The site being most sensitive to pixel localization was BAlte Mendel Strasse,^ which is located in close proximity to the settlement area of Bozen/Bolzano. Focusing on the LSTosr and LSTcam comparison, we found that LSTcam clearly deviate from LSTosr. ΔLST, the difference between LSTosr and LSTcam, showed a pronounced diel cycle, and ΔLST was negatively correlated (p < 0.001) with the dif- ference between Tpath and LSTosr, with Tpath being derived independently from microwave radiometer measurements. These findings show a strong influence of atmospheric prop- erties along the measuring path on the LSTcam measurements. Using a multiple linear regression model, we could show that the measured range of LSTcam data is amplified when account- ing for Tpath and mean LST are shifted either positively or negatively, depending on the temperature difference between LSTosr and Tpath. Discussion d Land surface temperatures as measured by ground-based TIR imagery (LSTcam). e Resulting LSTcam from model application (LSTcam corr). f Difference between LSTcam and LSTcam corr. g Temperature ranges of LSTcam and LSTcam corr for the entire scene. h Temperature ranges of LSTcam and LSTcam corr for the marked section in panels a–f. Grey shadings in g and h refer to min–max range, 90 % percentile, 50 % percentile (IQR), and the median (black line), respec- tively. i Histogram of the differences in panel f for the entire scene Fig. 7 Example 1 (2 August 2012, 11:30 CET)—a elevation model, as seen by ground-based TIR imagery. b Resulting atmospheric path lengths (APL) for each pixel. c Average path temperatures (Tpath) for the time the infrared images were taken. d Land surface temperatures as measured by ground-based TIR imagery (LSTcam). e Resulting LSTcam from model application (LSTcam corr). f Difference between LSTcam and LSTcam corr. g Temperature ranges of LSTcam and LSTcam corr for the entire scene. h Temperature ranges of LSTcam and LSTcam corr for the marked section in panels a–f. Grey shadings in g and h refer to min–max range, 90 % percentile, 50 % percentile (IQR), and the median (black line), respec- tively. i Histogram of the differences in panel f for the entire scene Int J Biometeorol (2017) 61:575–588 585 Fig. 8 Example 2 (24 August 2012, 03:00 CET)—a elevation model, as seen by ground-based TIR imagery. b Resulting atmospheric path lengths (APL) for each pixel. c Average path temperatures (Tpath) for the time the infrared thermograms were taken. d Land surface temperatures as measured by ground-based TIR imagery (LSTcam). e Resulting LSTcam from model application (LSTcam corr). f Difference between LSTcam and LSTcam corr. g Temperature ranges of LSTcam and LSTcam corr for the entire scene. h Temperature ranges of LSTcam and LSTcam corr for the marked section in panels a–f. Grey shadings in g and h refer to min–max range, 90 % percentile, 50 % percentile (IQR), and the median (black line), respectively. i Histogram of the differences in panel f for the entire scene l i d l LST T f LST d LST f h i Fig. 8 Example 2 (24 August 2012, 03:00 CET)—a elevation model, as seen by ground-based TIR imagery. b Resulting atmospheric path lengths (APL) for each pixel. Conclusions While atmospheric corrections on LST measurements are rou- tinely applied on remote sensing or airborne systems, such corrections had been neglected in many landscape-scale stud- ies using TIR cameras, despite atmospheric measurement path lengths of several hundred or thousand meters. Based on our intensive field measurements and on our modelling results, we were able to show that neglecting the atmospheric effect on ground-based TIR imagery does lead to substantial measure- ment errors. We could demonstrate that, depending on the temperature difference between the land surface and the over- lying air masses, these errors are relevant even at relatively short measurement paths and particularly for spatially varying LOS parameters due to an oblique view of the TIR camera. Furthermore, our results suggest that differences in LST on landscape scale are underestimated in both spatial and tempo- ral domains, due to the dampening effect of the atmosphere on the LST measurements. Given these conditions, any measurements of LST in mountain landscapes, not accounting for atmospheric effects, do not only result in inaccurate absolute LST data but also underestimate the LST variability, both in space (Fig. 7 and Fig. 8) and time, due to the diel cycle in LST-Tpath difference. Under these circumstances, the findings of, e.g., Scherrer and Körner (2010) may very likely even have underestimated the described high LST variability within their investigated area. One could argue that the atmospheric effects on LSTcam data are lower than the camera accuracies. However, in order to stay below a measurement error of 1.5 K (camera accuracy), the absolute differences between LSTosr and Tpath (ΔT) would have had to be lower than 4.6 and 2.1 K on average for our minimum APL (3260 m; Kaiserau) and our maximum APL (9038 m; Terlan), respectively (data not shown). Given the results shown in Fig. 5, ΔT reaches values as high as 15 K, which is well above the thresholds described above. Put an- other way, extrapolation of our data implies that an APL of less than 1000 m would be required to stay within the ±1.5 K range given by the accuracy of conventional TIR cameras. Acknowledgments Open access funding provided by University of Innsbruck and Medical University of Innsbruck. The research was kindly supported by the Province of Bozen-Bolzano (Autonome Provinz Bozen – Suedtirol, Abteilung Bildungsfoerderung, Universitaet und Forschung). We thank the Hydrographic Service of the Province of Bozen-Bolzano for providing radiometer data. Discussion Our findings of a distinct influence of the atmosphere on LST derived from ground-based TIR imagery are in accor- dance with numerous studies that report on the these influ- ences from satellite-based measurements down to ground- based measurements with APL of some hundred meters (Chandrasekhar 1960; Jacob et al. 2003; Meier et al. 2011; Norman et al. 1995; Voogt and Oke 2003). Meier et al. (2011) and Wawrzyniak et al. (2013), for example, report a magnitude of atmospheric effects during a diel cycle of up to 6.7 K in an urban environment and over natural environments, respectively, at path lengths lower than 800 m. These effects are particularly noticeable under the conditions of a high surface-to-path temperature difference, a pattern described, Comparing results of LSTosr with LSTcam of course holds the problem of finding the exact pixel covering the LSTosr site and the fact that these two methods differ in their spatial res- olution. To check the validity of our results, we performed a sensitivity analysis on our comparison of methods. To this end, we defined a pixel region for any LSTosr site within a thermal image with the pixel most likely covering the LSTosr site centered within a 5 times 5 pixel domain (center pixel surrounded by 24 pixels). Given these regions, we ran our 586 Int J Biometeorol (2017) 61:575–588 e.g., by Meier et al. (2011) as well. In the present study, this difference alone explained 81 % of the variation in LSTcam- LSTosr difference (p < 0.01) (Fig. 5). 1998). Our method has the advantage that we do not need the radiative transfer simulations, but due to the oblique view of the TIR camera at the landscape scale, we need to consider spatial variability of LOS parameters in contrast to a near nadir view of most satellite-based sensors. All single channel ap- proaches need accurate data on temperature and water vapor distribution in the atmosphere between the surface and the sensor. Therefore, and as our study shows, on-site data about the vertical distribution of at least atmospheric temperature and a DEM of the study region should be available for LST studies on landscape scale by means of ground-based TIR imagery. This should be considered in the conception of ex- perimental setups and field campaigns. Discussion Huge differences in temperature between a vegetated surface and the air above it may occur at times with (i) a high energy input, especially due to high incoming shortwave radiation (Kahmen et al. 2011; Lambers et al. 1998; Martin et al. 1999; Wilson et al. 1987), (ii) a low transpirational cooling due to water limitation (Camoglu 2013; Fuchs 1990; Gates 1964; Jackson et al. 1981), and (iii) low atmosphere-vegetation cou- pling (Jones 1992; McNaughton and Jarvis 1983). Due to the increase in solar radiation and a decrease in air temperature with increasing altitude and the low atmospheric coupling of short alpine vegetation (Goldberg and Bernhofer 2008; Jarvis and Mcnaughton 1986; Tappeiner and Cernusca 1996), mountain landscapes facilitate high surface to air tem- perature differences. 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https://openalex.org/W4302550364	https://www.econstor.eu/bitstream/10419/125599/1/827065094.pdf	English	null	Retirement Patterns of Couples in Europe	RePEc: Research Papers in Economics	2,014	cc-by	9,614	Hospido, Laura; Zamarro, Gema Suggested Citation: Hospido, Laura; Zamarro, Gema (2014) : Retirement patterns of couples in Europe, IZA Journal of European Labor Studies, ISSN 2193-9012, Springer, Heidelberg, Vol. 3, Iss. 12, pp. 1-18, https://doi.org/10.1186/2193-9012-3-12 Suggested Citation: Hospido, Laura; Zamarro, Gema (2014) : Retirement patterns of couples in Europe, IZA Journal of European Labor Studies, ISSN 2193-9012, Springer, Heidelberg, Vol. 3, Iss. 12, pp. 1-18, https://doi.org/10.1186/2193-9012-3-12 https://doi.org/10.1186/2193-9012-3-12 This Version is available at: https://hdl.handle.net/10419/125599 This Version is available at: https://hdl.handle.net/10419/125599 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. 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Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. http://creativecommons.org/licenses/by/2.0/ http://creativecommons.org/licenses/by/2.0/ http://creativecommons.org/ © Hospido and Zamarro; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2014 Abstract In this paper we study the retirement patterns of couples in a multi-country setting using data from the Survey of Health, Aging and Retirement in Europe. In particular we test whether women?s (men?s) transitions out of the labor force are directly related to the actual realization of their husbands? (wives?) transition, using the institutional variation in country-specific early and full statutory retirement ages to instrument the latter. Exploiting the discontinuities in retirement behavior across countries, we find a significant joint retirement effect for women of 21 percentage points. For men, the estimated effect is insignificant. Our empirical strategy allows us to give a causal interpretation to the effect we estimate. In addition, this effect has important implications for policy analysis. JEL Codes: J26, D10, C21 JEL Codes: J26, D10, C21 Keywords: Joint retirement; Social security incentives JEL Codes: J26, D10, C21 Keywords: Joint retirement; Social security incentives Keywords: Joint retirement; Social security incentives Laura Hospido1* and Gema Zamarro2 Correspondence: laura.hospido@bde.es 1Bank of Spain and IZA, Madrid, Spain Full list of author information is available at the end of the article Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 2014, 3:12 Open Access Retirement patterns of couples in Europe Laura Hospido1 and Gema Zamarro2 Laura Hospido1* and Gema Zamarro2 1 Introduction Continued improvements in life expectancy and fiscal insolvency of public pensions have led to an increase in pension entitlement ages in several countries, especially for women for whom eligibility ages for retirement pensions have been traditionally lower than for men. The success of such policies, however, relies on how responsive individuals are to changes in pension eligibility. In this paper we use longitudinal data from the Sur- vey of Health, Aging and Retirement in Europe (SHARE) to study the determinants of retirement decisions among European couples and how responsive each member of the couple is to their own eligibility to retirement pensions, as well as their partner?s eligi- bility induced retirement choice, after controlling for other factors that may affect their retirement decisions. Numerous studies have shown the importance of Social Security incentives for retire- ment decisions. The timing of retirement has been found to be in part determined by the incentives imbedded in the rules determining Social Security benefits, as well as employer-provided pension benefits (see Hurd 1990 and Lumsdaine and Mitchell 1999 for reviews). Likewise, other cross-national research published volumes edited by Gruber and Wise (1999, 2004) note that there is a strong negative correlation between labor force participation at older ages and the generosity of early retirement benefits. Finally, Coe and Zamarro (2011) find that official retirement ages in Europe are a strong predictor of retirement for men. However, these studies focused mostly on men and little is known about the determinants of women?s retirement decisions. Page 2 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Finally, this paper also contributes to the increasing literature that considers retirement as a decision concerning the couple, rather than the individual (Ruhm 1996; Gustman and Steinmeier 2000, 2004, 2009; Blau and Gilleskie et al. 2006; Coile 2004a, 2004b; Michaud 2003; Michaud and Vermeulen 2004; Casanova 2010; Stancanelli and van Soest 2012a, 2012b; Stancanelli 2012; Honoré and de Paula 2013), by providing empirical evidence of joint retirement behavior. The phenomenon of joint retirement refers to the coincidence in time of spouses? retirement and follows the observation that a significant proportion of spouses retire within less than one year of each other, independently of the age differ- ence between them. 1 Introduction The left graph of Figure 1 shows the histogram of the age differences between spouses using couples from the waves 1, 2, and 4 of SHARE. The average gap between the husband?s age and the wife?s age is of 2.2 years, being this difference quite stable across SHARE countries (with the only exception of Greece, where the average dif- ferential is of 4.4 years). The right graph shows the histogram of the differences between the age the husband stopped working and the age his wife did so. As expected by the joint retirement phenomenon, the peak at zero is large and much bigger than the proportion of couples with no age differences among its members. This hints at the presence of joint retirement behavior among couples in Europe. In this paper we then focus on the retirement patterns of couples and study the comple- mentarity of spouses? retirement patterns in continental Europe. This study complements the one of Banks et al. (2010) for England and the US who, focusing on men, found that British men are from 14 to 20 percentage points more likely to retire when their wife reaches state pension age at 60 than their American counterparts. Considering the numerous differences in the labor markets, health insurance and social plans between the UK and US and many European countries, there is no a priori reason to assume that their findings would still hold in Europe. In addition, in contrast with Banks et al. (2010), 0 5 10 15 Percent −10 −5 0 5 10 15 Husband Age − Wife Age 0 5 10 15 Percent −10 −5 0 5 10 15 Retirement Age Gap Figure 1 Age gaps between spouses. Notes: Source: SHARE (waves 1, 2, and 4). Retirement age gap = weighted mean of differences between the age of stop working for the husband and the age of stop working for his wife. Figure 1 Age gaps between spouses. Notes: Source: SHARE (waves 1, 2, and 4). Retirement age gap = weighted mean of differences between the age of stop working for the husband and the age of stop working for his wife. Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 3 of 18 we are interested in studying both women?s and men?s transitions out of the labor force and how they directly relate to the actual realization of their husbands? 1 Introduction (wives?) transition, using the institutional variation in country-specific early and normal retirement ages to instrument the latter. We find significant evidence of complementarity on spouses? transitions out of the labor force. The probability of women leaving the labor force increases in around 21 per- centage points when their husbands also stop working. The effect for men, however, is insignificant. Controlling for spouse?s working status reduces the impact of own eligibil- ity for retirement pensions on the probability of leaving the labor force. In particular, the effect for women is reduced in about 2 percentage points for full retirement pensions. Therefore, by ignoring joint retirement, governments would be overstating the impact of eligibility rules on retirement decisions. Our empirical strategy allows us to give a causal interpretation to these effects we estimate as we control for the potential endogeneity of spouse?s retirement decisions. The rest of the paper proceeds as follows. Section 2 describes the data and key variables for the analysis. Section 3 discusses the empirical reduced form model and identifica- tion strategy. In section 4 we present econometric results from estimating our empirical model. Finally we conclude in section 5. 2 Data This paper uses data from SHARE, a multidisciplinary and cross-national panel database of micro data on health, socioeconomic status and social and family networks of more than 40,000 individuals aged 50 or over. The main purpose of this survey is to provide detailed information about the living conditions of middle-aged and older people for several countries in Europe. There are currently four waves of data available in SHARE corresponding to the years 2004-2005, 2006-2007, 2008-2009, and 2010-2011. However, the third wave of SHARE (2008-2009) was devoted to a retrospective survey about life events of the respondents and did not collect all the information available in other waves. For this reason, this paper focuses on analysis of waves 1 (2004-2005), 2 (2006-2007), and 4 (2010-2011). The first wave of the SHARE dataset contains a balanced representation of the various European regions, ranging from Scandinavia (Denmark and Sweden), Cen- tral Europe (Austria, France, Germany, Switzerland, Belgium, and the Netherlands) and Mediterranean countries (Spain, Italy and Greece). Further data have been collected in 2005-06 and 2006-2007 in Israel. The Czech Republic, Poland and Ireland joined SHARE in the wave 2006-2007. However, Ireland only participated in the wave 2006-2007. Por- tugal also recently joined the SHARE team and participated in the survey during the last wave of data 2010-2011. To maximize the number of waves of data available we decided to focus our analysis on those OECD countries, for which information on retirement ages is available, who participated in the survey for at least two consecutive waves (i.e. Austria, Belgium, Czech Republic, Denmark, France, Germany, Greece, Italy, the Netherlands, Poland, Spain, Sweden, and Switzerland). 1 SHARE collects information on health variables (self-reported health, health condi- tions, physical and cognitive functioning, health behavior, use of health care facilities), biomarkers (grip strength, body-mass index, peak flow), psychological variables (psycho- logical health, well-being, life satisfaction), economic variables (current work activity, job characteristics, opportunities to work, retirement age, sources and composition of current Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 4 of 18 income, wealth and consumption, housing, education), and social support variables (assistance within families, transfers of income and assets, social networks, volunteer activities), both at the household and at the individual level. This gives the possibility to analyze a wide variety of questions related to population ageing and the quality of life of the elderly. 2.1 Sample This paper uses data from three waves of SHARE (waves 1, 2 and 4) for thirteen coun- tries (Austria, Belgium, Czech Republic, Denmark, France, Germany, Greece, Italy, the Netherlands, Poland, Spain, Sweden, and Switzerland). In particular, our sample consists of couples - married or living with a partner - who reported being working in either wave 1 or wave 2, with both members aged between 50 and 70, and with both members present in at least two consecutive waves. After dropping observations with incomplete records, our sample has 3,058 such couples.3 Given that our aim is to measure the causal effect of joint retirement we focus the analysis on working couples in one wave of data (waves 1 or 2) and study their retire- ment transitions in the subsequent wave (waves 2 or 4). However, it should be stressed that, for some countries, this sample would not be representative of the whole middle- age and older population, especially for women. This is so because, as shown in the Appendix, some European countries (notably the Mediterranean countries) have very high proportions of women who never worked (Figure 4 in the Appendix). Moreover, a large proportion of women who ever worked but stopped before age 50 did so at the early stages of their careers (Figure 5 in the Appendix) . Many of those early career stops are, however, not related to retirement decisions and so they are excluded from our analysis. 2 Data In addition, following Coe and Zamarro (2011) we supplemented the SHARE dataset with information regarding country and gender specific statutory ages of eligibility for early and full retirement pensions in order to construct instruments based on dummy variables indicating whether the individual is above the full or early retirement ages set in his country. Table 1 reports the statutory Early and Normal retirement ages in place in each country. Early and Normal retirement ages are based on OECD?s definitions and represent eligibility ages for early and full retirement pensions, respectively (see, OECD 2003, 2005a, 2005b, 2007, 2009, and 2011).2 As it can be seen in this table, the official retirement ages in Europe vary by country, and sometimes by gender, by as much as ten years. Note that for multiple countries in our study retirement ages have been increasing in the period of our analysis. This is the case, for example, in France, Germany or Greece, among others. In such cases, we abstract from the fact that these reforms might only affect certain cohorts in our analysis. We believe this could affect the relevance of our instruments but not its validity. However, as we show later in the paper our instruments continue to be relevant and have a significant effect on transitions out of the labor force, despite this simplification in its definition. 2007 arly (65) (60) 56-60) (65) (60) (63) (55) (60) (60) (60) (60) (61) (62) e, early retirem (2004), Preesm me of those pu 2.2 Definition of retirement We define retirement as making a transition out of work between two waves of data. That is, we consider a respondent as having retired if she reports working as her current job status in one wave and reports other working status (i.e. retired, unemployed, perma- nently sick or disabled, or homemaker) in the subsequent wave of data. In our sample, the E 62 60 60 ( 67 56-60 63 2007 2009 Early Normal Early Normal 5 (65) 65 (65) 65 (65) 65 (65) 0 (60) 65 (65) 60 (60) 65 (65) (56-60) 63 (59-63) 60 (59-60) 65 (62-65) 5 (65) 65 (65) 65 (65) 65 (65) 0 (60) 60 (60) 61 (61) 61 (61) 56 3 (63) 65 (65) 63 (63) 67 (67) 5 (55) 65 (65) 55 (55) 65 (65) 0 (60) 65 (60) 60 (60) 65 (60) 0 (60) 65 (65) 60 (60) 65 (65) 5 (60) 65 (60) 65 (60) 65 (60) 0 (60) 65 (65) 60 (60) 65 (65) 1 (61) 65 (65) 61 (61) 65 (65) 3 (62) 65 (64) 63 (62) 65 (64) ce, early retirement is possible from 56 under certain circumstances related to wo n (2004), Preesman (2006), and OECD (2005a). Slight differences can be found bet me of those publications and the law that was in place when individuals were fac Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 6 of 18 percentage of males transitioning out of the labor force is 29 per cent, while for women the percentage is 25. The proportion actually describing themselves as transitioning into retirement is 25 per cent for men, but only 17 per cent for women.4 Figure 2 presents percentages of respondents out of the labor force by age intervals and partner?s labor market status. We find that in our sample the fraction of workers that transition out of the labor force is higher, for both men and women and at every age interval, when the partner also makes such transition. Some other descriptive statistics for our sample of working couples can be found in the Appendix (Table 4 in the Appendix). The average age of men in our sample is 60 and 58 for women. Eleven per cent of men and a 6.5 percent of women are over the normal retirement age, while 33 and 21.5 percent, respectively, are over the early retirement age. 2.2 Definition of retirement Finally, educational attainment and health status are similar among males and females in our sample of couples. 3 Empirical model Dearly i is an indicator for eligibility for early retirement pensions, which is defined as: Dearly i = 1 if individual i?s age is above the early official retirement age in the country 0 , otherwise , and similarly Dnormal i is an indicator for eligibility for full retirement pensions defined as: Dnormal i = 1 if individual i?s age is above the full official retirement age in the country 0 , otherwise . Dearly j(i) and Dnormal j(i) are our external instruments for retirement decisions, that is, they are the exclusion restrictions that allow identification of the model. Note that, identifi- cation then relies on partner?s age being different than the individual?s age. As Figure 1 suggests, that is precisely the case in our data. In addition, in our analysis we control for the age difference between the two members of the couple to capture any unobserv- able characteristics at the couple level revealed by choosing a partner with a certain age difference.7 In practice what we assume is that - conditional on observables - whether the partner is eligible for retirement pensions only has an impact on the individual?s retirement decision through the partner?s retirement decision, as opposed to directly having an effect. Note that our exogeneity assumption does not imply that partner?s eligibility for retirement pensions does not affect ones? retirement decisions. The assumption is that it does so but only through the actual retirement decision of the partner. Under this assumption, our estimates of β are interpreted as the effect of the partner?s retirement, induced through eligibility for retirement pensions, on the individual?s retirement decision. Our econometric approach exploits the fact - illustrated by Figure 3 - that the regres- sor of interest (transition into retirement) is partly determined by a known discontinuous (non-linear and non-monotonic) function of an observed covariate (age) to control for the endogeneity of partner?s retirement decisions. This sort of identification strategy has a long tradition in social science and can be viewed as an application of a regression discontinuity design for evaluating the effect of joint retirement.8, 9 Finally, note that by estimating the equations for both members of the couple jointly we also take into account the potential correlation among unobservables across members of the couple.10 3 Empirical model We aim to determine the effect of having a partner leaving the labor force on the respon- dent?s probability of retirement. In particular, letRi be a binary indicator that takes value 1 if respondent i leaves the labor force, and let Rj(i) be another indicator that takes value 1 if her partner j(i) also makes such transition, conditional on both members being at work in the previous wave. Then, we consider a reduced form bivariate probit model, for transitions out of the labor force,5,6 of the following form: Ri = 1 βRj(i) + λDearly i + αDnormal i + Xi,jθ′ > ui (1) Rj(i) = 1 γ Dearly j(i) + δDnormal j(i) + Zi,jφ′ > ϵi (2) (1) (2) where i = {h, w} stands for husbands and wives, respectively, and (ui, ϵi) ∼N(0, ). 0 .2 .4 .6 .8 1 5055 5560 6065 6570 Husbands Wife working Wife not working 0 .2 .4 .6 .8 1 5055 5560 6065 6570 Wives Husband working Husband not working Figure 2 Retirement transitions by age intervals and partner?s labor market status.Notes: Sample size: Men=3,058; Women=3,058. Figure 2 Retirement transitions by age intervals and partner?s labor market status.Notes: Sample size: Men=3,058; Women=3,058. Page 7 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 In this framework, β is our main parameter of interest, Xi,j is a vector of explanatory variables containing demographic information for both members of the couple, and Zi,j = Xi,j, Dearly i , Dnormal i . The vector of explanatory variables Xi,j includes a series of controls for the individual and partner?s characteristics, such as the respondent?s age, the age difference between the two members of the couple, level of education and health status of each member, family composition (whether they have children and grandchildren), country and survey wave dummies. 4 Estimation results In this section we present the results of jointly estimating the system of equations (1-1). Table 2 reports, separately for men and women, average marginal effects of estimates of probit models for the probability of leaving the labor force, given that both spouses were Hospido and Zamarro IZA Journal of European Labor Studies Page 8 of 18 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 8 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 0 .2 .4 .6 .8 1 −10 −5 0 5 10 Years to full retirement age Figure 3 Probability of transitions out of the labor force by age. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=17,883; Women=16,119. Figure 3 Probability of transitions out of the labor force by age. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=17,883; Women=16,119. Figure 3 Probability of transitions out of the labor force by age. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=17,883; Women=16,119. Figure 3 Probability of transitions out of the labor force by age. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=17,883; Women=16,119. working in the previous wave. The set of controls included in the regressions is the fol- lowing: dummy variables for the respondent being eligible for early or full retirement, the respondent?s age, the age difference between the two members of the couple,11 coun- try and survey wave dummies, education variables for the two spouses, information on whether the couple has children and grandchildren as a measure of care necessities, and health status controls for both spouses, lagged one period to lessen endogeneity concerns. Within each section of the table we present results of models that ignore the possibility of joint retirement by excluding information on the current working status of the spouse, and preferred bivariate probit models where we include this variable and instrument it with the dummies for spouse?s eligibility for retirement pensions. Our results show that there is a significative joint retirement effect for women, of 21 percentage points. For men, the estimated effect is insignificant. These results are similar in size to those found by Banks et al. (2010) for British men. Introducing information on working status of the spouse reduces the impact of own eligibility for retirement pensions for women in about 2 percentage points. 4 Estimation results Therefore, by ignoring joint retirement, govern- ments would be overstating the impact of eligibility rules on retirement decisions. The remainder of the variables have the expected effects. Higher levels of education lower the probability of leaving the labor force but only for men, whereas bad health has a positive impact on the probability of leaving the labor force only for women. Finally, having grand- children increases the probability of leaving the labor force for both men and women, while having children reduces the probability of retirement only for men.12 In order for the official retirement ages to be valid instruments, they must be exoge- nous and relevant. With respect to the exogeneity assumption, we assume that if the husband (wife) reaches the statutory retirement age, his (her) spouse?s retirement decision is only affected through his (her) own transition. This assumption is not testable. Regard- ing relevance, statutory retirement ages must be related to actual retirement behavior. To illustrate this latter point we estimated probit regressions of the individual probabil- ity of leaving the labor force, separately for husbands and wives. 4 Estimation results This set of regressions Page 9 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Table 2 Bivariate probit estimates Probability of leaving the labor force Men Women Partner leaving the labor force 0.026 0.212* (0.094) (0.063) Age>early 0.046 0.045 -0.001 -0.007 (0.021) (0.022) (0.023) (0.022) Age>full 0.165* 0.168* 0.143* 0.125* (0.033) (0.034) (0.035) (0.034) Age 0.045* 0.044* 0.047* 0.032*** (0.003) (0.006) (0.003) (0.006) Age difference<0 0.032 0.020 -0.039* 0.066* (0.023) (0.049) (0.022) (0.035) 0⩽Age difference<1 0.054 0.045 -0.009 0.058 (0.024) (0.039) (0.024) (0.029) 1⩽Age difference<2 0.057* 0.048 0.007 0.060 (0.022) (0.036) (0.023) (0.026) 2⩽Age difference<4 0.039* 0.035 -0.031 0.005 (0.020) (0.023) (0.022) (0.024) High education -0.046 -0.047 -0.029 -0.018 (0.021) (0.021) (0.021) (0.021) Medium education 0.012 0.012 0.006 0.011 (0.018) (0.018) (0.018) (0.017) Partner high education -0.045 -0.044 0.018 0.028 (0.021) (0.021) (0.022) (0.021) Partner medium education -0.021 -0.021 0.020 0.013 (0.018) (0.018) (0.018) (0.017) Having children -0.055* -0.056* 0.030 0.033 (0.031) (0.031) (0.034) (0.032) Having grandchildren 0.073* 0.072* 0.047*** 0.028* (0.016) (0.017) (0.016) (0.016) Bad health in previous wave 0.022 0.021 0.071* 0.071* (0.022) (0.022) (0.019) (0.019) Partner bad health in previous wave 0.005 0.002 0.039* 0.031 (0.020) (0.023) (0.020) (0.021) Log-likelihood -2524.83 -2524.21 -2524.83 -2513.37 ρ 0.361 0.302 0.361 -0.195 (0.036) (0.228) (0.036) (0.191) LR test of ρ = 0 82.13 1.55 82.13 0.99 [0.00] [0.21] [0.00] [0.32] Notes: N. obs=3,058. Age difference measured as husband age minus wife age. All specifications include country and survey wave dummies. Delta-method standard errors clustered at the individual level in parentheses. Significant at the 10%, * 5%, and *** 1% level. ρ = corr(uh,ϵh). p-values in squared brackets. Notes: N. obs=3,058. Age difference measured as husband age minus wife age. All specifications include country and survey wave dummies. Delta-method standard errors clustered at the individual level in parentheses. Significant at the 10%, * 5%, and * 1% level. ρ = corr(uh,ϵh). p-values in squared brackets. would represent a standard first-stage step in a two-stage estimation procedure such as an Instrumental Variable (IV) model.13 Estimated marginal effects for these regressions can be found in the Appendix (Table 5 in the Appendix). Our results show that eligibil- ity for retirement pensions are a significant predictor of retirement decisions both for husbands and wives. 4 Estimation results In terms of the model in equations (1-1), these results confirm that Dearly j(i) and Dnormal j(i) affect Rj(i), after controlling for Dearly i and Dnormal i , and the rest of the control variables.14 Page 10 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 To get a better insight of the effect of policies on pension entitlement ages on retirement behaviors of couples, we also estimate bivariate models dividing the sample in two groups of countries. Group 1 includes those countries with a low gap in participation rates by gender, while group 2 contains those countries with high differentials in the participation rates between men and women. The mean gender gap in employment/population ratios for individuals aged 50-64 years in the thirteen countries considered in the analysis is 17.13, as reported in the Appendix (Table 6 in the Appendix). Below that number are the countries with low gender gaps, that is, Belgium, Denmark, France, Germany, Poland, and Sweden; whereas above the mean, we find countries with high gender gaps like Austria, Czechia, Greece, Italy, Netherlands, Spain, or Switzerland. The estimated marginal effects from these regressions are reported in Table 3. As before, the estimated joint retirement effect is insignificant for men, both for groups 1 and 2. On the contrary, for women we find that the significative joint retirement effect is wholly due to those countries where the differences by gender in participation rates are small (that is, group 1, for which the estimated effect is 28 percentage points). On the contrary, for women in countries with large differences in participation rates by gender, the estimated effect of joint retirement is not statistically different from zero. Finally, to assess the robustness of our results to different definitions of retirement, we also estimated models for the probability that the respondent describes herself as retired as opposed to out of the labor force. The results of these regressions can be found in the Appendix (Table 7 in the Appendix). Our results are still robust to this alternative definition of the dependent variable. For women, we find a significant joint retirement effect, although the magnitude of the effect gets reduced to about half the size (from 21 to 10 percentage points). For men we find an effect similar in magnitude, but again insignificant. 4 Estimation results Another difference with previous results is that lagged bad health does not seem to have an impact on retirement decisions for women in this case. This suggests that bad health shocks might lead women to rather leave the labor force without actually retiring. More research is needed to better understand the differences between women?s transitions out of work to self-reported retirement or to homemaking. 5 Conclusions Continued improvements in life expectancy and fiscal insolvency of public pensions have led to an increase in pension entitlement ages in several countries. For example, the nor- mal retirement age in the US is currently rising from 65 to 67 for successive birth cohorts. England, Austria, Germany and Italy are also phasing in increases in their retirement ages. However, the success of such policies relies on how responsive individuals are to such changes in pension eligibility. In this paper we use longitudinal data from SHARE to study the determinants of retirement decisions among European couples and how responsive each member of the couple is to their own eligibility to retirement pensions, as well as their partner?s eligibility induced retirement choice, after controlling for other factors that may affect their retirement decisions. Our empirical strategy exploits the discontinuities in retirement behavior across coun- tries to control for the endogeneity of partner?s labor participation decisions. This allows us to give a causal interpretation to the effects we estimate. Our results show a significative joint retirement effect for women of 21 percentage points. For men, the estimated effect is insignificant. 5 Conclusions Page 11 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Table 3 Bivariate probit estimates Probability of leaving the labor force Men Women (By participation rates gap) Group 1 Group 2 Group 1 Group 2 Partner leaving the labor force 0.012 -0.119 0.277* 0.107 (0.085) (0.134) (0.048) (0.175) Age>early 0.032 0.066* -0.015 -0.040 (0.028) (0.037) (0.026) (0.042) Age>full 0.150* 0.208* 0.094 0.157 (0.046) (0.054) (0.038) (0.065) Age 0.046* 0.043* 0.029* 0.038* (0.006) (0.008) (0.006) (0.012) Age difference<0 0.025 0.089 0.073 0.051 (0.046) (0.084) (0.031) (0.089) 0⩽Age difference<1 0.078 0.053 0.011 0.143* (0.035) (0.088) (0.029) (0.077) 1⩽Age difference<2 0.036 0.119* 0.049* 0.086 (0.034) (0.064) (0.027) (0.055) 2⩽Age difference<4 0.041 0.047 -0.003 0.019 (0.026) (0.044) (0.026) (0.050) High education -0.056 -0.031 0.013 -0.009 (0.028) (0.037) (0.024) (0.040) Medium education 0.017 -0.014 0.066* -0.045 (0.024) (0.029) (0.021) (0.029) Partner high education -0.071* 0.018 0.075* -0.049 (0.025) (0.039) (0.025) (0.037) Partner medium education -0.038 -0.004 0.041 -0.012 (0.025) (0.029) (0.021) (0.030) Having children -0.024 -0.047 -0.015 0.072 (0.046) (0.045) (0.044) (0.047) Having grandchildren 0.068* 0.093* -0.008 0.079 (0.019) (0.031) (0.018) (0.033) Bad health in previous wave 0.010 0.029 0.096* 0.024 (0.027) (0.036) (0.021) (0.038) Partner bad health in previous wave -0.037 0.086* 0.045* 0.005 (0.030) (0.032) (0.023) (0.038) Log-likelihood -1632.39 -847.63 -1619.35 -847.11 ρ 0.370 0.643 -0.410 0.116 (0.195) (0.279) (0.173) (0.443) LR test of ρ = 0 2.97 2.57 4.35 0.07 [0.08] [0.11] [0.04] [0.79] N. obs 2,040 1,018 2,040 1,018 Notes: Group 1 includes Belgium, Denmark, France, Germany, Poland, and Sweden. Group 2 Austria, Czechia, Greece, Italy, Netherlands Spain or Switzerland Age difference as husband age minus wife age All specifications include country and Table 3 Bivariate probit estimates Notes: Group 1 includes Belgium, Denmark, France, Germany, Poland, and Sweden. Group 2 Austria, Czechia, Greece, Italy, Netherlands, Spain, or Switzerland. Age difference as husband age minus wife age. All specifications include country and wave dummies. Delta- method standard errors clustered at the individual level in parentheses. Significant at 10%, * 5%, and *** 1%. ρ = corr(uh,ϵh). p-values in squared brackets. Notes: Group 1 includes Belgium, Denmark, France, Germany, Poland, and Sweden. Group 2 Austria, Czechia, Greece, Italy, Netherlands, Spain, or Switzerland. Age difference as husband age minus wife age. All specifications include country and wave dummies. Delta- method standard errors clustered at the individual level in parentheses. Significant at 10%, * 5%, and *** 1%. ρ = corr(uh,ϵh). p-values in squared brackets. 5 Conclusions We also compare our estimates with models that do not control for the partner?s labor participation decisions and found that introducing information on working status of the spouse reduces the impact of own eligibility for retirement pensions for women in about 2 percentage points. Therefore, by ignoring joint retirement, governments would be overstating the impact of eligibility rules on retirement decisions. Page 12 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Finally, our results are still robust to using self-reported retirement status as an alterna- tive definition of the dependent variable. In this case, we find a significant joint retirement effect for women, although the magnitude of the effect gets reduced to about half the size. For men, the estimated effect remains insignificant. As recent pension reforms that increase pension entitlement ages get established and new waves of data get collected, it would be good to analyze how these reforms are affecting retirement patterns of men and women. Lastly, additional waves of data would allow for the estimation of panel data models that could better control for unobserved heterogeneity affecting transitions out of the labor force. Endnotes 1 Note that Israel is excluded from the group of countries we analyze because it did not join OECD until 2010 and by that time they did not participate in SHARE anymore. 2 In the case of the Netherlands, the modelling at age 60 refers to a ?typical? occupational early retirement scheme. However since the early 1990s these schemes have been progressively transformed into less generous, fully-funded, systems. As a result of these transformations, early retirement ages may have a lower incentive effect there than in other countries considered. 2 In the case of the Netherlands, the modelling at age 60 refers to a ?typical? occupational early retirement scheme. However since the early 1990s these schemes have been progressively transformed into less generous, fully-funded, systems. As a result of these transformations, early retirement ages may have a lower incentive effect there than in other countries considered. 3 The distribution of number of couples by country is as follows: 70 couples in Austria, 350 couples in Belgium, 134 couples in Czechia, 454 couples in Denmark, 376 couples in France, 294 couples in Germany, 88 couples in Greece, 128 couples in Italy, 334 couples in the Netherlands, 64 couples in Poland, 98 couples in Spain, 502 couples in Sweden, and 166 couples in Switzerland. 4 Note that 4% of women in our couples reported transitioning from work to homemaker. This is in contrast with only 0.4% of men that reported such transition. Not to lose these transitions from women, we focus on transitions out of the labor force. Transitions to unemployment or disability were lower and did not show gender differences (1.7 % of men reported transitioning to disability vs. 1.3% of women; 2.3% of men reported transitioning to unemployment vs. 2.2 % of women). In any case, in the empirical analysis we also study only transitions to self-reported retirement status as the dependent variable and results are robust to this alternative definition. 4 Note that 4% of women in our couples reported transitioning from work to homemaker. This is in contrast with only 0.4% of men that reported such transition. Not to lose these transitions from women, we focus on transitions out of the labor force. Transitions to unemployment or disability were lower and did not show gender differences (1.7 % of men reported transitioning to disability vs. 1.3% of women; 2.3% of men reported transitioning to unemployment vs. Endnotes 2.2 % of women). In any case, in the empirical analysis we also study only transitions to self-reported retirement status as the dependent variable and results are robust to this alternative definition. 5 It should be pointed out that our approach, focusing on transitions out of the labor force for working couples on waves 1 or 2, is equivalent to a discrete duration model for durations in the labor force where we assume that coefficients are constant across durations. 6 In our sample, 550 out of the 3,058 couples are observed working both in waves 1 and 2 and so they contribute twice to our sample. To take this into account we obtain cluster robust standard errors at the respondent level. Note that the limited number of couples that appear in both waves and the limited number of waves do not allow us to estimate more sophisticated panel models to control for unobserved heterogeneity. 7 For instance, those who have partners who are much younger than themselves might have a certain personality that could reflect in their retirement decisions. This could be confounded with the effect of having a spouse that does not retire if not controlled for. 8 For literature reviews of regression discontinuity methods see Imbens and Lemieux 2007, Van der Klaauw 2008, or Lee and Lemieux 2010. For applications of regression discontinuity to the retirement decision see also Battistin et al. 2009, and Stancanelli and van Soest 2012a, 2012b. 9 It should be pointed out that, if the effect of partner?s retiring on own retirement choices is heterogeneous among individuals, our results would then be interpreted as the average effect of those whose partners change their retirement decisions when they become eligible for retirement pensions. As eligibility for retirement pensions, especially for early retirement, depends on many cases of the labor history of the individual, our Page 13 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 estimates would be more representative of effects for those whose partners have longer labor histories which might not necessarily coincide with those for the whole population. estimates would be more representative of effects for those whose partners have longer labor histories which might not necessarily coincide with those for the whole population. 10 An alternative model would consist on an Instrumental Variables (IV) discrete choice model estimated separately for men and women. Endnotes However, this approach would be less efficient than the simultaneous discrete choice model models we estimate and it would not account for the potential correlation among unobservables across spouses. 11 The age of the respondent is included as a continuous variable (measuring age in months), whereas the age gap between spouses enters as dummies. 12 We also estimated models controlling for household income in the previous wave and household wealth but this did not change our main results. Estimates for these models are available from the authors upon request. 13 In practice, we follow a more efficient approach and estimate the whole bivariate model by maximum likelihood in a single step. 14 The estimated model in this paper is a simultaneous equation binary choice model where partner?s eligibility for early and full retirement pensions is used as exclusion restrictions to identify the system. As such, the reduced form probit models that we estimate just proxy what it would be a first step to assess the relevance of our instruments. Moreover, in order to also approximate traditional Hausman/Sargan test for the joint validity of the instruments, we have tried and estimate reduced form linear models for the probability of individual i leaving the labor force, separately for men and women, by Two-Stage Least Squares. In these models, the partner?s transition out of the labor force, Ri(i), is instrumented using the indicators Dearly j(i) and Dnormal j(i) . For these latter regressions, we obtained the Sargan test of overidentifying restrictions. For men the corresponding p-value for the test was 0.274, whereas for women was 0.869; meaning that in both cases we can not reject the null hypothesis that the over-identifying restrictions are valid. 14 The estimated model in this paper is a simultaneous equation binary choice model where partner?s eligibility for early and full retirement pensions is used as exclusion restrictions to identify the system. As such, the reduced form probit models that we estimate just proxy what it would be a first step to assess the relevance of our Notes: 3,058 observations for men and 3,058 observations for women. Standard deviations of non-binary variables in parentheses. Significant at the * 10%, 5%, and * 1% level. Appendix Given the aim of the analysis, we focus on working couples only. Table 4 reports descriptive statistics by gender for our sample of working couples. However, for some countries, this sample would not be representative of the whole middle-age and older population. On the one hand, as shown in Figure 4, some European countries have very high proportions of women who never worked. In Table 4 Descriptive statistics by gender [1] Men [2] Women Dif=[1]-[2] Age 60.040 58.376 1.664* (3.792) (3.618) Over Early Age 0.329 0.215 0.114* Over Normal Age 0.109 0.065 0.044* Out of the labor force 0.294 0.253 0.041* Declared as retired 0.250 0.173 0.077* Bad Health 0.151 0.169 -0.018 Low Education 0.248 0.263 -0.015* Medium Education 0.389 0.366 0.023** High Education 0.355 0.361 -0.006* Dummy of having children 0.942 Dummy of having grandchildren 0.587 Notes: 3,058 observations for men and 3,058 observations for women. Standard deviations of non-binary variables in parentheses. Significant at the * 10%, 5%, and * 1% level. Table 4 Descriptive statistics by gender Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 14 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 0 .1 .2 .3 .4 AT BE CZ DK FR DE GR IT NL PO ES SE SW Country Men Women Figure 4 Fraction of individuals who never worked. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=33,387; Women=41,714. Brackets represent 95% confidence intervals. 0 .1 .2 .3 .4 AT BE CZ DK FR DE GR IT NL PO ES SE SW Country Men Women Figure 4 Fraction of individuals who never worked. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=33,387; Women=41,714. Brackets represent 95% confidence intervals. Figure 4 Fraction of individuals who never worked. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=33,387; Women=41,714. Brackets represent 95% confidence intervals. addition, a large proportion of women who ever worked but stopped before age 50 did so at the early stages of their careers. For instance, in Figure 5 we can see bimodal histogram shapes for women in countries like Belgium, Italy, the Netherlands or Spain. Table 5 reports estimated marginal effects from probit regressions of the individ- ual probability of leaving the labor force, separately for husbands and wives. Notes: N. obs=3,058. Age difference measured as husband age minus wife age. All specifications include country and wave dummies. Standard errors clustered at the individual level in parentheses. Significant at 10%, * 5%, *** 1%. Appendix This set 0 .05 .1 .15 .2 Density 10 30 50 70 Age Men 0 .05 .1 .15 .2 Density 10 30 50 70 Age Women Whole sample 0 .1 .2 .3 Density 10 30 50 70 Age Men 0 .1 .2 .3 Density 10 30 50 70 Age Women Austria 0 .05 .1 .15 .2 Density 10 30 50 70 Age Men 0 .05 .1 .15 .2 Density 10 30 50 70 Age Women Belgium 0 .1 .2 .3 .4 Density 10 30 50 70 Age Men 0 .1 .2 .3 .4 Density 10 30 50 70 Age Women Czechia 0 .05.1.15.2 Density 10 30 50 70 Age Men 0 .05.1.15.2 Density 10 30 50 70 Age Women Denmark 0 .1 .2 .3 Density 10 30 50 70 Age Men 0 .1 .2 .3 Density 10 30 50 70 Age Women France 0 .1 .2 .3 Density 10 30 50 70 Age Men 0 .1 .2 .3 Density 10 30 50 70 Age Women Germany 0 .05 .1 .15 .2 Density 10 30 50 70 Age Men 0 .05 .1 .15 .2 Density 10 30 50 70 Age Women Greece 0 .05 .1 .15 .2 Density 10 30 50 70 Age Men 0 .05 .1 .15 .2 Density 10 30 50 70 Age Women Italy 0 .05 .1 .15 .2 Density 10 30 50 70 Age Men 0 .05 .1 .15 .2 Density 10 30 50 70 Age Women Netherlands 0 .05 .1 .15 .2 Density 10 30 50 70 Age Men 0 .05 .1 .15 .2 Density 10 30 50 70 Age Women Poland 0 .1 .2 .3 Density 10 30 50 70 Age Men 0 .1 .2 .3 Density 10 30 50 70 Age Women Spain 0 .1 .2 .3 .4 Density 10 30 50 70 Age Men 0 .1 .2 .3 .4 Density 10 30 50 70 Age Women Sweden 0 .2 .4 .6 Density 10 30 50 70 Age Men 0 .2 .4 .6 Density 10 30 50 70 Age Women Switzerland Figure 5 Stop working age. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=32,926; Women=34,260. Figure 5 Stop working age. Notes: Source: SHARE (waves 1, 2 and 4). Sample size: Men=32,926; Women=34,260. Appendix Hospido and Zamarro IZA Journal of European Labor Studies Page 15 of 18 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 15 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Table 5 Probit estimates Probability of leaving the labor force Men Women Age>early 0.056* -0.002 (0.030) (0.028) Age>full 0.284* 0.162* (0.058) (0.060) Partner age>early 0.008 -0.001 (0.029) (0.026) Partner age>full -0.104 0.036 (0.034) (0.042) Age 0.059* 0.055* (0.005) (0.004) Age difference<0 0.079 -0.033 (0.037) (0.032) 0⩽Age difference<1 0.097* -0.002 (0.037) (0.033) 1⩽Age difference<2 0.096* 0.022 (0.033) (0.032) 2⩽Age difference<4 0.062 -0.029 (0.028) (0.026) High education -0.062 -0.032 (0.025) (0.024) Medium education 0.010 0.010 (0.022) (0.022) Partner high education -0.052** 0.022 (0.025) (0.027) Partner medium education -0.028 0.021 (0.022) (0.022) Having children -0.072* 0.029 (0.044) (0.037) Having grandchild 0.086* 0.054* (0.019) (0.018) Bad health previous wave 0.024 0.091*** (0.028) (0.027) Partner bad health previous wave 0.008 0.050* (0.025) (0.027) Pseudo R2 0.303 0.267 Notes: N. obs=3,058. Age difference measured as husband age minus wife age. All specifications include country and wave Table 5 Probit estimates of regressions would represent a standard first-stage step in a two-stage estimation procedure such as an Instrumental Variable (IV) model. Results show that eligibility for retirement pensions are a significant predictor of retirement decisions both for men and women. Table 6 shows gender gaps in employment/population ratios, by country, for individuals aged 50-64 years. Finally, Table 7 shows estimation results of models for the probability that the respon- dent describes herself as retired as opposed to out of the labor force. Our results are robust to this alternative definition of the dependent variable. Author details 1 1Bank of Spain and IZA, Madrid, Spain. 2Dornsife Center for Economic and Social Research University of Southern California, Los Angeles, CA, USA. Received: 17 July 2013 Accepted: 18 January 2014 Published: 20 Jun 2014 Acknowledgements We thank Jorge Alonso, Marco Angrisani, Manuel Bagues, Olympia Bover, Pedro Mira, two anonymous referees, and the seminar participants at the Bank of Spain, the COSME Workshop in Madrid, the 2nd International SHARE User Conference in Mainz, the SAEe in Madrid and in Santander, the ESPE Meeting in Hangzhou, the Netspar International Pension Workshop in Amsterdam, and the ESEM meetings in Malaga for useful comments. All remaining errors are our own. This paper uses data from SHARE wave 4 release 1.1.1, as of March 28th 2013 or SHARE wave 1 and 2 release 2.6.0, as of November 29th 2013 or SHARELIFE release 1, as of November 24th 2010. The SHARE data collection has been primarily funded by the European Commission through the 5th Framework Programme (project QLK6-CT-2001-00360 in the thematic programme Quality of Life), through the 6th Framework Programme (projects SHARE-I3, RII-CT-2006-062193, COMPARE, CIT5- CT-2005-028857, and SHARELIFE, CIT4-CT-2006-028812) and through the 7th Framework Programme (SHARE-PREP, N◦211909, SHARE-LEAP, N ◦227822 and SHARE M4, N◦261982). Additional funding from the U.S. National Institute on Aging (U01 AG09740-13S2, P01 AG005842, P01 AG08291, P30 AG12815, R21 AG025169, Y1-AG-4553-01, IAG BSR06-11 and OGHA 04-064) and the German Ministry of Education and Research as well as from various national sources is gratefully acknowledged (see www.share-project.org for a full list of funding institutions). The opinions and analyses are the responsibility of the authors and, therefore, do not necessarily coincide with those of the Bank of Spain or the Eurosystem. Responsible editor: Martin Kahanec Competing interests h l f Competing interests The IZA Journal of European Labor Studies is committed to the IZA Guiding Principles of Research Integrity. The authors declare that they have observed these principles. Competing interests The IZA Journal of European Labor Studies is committed to the IZA Guiding Principles of Research Integrity. The authors declare that they have observed these principles. Appendix Age difference measured as husband age minus wife age. All specifications include country and survey wave dummies. Delta-method standard errors clustered at the individual level in parentheses. Significant at 10%, * 5%, and * 1%. ρ = corr(uh, ϵh). p-values in squared brackets. 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J Lab Econ 18(3):503?545 Gustman A, Steinmeier T (2004) Social security, pensions and retirement behaviour with the family. J Appl Econometrics 19:723–737 Gustman A, Steinmeier T (2009) Integrating retirement models. NBER Working Paper 15607. http://www.nber.org/ papers/w15607 Gruber J, Wise D (1999, 2004) Social security programs and retirement around the world. University of Chicago Press, Chicago Honoré B, de Paula A (2013) Interdependent durations in joint retirement, cemmap working paper CWP05/13. http:// www.cemmap.ac.uk/cemmap/publication/id/6632 www.cemmap.ac.uk/cemmap/publication/id/6632 Hurd MD (1990) Research on the elderly: economic status, retirement, and consumption and savings. J Econ Lit 28:565–637 Imbens G, Lemieux T (2007) Regression discontinuity design: a guide to practice. J Econometrics 14 Lee DS Lemieux T (2010) Regression discontinuity designs in economics J Econ Lit 48(2):281?355 Imbens G, Lemieux T (2007) Regression discontinuity design: a guide to practice. J Econometrics 142:615 Imbens G, Lemieux T (2007) Regression discontinuity design: a guide to practice. J Econometrics 14 Lumsdaine RL, Mitchell OS (1999) New Developments in the Economic Analysis of Retirement. Appendix Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 16 of 18 Table 6 Employment/population ratios (50-64 years) Table 6 Employment/population ratios (50-64 years) Country Total [1] Men [2] Women Dif=[1]-[2] Austria 39.22 50.57 28.23 22.34 Belgium 45.26 52.74 37.88 14.86 Czechia 50.01 62.17 39.60 22.57 Denmark 67.52 72.81 62.48 10.33 France 53.58 57.22 50.26 6.96 Germany 56.99 62.80 51.34 11.46 Greece 51.04 73.47 30.24 43.23 Italy 38.22 51.48 26.14 25.34 Netherlands 55.94 65.50 46.23 19.27 Poland 31.79 40.57 23.98 16.59 Spain 46.79 63.53 31.10 32.43 Sweden 76.35 79.26 73.45 5.81 Switzerland 71.18 80.75 61.71 19.04 Overall 50.65 59.48 42.35 17.13 Notes: Source: SHARE (waves 1, 2, and 4). Sample size: Men=16,261; Women=20,176. Weighted means. Table 7 Bivariate probit estimates Probability of retiring Men Women Partner leaving the labor force 0.104 0.097 (0.064) (0.041) Age>early 0.043 -0.001 (0.018) (0.018) Age>full 0.096* 0.058 (0.028) (0.026) Age 0.043* 0.037*** (0.005) (0.004) Age difference<0 -0.052 0.051 (0.037) (0.031) 0⩽Age difference<1 -0.005 0.047* (0.030) (0.025) 1⩽Age difference<2 0.016 0.051 (0.026) (0.022) 2⩽Age difference<4 0.004 0.014 (0.019) (0.021) High education -0.029 0.026 (0.020) (0.016) Medium education 0.024 -0.003 (0.016) (0.014) Partner high education -0.028 0.002 (0.020) (0.017) Partner medium education -0.023 0.014 (0.016) (0.014) Having children -0.047 0.037 (0.029) (0.028) Having grandchildren 0.081* 0.024* (0.015) (0.013) Bad health in previous wave -0.030 0.017 (0.019) (0.015) Partner bad health in previous wave -0.010 0.015 (0.017) (0.017) Hospido and Zamarro IZA Journal of European Labor Studies Page 17 of 18 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies Page 17 of 18 2014, 3:12 http://www.izajoels.com/content/3/1/12 Page 17 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Table 7 Bivariate probit estimates (Continued) Log-likelihood -1845.45 -1838.39 ρ 0.179 0.121 (0.207) (0.174) LR test of ρ = 0 0.72 0.47 [0.40] [0.49] Notes: N. obs=3,058. Age difference measured as husband age minus wife age. All specifications include country and survey wave dummies. Delta-method standard errors clustered at the individual level in parentheses. Significant at 10%, * 5%, and *** 1%. ρ = corr(uh, ϵh). p-values in squared brackets. Table 7 Bivariate probit estimates (Continued) Notes: N. obs=3,058. Cite this article as: Hospido and Zamarro: Retirement patterns of couples in Europe. IZA Journal of European Labor Studies 10.1186/2193-9012-3-12 2014, 3:12 References In: Ashenfelter O, Card D (eds) Handbook of Labor Economics, 3C, Amsterdam, North Holland, pp 3261?3307 Lumsdaine RL, Mitchell OS (1999) New Developments in the Economic Analysis of Retirement. In (eds) Handbook of Labor Economics, 3C, Amsterdam, North Holland, pp 3261?3307 Page 18 of 18 Hospido and Zamarro IZA Journal of European Labor Studies 2014, 3:12 http://www.izajoels.com/content/3/1/12 Michaud P (2003) Joint labor supply dynamics of older couples, IZA DP 832. http://ftp.iza.org/dp832.pdf Michaud P, Vermeulen F (2004) A collective retirement model: identification and estimation in the presence of externalities IZA DP 1294 http://ftp iza org/dp1294 pdf Natali D (2004) The Pension System Observatoire Social Européen. Research Project. La Methode Ouverte de Coordination (MOC) en matiere des pensions et de l?integration Europeenne. Service Public Fédéral Sécurité Sociale. OECD (2003) Economic Survey of Austria. chapter 3: Pensions. http://www.oecd.org/dataoecd/5/16/27424371.pdf OECD (2005a) Vieillissement et politiques de l?emploi. France, Ageing and Employment Policies. http://www.oecd.org/ dataoecd/52/58/34591763.pdf p OECD (2005b) OECD Pensions at a Glance 2005: public policies across OECD countries. OECD Publishing. http://www. oecd-ilibrary.org/finance-and-investment/oecd-pensions-at-a-glance-2005_pension_glance-2005-en OECD (2007) Pensions at a Glance 2007: public policies across OECD countries. OECD Publishing. http://www.oecd- ilibrary.org/finance-and-investment/oecd-pensions-at-a-glance-2007_pension_glance-2007-en OECD (2009) Pensions at a Glance 2009: retirement-income systems in OECD countries. OECD Publishing. http://www. oecd-ilibrary.org/finance-and-investment/pensions-at-a-glance-2009_pension_glance-2009-en OECD (2011) Pensions at a Glance 2011: retirement-income systems in OECD and G20 countries. OECD Publishing. http://www.oecd-ilibrary.org/finance-and-investment/pensions-at-a-glance-2011_pension_glance-2011-en Preesman L (2006) Dutch to abolish civil service retirement age. IPE International Publishers Ltd., Netherlands Ruhm CJ (1996) Do pensions increase the labor supply of older men?. J Pub Econ 59(2):157?175 Stancanelli E (2012) Spouses? retirement and hours outcomes: evidence from twofold regression discontinuity with differences-in-differences, IZA DP No. 6791. http://ftp.iza.org/dp6791.pdf Stancanelli E, van Soest A (2012a) Retirement and home production: a regression discontinuity approach. Am Econ Rev 102(3):600–605 Stancanelli E, van Soest A (2012b) Joint leisure before and after retirement: a double regression discontinuity approach. IZA DP 6698. http://ftp.iza.org/dp6698.pdf Sundén A (2004) The future of retirement in Sweden. PRC WP 2004-16. Pension Research Council Working Paper, Pension Research Council. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.201.9427&rep=rep1&type=pdf The Bertelsmann Foundation, International Reform Monitor (2010) Country info. http://www.bertelsmann-stiftung.de/ cps/rde/xchg/SID-465C2575-83F7BF1C/bst_engl/hs.xsl/54224_104034.htm Van der Klaauw, W (2008) Regression-discontinuity analysis: a survey of recent developments in economics. Labour 22(2):219?245. http://onlinelibrary.wiley.com/doi/10.1111/j.1467-9914.2008.00419.x/abstract Van der Klaauw, W (2008) Regression-discontinuity analysis: a survey of recent developments in economics. Labour 22(2):219?245. http://onlinelibrary.wiley.com/doi/10.1111/j.1467-9914.2008.00419.x/abstract Cite this article as: Hospido and Zamarro: Retirement patterns of couples in Europe. 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https://openalex.org/W4313578592	https://periodicos.ufba.br/index.php/revistagerminal/article/download/51502/28267	Portuguese	null	A sobrevida da agenda pós-moderna e seus malabarismos teórico-políticos	Germinal	2,022	cc-by	11,707	Sandra Soares Della Fonte1 Resumo: Pontuam-se aspectos da construção e das repercussões do discurso pós-moderno no campo educacional brasileiro para evidenciar, à semelhança do que Moraes fez no início dos anos 2000, o seu renovado conservadorismo. Para tanto, ressaltam-se a estruturação desse discurso como uma agenda e alguns de seus rearranjos, em especial os que derivam de seus intelectuais de esquerda. Discutem-se ainda as reações do campo educacional brasileiro diante desses rearranjos. A relação orgânica entre neoliberalismo e neoconservadorismo, assim como pandemia mundial de Covid-19 têm tornado mais evidentes e profundas as aporias e (des)caminhos dessa agenda. A crítica a qualquer relativismo da verdade e depreciação da ciência tem sido assumida por vários segmentos sociais progressistas, fato que, por um lado, favorece a luta contra argumentações da agenda pós, por outro, pode se reverter em força para a renovação de seu conservadorismo. Palavras-chave: Agenda pós-moderna. Educação. Renovado conservadorismo. Resumen: Se destacan aspectos de la construcción y repercusiones del discurso posmoderno en el campo educativo brasileño para mostrar, de manera similar a lo que hizo Moraes a principios de la década de 2000, su renovado conservadurismo. Para ello, se destaca la estructuración de este discurso como agenda y algunos de sus reordenamientos, especialmente los que se derivan de sus intelectuales de izquierda. También se discuten las reacciones del campo educativo brasileño frente a estos reordenamientos. La relación orgánica entre el neoliberalismo y el neoconservadurismo, así como la pandemia mundial del Covid-19, han hecho más evidentes y profundas las aporías y (mal)camino de esta agenda. La crítica a cualquier relativismo de la verdad y desvalorización de la ciencia ha sido asumida por varios segmentos sociales progresistas, hecho que, por un lado, favorece la lucha contra los argumentos de la post agenda, por otro lado, puede revertir con fuerza a la renovación de su conservadurismo. Palabras clave: Agenda posmoderna. Educación. Conservadurismo renovado. Abstract: Aspects of the construction and repercussions of the postmodern discourse in the Brazilian educational field are pointed out to show, similarly to what Moraes did in the early 2000s, its renewed conservatism. To do so, the structuring of this discourse as an agenda and some of its rearrangements are highlighted, especially those derived from its leftist intellectuals. The reactions of the Brazilian educational field to these rearrangements are also discussed. A SOBREVIDA DA AGENDA PÓS-MODERNA E SEUS MALABARISMOS TEÓRICO- POLÍTICOS A SOBREVIDA DA AGENDA PÓS-MODERNA E SEUS MALABARISMOS TEÓRICO- POLÍTICOS THE SURVIVAL OF THE POST-MODERN AGENDA AND ITS THEORETICAL- POLITICAL JUGGLE DOI: https://doi.org/10.9771/gmed.v14i3.51502 Debate Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. Debate of this agenda. Criticism of any relativism of truth and depreciation of science has been assumed by several progressive social segments, a fact that, on the one hand, favors the fight against arguments of the postmodern agenda, on the other hand, it can revert in force to the renewal of its conservatism. Keywords: Postmodern agenda. Education. Neoconservatism. A Maria Célia Marcondes de Moraes cuja contribuição ainda nos inspira! Introdução A filiação à Pedagogia histórico-crítica evoca a defesa do projeto de uma formação humana ampla, que busca enriquecer o existir humano a partir da apropriação ativa do patrimônio cultural, tendo como horizonte um projeto societário de superação do capital. Esse compromisso implica um duplo movimento: criticar as correntes burguesas no campo educacional; e estruturar fundamentos para a sua própria proposição. Por certo, são ações interligadas. Porém, isso não descarta a necessidade de, em alguns momentos, por motivos diversos, priorizarmos uma dessas tarefas. A crítica às correntes burguesas no campo educacional tem-se revelado uma tarefa mais complexa que outrora. Isso se deve a várias razões. Pontuamos aqui três delas. A primeira se refere à observação de Saviani (2007, p. 426) relativa à dificuldade de caracterizar as ideias pedagógicas hegemônicas no país a partir dos anos de 1990: Não é fácil caracterizar em suas grandes linhas essa nova fase das ideias pedagógicas. Isso porque se trata de um momento marcado por descentramento e desconstrução das ideias anteriores, que lança mão de expressões intercambiáveis e suscetíveis de grande volatilidade. Não há, pois, um núcleo que possa definir positivamente as ideias que passam a circular já nos anos de 1980 e que se tornam hegemônicas na década de 1990. Daí que sua denominação tenda a se fazer lançando mão das categorias precedentes às quais se antepõem prefixos do tipo “pós” ou “neo” (SAVIANI, 2007, p. 426). O autor ainda observa que, a partir do deslocamento do arranjo produtivo para o toyotismo e da ofensiva neoliberal, o neoprodutivismo emerge como uma nova versão da teoria do capital humana. Suas variantes (neo-escolanovismo, neoconstrutivismo e neotecnicismo) se alimentam do clima cultural pós-moderno. Isso gera um cenário peculiar no campo das teorizações educacionais. Como observa Della Fonte (2011), o refluxo das teorizações educacionais críticas a partir dos anos de 1990 no país teve como contrapartida o desenvolvimento, no campo acadêmico, de um pensamento de direita renovado e refinado em suas formulações, surpreendentemente abraçado por intelectuais do campo político de esquerda. Em geral, essa tendência aglutina formas teóricas diversificadas, com inúmeras influências e autores distintos, mas que convergem em uma perspectiva denominada pós-ista (MORAES, 1996). Algumas de suas formulações contestam os rumos neoliberais e neoconservadores e, ao mesmo tempo, alimentam certos aspectos da lógica neoprodutivista, mesmo que à revelia das motivações políticas de parcela de seus intelectuais.2 74 erminal: marxismo e educação em debate, Salvador, v.14, n.3, p. Sandra Soares Della Fonte1 The organic relationship between neoliberalism and neoconservatism, as well as the world pandemic of Covid-19, have become more evident and profound the aporias and (mis)paths 73 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate Esse encontro, muitas vezes a contragosto de seus adeptos, funciona como um elemento que complexifica a crítica às correntes burguesas no campo educacional. Soma-se a isso uma terceira razão: essa ampla tendência pós-ista apresenta-se de forma mutante; seus rumos, suas ressignificações e refinamentos expressam tanto uma dinâmica interna ao campo teórico (de enfrentar as críticas, suas próprias aporias e/ou aprofundar seus argumentos), como um movimento articulado às mutações do capitalismo contemporâneo, inclusive em suas especificidades nacionais. Neste artigo, pontuamos alguns aspectos da construção e das repercussões do discurso pós no campo educacional brasileiro para evidenciar, à semelhança do que Moraes fez no início dos anos 2000, o renovado conservadorismo dessa agenda. Para discutir isso, talvez precisemos retomar, de início, como e por que esse conglomerado teórico se articula como uma agenda. Introdução 73-94, dez. 2022. ISSN: 2175-5604 Debate possibilitou a ampliação posterior desse pensamento na forma de uma agenda, isto é, como um conjunto de temas, problemas e compromissos compartilhado por grupos diversos. Ao inaugurar a discussão do pós-moderno no campo filosófico, Lyotard rompe o tratamento disciplinar do tema e trata a pós-modernidade como uma mudança geral na condição humana. Para Anderson (1999), esse aspecto possibilitou uma ressonância maior do livro em relação a todas as reflexões anteriores sobre o assunto. Nessa obra, Lyotard (2000) desenvolve a concepção de pós-moderno que se tornaria clássica. No contexto de crítica dos relatos iluministas e de defesa relativista de paridade de saberes, o autor (2000, p. XVI) define o pós-moderno como “a incredulidade em relação aos metarrelatos” utilizados para sustentar o ideal de emancipação e de superioridade da ciência diante dos demais conhecimentos. Como mencionado, a elaboração lyotardiana já continha o potencial de constituição de uma agenda, tendo em vista o caráter amplo e interdisciplinar conferido ao tema. Esse potencial se efetivou com o círculo de debate que se instaurou em função dessa formulação originária. Anderson lembra que, depois do impulso da ideia lyotardiana de pós-modernidade no final dos anos de 1970, a primeira reação abrasiva e elaborada a partir do campo da esquerda em relação ao tema foi a de Jürgen Habermas, em seu discurso de recebimento do prêmio Adorno da municipalidade de Frankfurt em 1980, por mais que Habermas (1993, p. 98) aponte que seu ponto de partida tenha sido a Bienal de Arquitetura em Veneza em 1980. Ao colocar a questão se se deve declarar o projeto inteiro da modernidade uma causa perdida ou se podemos nos agarrar às intenções, mesmo que frágeis, do Iluminismo, Habermas responde: é cedo para se renunciar à modernidade, pois o seu projeto ainda está inacabado (HABERMAS, 1993). Seu horizonte é a defesa de fundação pragmático-transcendental. As condições de possibilidade do debate, do agir comunicativo implicam regras que determinam o círculo dos participantes potenciais, como inclusão de todos os sujeitos, sem exceção, que disponham de capacidade de participar de argumentações, chances iguais de contribuir para a argumentação e condições de comunicação que tornam possível o prevalecer de um direito sem qualquer repressão. Essa é a forma ideal da comunicação; as regras do discurso não são simplesmente convenções, mas, segundo o autor, pressuposições universais inevitáveis. A intervenção de Habermas fez crescer a envergadura do debate sobre o pós-moderno. A formulação originária do discurso pós-moderno e sua primeira ampliação como agenda De seu nascimento, sem maiores repercussões no mundo hispânico, na década de 1930 (ANDERSON, 1999), até a sua ampla difusão a partir dos anos de 1970, o termo pós-moderno percorreu uma jornada na qual abandonou seu sentido originalmente estético e assumiu uma conotação histórica na filosofia, na ética, na cultura, nas ciências sociais e humanas. O ponto de inflexão dessa trajetória encontra-se na segunda metade da década de 1970, quando o termo pós-moderno ganhou uma precisa formulação conceitual. Como observa Connor (1993), o livro A condição pós-moderna de Lyotard, publicado em 1979 e traduzido para o inglês em 1984, é um marco, pois ofereceu uma confirmação interdisciplinar aos diagnósticos disciplinares que eram dados sobre o pós-moderno até então. A exposição de Lyotard busca responder a posição do saber nas sociedades mais desenvolvidas (pesquisa proposta pelo Conselho das Universidades junto ao governo de Quebec, a pedido do seu presidente). Embebido pelo clima de grande reação às tradições marxistas e comunista na França, em especial, à noção de totalidade (JAMESON, 2004, p. X), e tributário das reflexões de Daniel Bell, Alain Touraine e Ihab Hassan, o filósofo francês explica que o advento do pós-moderno se relaciona a amplas mudanças ocorridas a partir do final dos anos de 1950: o saber muda de estatuto ao mesmo tempo em que as sociedades entram na “idade pós-industrial” e a cultura na “era pós- moderna” (LYOTARD, 2000, p. 3). Essa é a resposta lyotardiana em meio a um período crítico do capital quando o esgotamento da sua expansão econômica após a 2ª Guerra Mundial e a configuração do Estado de Bem-Estar se tornaram nítidos. Em meio a essa crise, a recuperação de orientações neoliberais, a flexibilização produtiva e a substituição da automação baseada na eletromecânica pela aparelhagem eletrônica (informática e robótica) se transformaram nas bases principais de recomposição capitalista. Segundo Callinicos (2002), o status de A condição pós-moderna consiste no entrelaçamento entre arte pós-moderna, filosofia pós-estruturalista e teoria social pós-industrial, fato que, ao nosso ver, 75 erminal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate Segundo Anderson (1999), Habermas ajudou a selar a autoridade filosófica do debate sobre o pós-moderno. Mas essa validação complementou-se com a intervenção da academia estadunidense. No mesmo ano em que Lyotard publicou, na França, A condição pós-moderna, o neopragmático Richard Rorty lançou, nos EUA, Filosofia e espelho da natureza, no qual apresentou argumentos relacionados em especial à produção do conhecimento, que foram importantes para o desenvolvimento de aspectos filosóficos do pós-moderno. Rorty (1994) não chega a negar a existência da realidade, mas nega a possibilidade de a ela ter acesso fora do âmbito pragmático de descrições culturais particulares. O autor afirma que a noção de um espelho da natureza desanuviado, de um ser humano que conhece o fato, é uma imagem de Deus. Assim, “[...] a objetividade deveria ser vista como conformidade às normas de justificação (para asserções para ações) que encontramos sobre nós” (RORTY, 1994, p. 355). A contribuição de Rorty no debate sobre o pós-moderno ocorreu a partir de sua defesa de um pragmatismo cultural contextualista. Esse diálogo triangular não só legitimou o pós-moderno como tema da filosofia contemporânea, como propiciou a constituição dessa tendência como uma agenda, tendo em vista que, apesar de motivações e argumentações distintas, esses três intelectuais moviam-se dentro do amplo espectro da virada linguística3 de inspiração pragmática. Nesse sentido, o conhecimento não retrata a objetividade, mas as regras e jogos culturais (transcendentais ou contextuais) que constituem os acordos linguísticos. Há, nesses posicionamentos, um antirrealismo, não porque se nega a realidade objetiva; mas não reconhece essa realidade como passível de ser apreendida por um esforço gnosiológico. Há que se reconhecer também que, já nesses primórdios, essa agenda abrangeu controvérsias alimentadas pela prática social histórica. Em 1979, Lyotard era peremptório em suas posições a favor da deslegitimação do relato de emancipação, da equivalência de saberes, da relativização da verdade e da pluralidade ontológica. Sua ênfase no “determinismo local” (2000, p. XVI), na diferença e incomensurabilidade dos jogos de linguagem representavam, segundo ele, uma guerra contemporânea ao “terrorismo da noção de totalidade” (LYOTARD, 1993, p. 46). Difícil não se indagar “[...] por que então, justo no momento histórico em que esse sistema estava se tornando mais „total‟ que nunca, alguns intelectuais radicais começaram a denunciar toda a noção de totalidade como um sonho ruim” (EAGLETON, 1998, p. 125). Sua afirmação da incompletude do projeto moderno reage contra o relativismo e reforça a vitalidade de noções como razão, universalidade e emancipação, mesmo que em um horizonte pragmático- discursivo. Além disso, apresentar o encontro do pós-moderno com proposições antimodernas como uma aliança no interior do conservadorismo foi algo inédito e desconcertante para as análises do momento. Como indica Callinicos (2002, p. 93), “A idéia de que o pós-estruturalismo devia ser visto como um parente da nostalgia conservadora de uma ordem orgânica pré-capitalista encontrou muita resistência nos países de fala inglesa, onde a recepção de Foucault, Deleuze e (em menor medida) Derrida ocorreu principalmente por intelectuais de esquerda”. 76 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 A disseminação da agenda pós e de seus impasses A partir desse debate inaugural, essa agenda assumiu vários caminhos. Ao longo dos anos de 1980, houve a penetração do pós-moderno em vários círculos de discussões sobre diferentes temas e a partir de diversos campos do conhecimento. A análise do pós-moderno diluiu-se em discussões específicas de algumas áreas. Na área da História, por exemplo, ela deu o tom de polêmicas acerca da relação Literatura/História e fato/ ficção, da micro-história (cf. DOSSE, 1992) e mesmo das revisões historiográficas sobre a Revolução Francesa (cf. HOBSBAWM, 1996; DOSSE, 1992). A questão da cultura, aos poucos, tornou-se proeminente. Temas como “o Outro”, a diferença/identidade cultural, a relação Oriente e Ocidente, foram trazidos e problematizados pelos Estudos Culturais ingleses, pelo multiculturalismo norte-americano e pós-colonialismo. Por sua vez, a discussão sobre a cultura realizada por essas perspectivas se entrecruzou fortemente com os debates da “sociologia da ciência”, especialmente com o chamado “construcionismo social” e sua defesa de simetria entre ciência e não- ciência, verdade e falsidade, sociedade e natureza (cf. LATOUR, 1994). Contudo, a intensidade do debate sobre o pós-moderno não diz respeito apenas a sua disseminação cada vez mais ampliada na academia; houve também um aumento da sua tensão, muito alimentada não apenas por motivações gnosiológicas, mas também políticas e éticas. Basta lembrar a “impostura” de Alan Sokal4 (com sua repercussão internacional) e toda uma onda de revisionismo ou negacionismo do Holocausto, envolvendo, por exemplo, a “querela dos historiadores alemães” na segunda metade da década de 1980 cuja polêmica mais representativa se deu entre Ernst Nolte e Habermas (cf. SCHURSTER; LEITE, 2021) e o debate, também no campo historiográfico, entre Hayden White e Carlo Ginzburg sobre a relação história, ficção, fato, verdade e relativismo nos anos de 1990 (cf. CUNHA, 2014). Entre a segunda metade da década de 1980 e primeira metade dos anos de 1990, o debate sobre o pós-moderno teve um novo ingrediente, sendo atravessado pelos anúncios comemorativos de “fim da história”, como o de Francis Fukuyama em 1989, intensificados com a queda do Muro de Berlim (1989-1991). Cabe, no entanto, destacar que, já no final dos anos de 1990, o termo pós-moderno estava bastante escoriado. Além da sua rejeição por alguns pós-estruturalistas, Lyotard reforçou sua relação ambígua esse vocábulo (cf. LYOTARD, 1996 [1993]). Por sua vez, Rorty (1999 [1995], p. Debate Dessa forma, “Não buscar a totalidade representa apenas um código para não se considerar o capitalismo” (EAGLETON, 1998, p. 20). A posição de Lyotard era por demais celebratória ou cínica diante de um contexto de recomposição mundial do capitalismo. Por essa razão, já em surgimento, observa-se, na agenda pós, um mal-estar com a renúncia de ideais de emancipação e autonomia (cf. LYOTARD, 2000); em função disso, desenvolve-se uma espécie de contestação social que avista um leque de variações, mas nos marcos do capitalismo: a defesa de utopia liberal como pertencente à esquerda reformista (cf. RORTY, 2001) ou de uma relação entre ação comunicativa e reformismo radical a partir do vínculo democracia e soberania popular no seio de uma esquerda não-comunista (HABERMAS, 1991). 77 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate Debate A novidade que a virada para o século XXI trouxe foi a de reforçar essa atmosfera de desfalecimento do pós-moderno. Considerando a popularização do termo, não é de surpreender que os anúncios mais marcantes desse processo tenham vindo de jornais, mas após um evento bastante peculiar: o ataque às Torres Gêmeas em 11 de setembro de 2001 nos Estados Unidos. Em 22 de setembro de 2001, Rothstein (2001) afirma, em The New York Times, que a destruição do World Trade Center e o ataque ao Pentágono podem ter efeitos similares de desafiar ética e intelectualmente o pós-modernismo e o pós-colonialismo. As asserções pós-modernistas de não existência de validade objetiva para a verdade e o julgamento ético e a defesa pós-colonialista de que os princípios universalistas do Ocidente são construtos ideológicos “[...] parecem peculiares quando tentam prestar contas do recente ataque. Essa destruição dá a impressão de implorar por uma perspectiva ética transcendental. E mesmo o relativismo mais suave parece preocupante […]” (ROTHSTEIN, 2001, p. A17). Dias depois, em artigo da Chicago Tribune, a jornalista Julia Keller afirma, na mesma direção: “O que se encontra nos escombros na mais profunda Manhattan, no corte profundo no Pentágono e no campo no sudeste da Pensilvânia pode ser, entre tantas outras coisas, isso: o fim do pós- modernismo […]” (KELLER, 2001, p. 2). Como lembra Moraes (2004), esse debate ganhou espaço, no Brasil, no Caderno Mais!, do jornal Folha de São Paulo, de 2 de novembro de 2003, tendo como mote o lançamento do livro After theory de Terry Eagleton (2003). Para o filósofo e crítico literário britânico, “com a nova narrativa global do capitalismo, juntamente com assim chamada guerra contra o terror, é bem possível que o estilo de pensamento conhecido como pós-modernismo esteja chegando ao fim” (EAGLETON, 2003, p. 221). As duas décadas que nos separam desse anúncio nos permite afirmar que a agenda pós- moderna ainda está distante de óbito ou, se, de fato, ela faleceu nos escombros das Torres Gêmeas, sua ressurreição foi muito rápida. Della Fonte (2006, p. 26) chegou a afirmar no esteio dessa reflexão: “o tema do pós-moderno e a sua vitalidade parece renovar-se mesmo quando se anuncia a sua morte”. A disseminação da agenda pós e de seus impasses 13-14) afirmou que, apesar de ter utilizado o pós-moderno com certa frequência (e o fez no sentido lyotardiano de incredulidade quanto às metanarrativas), preferiria não tê-lo feito: “O termo pós- moderno tem sido tão superutilizado que vem causando mais confusões do que trazendo esclarecimentos”. Em seu lugar, ele (1999, p. 13-20) optou pela expressão “filosofia pós-nietzschiana”. Já Latour (1994) sugeriu que nunca fomos modernos e, portanto, não há sentido em falar em pós- moderno. 78 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate Ao seguir essa linha de argumentação, Della Fonte (2010) observa que agrupar diversas correntes intelectuais contemporâneas com trajetórias históricas próprias e categorias e conceitos peculiares em uma agenda ampla é passível de críticas. Contudo, evidencia a autora, há inúmeras tentativas, não apenas de críticos, mas inclusive de estudiosos que se identificam com essa perspectiva, de indicar possíveis aproximações ou eixos comuns. Assim, pontua: Com o termo “agenda pós-moderna”, acompanho esse movimento e sugiro um eixo comum compartilhado por essas diferentes perspectivas. Não se trata de igualá-las em suas proposições, mas de indicar que, na diversidade de seus argumentos, podem ser identificadas algumas aproximações e semelhanças que, a meu ver, manifestam a atmosfera político-intelectual vigente (DELLA FONTE, 2010, p. 50). Wood (1995, 1999) caracteriza a agenda como expressão da trajetória de uma parcela de intelectuais de esquerda cuja consciência política de reforma ou “humanização” do capitalismo se formou a partir do término da 2ª Guerra mundial e se atualizou com as reconfigurações do capitalismo no final do século XX. Considera que, mesmo tendo versões mais ou menos extremas, é possível dizer que a agenda pós-moderna se interessa por temas como linguagem, cultura e discurso. A linguagem é tudo que se pode conhecer do mundo, não se teria acesso a qualquer outra realidade (por mais que se possa admitir a existência de uma realidade objetiva): “[...] e, uma vez que todos nós somos dela cativos, nenhum padrão externo de verdade, nenhum referente externo para o conhecimento existe para nós, fora dos „discursos‟ específicos em que vivemos” (WOOD, 1999, p. 11). Nesse sentido, a ciência não pode pretender aproximar-se ou apreender um mundo externo; ela seria um discurso vinculado aos princípios imperialistas e opressivos, pilares da sociedade ocidental. Assim, em contraste com conhecimentos tidos como totalizantes e valores universais: “[...] os pós-modernistas enfatizam a „diferença‟: identidades particulares, tais como sexo, raça, etnia, sexualidade; suas opressões e lutas distintas, particulares e variadas; e „conhecimentos‟ particulares, incluindo mesmo ciências específicas de alguns grupos étnicos” (WOOD, 1995, p. 8). No Brasil, Maria Célia Marcondes de Moraes (1996, p. 47) destaca que esse amplo grupo, além de contrapor-se às propostas da Ilustração, associadas usualmente ao “moderno”, apresenta-se como “[...] arauto da indeterminação total, do caráter fragmentário, desintegrado, heterogêneo, descontínuo e plural do mundo físico e social [...]”. Agenda pós-moderna e seus eixos Apesar dos ecos analíticos da obra de Lyotard reverberarem até os dias atuais, a agenda pós- moderna está longe de se limitar a suas formulações teóricas inaugurais. Ao tratar o discurso pós- moderno como uma agenda, Wood (1995) busca evidenciar um rol de compromissos e temas que perpassam uma vasta gama de tendências intelectuais e políticas que surgiram em anos recentes. Portanto, o termo agenda pós-moderna tem o mérito de ampliar o foco de análise, uma vez que não remete a uma doutrina homogênea em termos de unidade conceitual. Nesse sentido, ela não se reduz ao que classicamente ficou conhecido como discurso pós-moderno: o pensamento lyotardiano. 79 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 A tentativa de sobrevivência de uma esquerda pós-moderna A capacidade da agenda pós de ressignificar-se e renovar-se comprovam o que Moraes, nos inícios dos anos 2000, chamava de seu “renovado conservadorismo” e permanência de sua vitalidade: Assim, se é difícil conceber a sobrevida de um pensamento pós-moderno tal como manifesto por seus primeiros representantes – que, de modo geral, asseveravam a derrota do projeto iluminista de emancipação, a mentira do progresso histórico, a fragilidade do sujeito, a impossibilidade da verdade, a negação do real, a recusa de fundamentos, rejeitavam as grandes narrativas, denunciavam a ciência e a racionalidade ocidental como imbricadas à estrutura de poder, à razão instrumental e à dominação –, é nítida a sobrevida de facetas da agenda pós-moderna como, por exemplo, suas vertentes culturalistas e neopragmáticas (MORAES, 2004, p. 340). O diagnóstico da autora ainda é válido após quase duas décadas. Mas a ele precisamos acrescentar outras reflexões. Mesmo sem renunciar ao seu antirrealismo, de modo aberto ou velado (cf. MEDEIROS, 2021), essa agenda tem aglutinado, cada vez mais, posições ético-políticas divergentes. Parece-nos que sua sobrevida tem decorrido da insistência de alguns de seus segmentos de esquerda. Desaceleremos para compreender essa hipótese. A constatação de Moraes em relação a vertentes culturalistas e neopragmáticas na década de 1990 e início dos anos 2000 tinha como alvo principal Richard Rorty. Como observa Medeiros (2021), dentre os vários pensadores da agenda pós, o neopragmático ocupa um lugar de destaque: Primeiro, Rorty é um filósofo extraordinariamente dotado, instruído, com um saber enciclopédico na área. Segundo, sua filosofia, por mais controversa que seja, é reconhecidamente coerente, difícil de ser atacada. Por fim, Rorty se apresenta como um pensador de esquerda (RORTY, 2002, p. 28) – um ralwsiano de esquerda – ao mesmo tempo em que constrói uma filosofia com o propósito confesso de defender e embasar o que ele chama de democracias do Ocidente, um eufemismo para designar o capitalismo neoliberal (MEDEIROS, 2021, p. 455). Primeiro, Rorty é um filósofo extraordinariamente dotado, instruído, com um saber enciclopédico na área. Segundo, sua filosofia, por mais controversa que seja, é reconhecidamente coerente, difícil de ser atacada. Por fim, Rorty se apresenta como um pensador de esquerda (RORTY, 2002, p. 28) – um ralwsiano de esquerda – ao mesmo tempo em que constrói uma filosofia com o propósito confesso de defender e embasar o que ele chama de democracias do Ocidente, um eufemismo para designar o capitalismo neoliberal (MEDEIROS, 2021, p. 455). Debate Desse modo, em um contexto de grande avanço tecnológico-informacional e de afirmação da ideologia e de políticas neoliberais, essa agenda expressa a atmosfera político-intelectual com bases pós-metafísicas e da virada linguística (DELLA FONTE, 2010). Se, por um lado, a adoção do termo agenda mostra a complexidade que tal tendência alcançou ao aglutinar, em seu grande guarda-chuva, diversas construções teóricas, por outro, as feições dessa agenda não estão estáticas, o que torna seu tratamento ainda desafiador. Como pontua Moraes (2004), “[...] como qualquer pensamento, os que compõem a agenda pós-moderna, modismos ou não, transmutam-se no decorrer da história” (MORAES, 2004, p. 340). Seduzido pela “virada lingüística”, supõe “[...] que há uma anterioridade da linguagem em relação ao mundo real e, assim, o que se pode experimentar como 'realidade' nada mais seria do que um constructo ou um 'efeito' do sistema particular de linguagem ao qual pertencemos” (MORAES, 1996, p. 50). De acordo com Ahmad (1997), longe de indicar uma nova época histórica, a “condição-pós” expressa um clima intelectual generalizado, senão dominante, do pensamento social e político euro- americano. Sua peculiaridade reside não apenas no alívio, mas na euforia diante do colapso (considerado definitivo) dos três principais projetos de emancipação universal do século XX: o nacionalismo anti-imperialista, a democracia social de esquerda e o comunismo. 80 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate organismos multilaterais e no conjunto de reformas da educação brasileira nos últimos anos” (MORAES, 2009, p. 323-324). Se considerarmos a atual Base Nacional Comum Curricular e Reforma do Ensino Médio, não há dúvidas de que Rorty continua uma presença ausente. Contudo, mesmo com proximidades das reflexões neopragmáticas, intelectuais pós-modernos do campo democrático antineoliberal tem se incomodado com seu etnocentrismo manifesto. Em um dos capítulos do livro Epistemologias do Sul, organizado por Boaventura de Sousa Santos e Meneses, Maldonado-Torres (2010, p. 357) assevera que “Nos EUA foi mais o patriotismo do que, propriamente, o cristianismo que serviu de alicerce aos liberalistas pragmáticos de tendência esquerdizante, como Richard Rorty e Cornel West”. Como manter as máximas da virada linguística de cunho pragmático sem cair, por um lado, no relativismo e, por outro, no etnocentrismo? A resposta rortyana contra o relativismo não agradou à essa esquerda pós-moderna que tem revisitado as polêmicas e os impasses dessa agenda, como o abandono dos ideais emancipatórios, o esvaziamento da crítica, a incomensurabilidade dos jogos de linguagem e o relativismo. Em outros termos, tem elaborado novas respostas a temas, no mínimo, desconfortáveis para essa agenda. Em função disso e sem perder seus inimigos externos (o “moderno”, o Iluminismo, as tradições marxistas), essa agenda passou a se diversificar ainda mais. Contra o relativismo, essa esquerda tem se posicionado em prol dos oprimidos e comunidades subalternas (McLAREN, 2000, 2005), das epistemologias do Sul (SANTOS; MENESES, 2010), da luta contra a dominação neocolonial (SAID, 1990) e da articulação das lutas sociais em nível global. Essas várias adaptações e renovações tem atualizado a agenda pós, mas sem abandonar o vínculo com a virada linguística e o descrédito da ciência. Basta lembrar da emblemática visão de ciência de Boaventura de Souza Santos para quem: Sendo um modelo global, a nova racionalidade científica é também um modelo totalitário na medida em que nega o caráter racional a todas as formas de conhecimento que não se pautarem pelos seus princípios epistemológicos e pelas suas regras metodológicas (SANTOS, 2008, p. 21). A ciência moderna não é a única explicação possível da realidade e não há sequer qualquer razão científica para a considerar melhor que as explicações alternativas da metafísica, da astrologia, da religião, da arte ou da poesia. A razão por que privilegiamos hoje uma forma de conhecimento assente na previsão e no controlo dos fenómenos nada tem de científico. A tentativa de sobrevivência de uma esquerda pós-moderna Moraes (2004, p. 345) já considerava a sofisticação do neopragmatismo rortyano, segundo suas palavras, “o mais vivo e penetrante” de todos os rumos teóricos e práticos dessa agenda. Sua reflexão, porém, era incisiva: considerando a predominância de uma utopia praticista, do privilégio da eficácia, da manipulação do tópico e imediato, enfim, da lógica pragmática na vida social e na educação, Rorty revela-se como “[...] “presença ausente” em boa parte dos documentos elaborados pelos 81 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. Debate com atuação política no campo democrático. Mas indica que sua leitura simplória do marxismo representa “receita nova, pudim velho”: “Correndo o risco de cometer uma indelicadeza formal, a leitura de Marx por Sousa Santos pode ser caracterizada como uma receita nova – com ingredientes como Modernidade, paradigma etc. que culmina na feitura de um pudim cujo gosto se conhece há muito determinismo, evolucionismo, reducionismo econômico” (NETTO, 2004, p. 237). Para o autor, o seu antimarxismo influencia na própria intenção de representar um novo paradigma de oposição: “a sua análise da teoria marxiana é de todo incompetente para fundar uma interpretação que dê conta, minimamente, da fecundidade ou não daquela teoria para enfrentar os grandes desafios contemporâneos” (NETTO, 2004, p. 238). Por sua vez, ao discutir o orientalismo do intelectual e ativista político palestino- estadunidense Edward Said e sua posição contra o neocolonialismo europeu, Medeiros (2021, p. 453) observa: “causa certa pelas linhas tortas”. Essa parece ser uma síntese acertada acerca dessa esquerda pós-moderna. Sob esse aspecto, seguindo a provocação de Della Fonte (2006), talvez seja mais apropriado recorrer àquilo que Lukács usou para descrever tanto a sua passagem do neokantismo para o hegelianismo em seu livro A teoria do romance, quanto para evidenciar sua crítica à filosofia sartreana: os pós-modernos aglutinam uma “ética de esquerda” a uma “epistemologia de direita”, ou seja, uma ética progressista a uma exegese convencional da realidade. Novamente, Lukács convida a pensar o quanto a objetivação do pensamento filosófico, muitas vezes, não obedece às intenções dos seus autores, assim como ocorre no âmbito literário. Mas aqui um novo cuidado deve ser tomado: a ética de esquerda que se encontra no Lukács d‟A teoria do romance ainda sustenta a noção de revolução, mesmo que inclinado à cambiante atitude política e filosófica do anticapitalismo romântico. Com o término da Segunda Guerra e a derrota de Hitler, a ética de esquerda diluiu-se e cedeu lugar a uma forma de “conformismo disfarçado de não-conformismo” (LUKÁCS, 2000, p. 18). É muito mais nesse sentido que se pode falar da fusão de uma “ética de esquerda” e uma “epistemologia de direita” por essas frações da “agenda pós”. É um juízo de valor (Em outro texto, no mesmo volume (SANTOS, 2008, p. 83-84). Sendo um modelo global, a nova racionalidade científica é também um modelo totalitário na medida em que nega o caráter racional a todas as formas de conhecimento que não se pautarem pelos seus princípios epistemológicos e pelas suas regras metodológicas (SANTOS, 2008, p. 21). A ciência moderna não é a única explicação possível da realidade e não há sequer qualquer razão científica para a considerar melhor que as explicações alternativas da metafísica, da astrologia, da religião, da arte ou da poesia. A razão por que privilegiamos hoje uma forma de conhecimento assente na previsão e no controlo dos fenómenos nada tem de científico. É um juízo de valor (Em outro texto, no mesmo volume (SANTOS, 2008, p. 83-84). Evitando o vínculo com a tradição marxista, mas sem ratificar a celebração do fim da história, segmentos da agenda têm “salvado” seu verniz progressista. Disso decorreram proposições, como a de um “multiculturalismo pós-moderno crítico” ou revolucionário (cf. McLAREN, 2000, 2005) ou um “pós-modernismo inquietante ou de oposição” (cf. SANTOS, 1997, p. 35). Como avaliar o rumo assumido por essa parcela da agenda? Dentre os vários caminhos possíveis para tratar essa questão, lembramos duas análises específicas. José Paulo Netto (2004) reconhece a magnitude e renome de Boaventura de Sousa Santos como um intelectual progressista 82 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. Debate de amalgamá-las (cf. SILVA, 1993, 1996, 2002), as teorias educacionais pós-críticas ganharam identidade própria no final da década de 1990 e, assim, explicitaram sua contraposição às teorias educacionais críticas (cf. CORAZZA, 1999; SANTOS, 2000; PARAÍSO, 2004). Foi dentro desse espírito que o pós-moderno se disseminou, de maneira capilar, em diversos campos investigativos da pesquisa educacional brasileira (cf. PARAÍSO, 2004). O termo pós-crítico não é apenas vago e impreciso. Ele porta, em sua imanência, um embaraço insolúvel. Abdicar da ou esvaziar a função crítica é um impasse sem solução dentro do horizonte intelectual progressista. Como alerta Duayer (2003, p. 5), [...] doutrinas que à primeira vista propugnam a relatividade, a indiferenciação, a equiparação de crenças, a total tolerância e o absurdo pluralismo, negam, pela lógica de sua própria construção, a possibilidade de crítica e, ipso facto, alimentam toda sorte de dogmatismo. A defesa da qualificação pós-críticas para as teorias educacionais alinhadas à agenda pós- moderna tem se mostrado frágil por várias razões. Por exemplo, ela torna inexata a equação compromissos com grupos sociais oprimidos e com algum tipo de mudança social e descarte do lugar de criticidade e da função de criticar. A crítica é um exercício fundamental para a tomada de posição e para o abandono do relativismo. Há que se considerar também um outro problema: a qualificação de pós-críticas para esse aglomerado de tendências intelectuais da educação não tem suporte em alguns filósofos e cientistas sociais aos quais recorrem as teorias educacionais pós-críticas. Longe de autorizar ou validar essa nomenclatura, esses autores não renunciaram à crítica e inclusive defendem uma “teoria crítica pós- moderna” (BOAVENTURA, 1999, p. 204) ou um “pensamento crítico” sob a forma de “uma ontologia de nós mesmos, de uma ontologia da atualidade” (FOUCAULT, 1994, p. 687-688). Ainda existe outro fenômeno curioso: estudiosos da educação de tradição anglófona (alguns dos quais apropriados pelas teorizações educacionais pós-críticas no Brasil) também não legitimam a nomenclatura pós-crítica, pois defendem uma concepção dilatada e profusa de pedagogia crítica, acreditando não só que ela envolve discursos pós-modernos, como também tem se beneficiado desses discursos. Movimentos emancipatórios “anti-racistas e pós-coloniais, formas radicais de multiculturalismo, homossexuais e lésbicas, múltiplas vozes feministas, neomarxistas e socialistas democratas, „verdes‟ etc.” podem assumir uma “unidade descentrada”: “Múltiplos projetos progressistas, múltiplas „pedagogias críticas‟ são articuladas” (APPLE, 2002, p. 135). Rearranjos do campo educacional brasileiro diante dos rumos da agenda pós-moderna Apesar de sua formulação filosófica datar do final da década de 1970, a chamada agenda pós adentrou a produção do conhecimento no campo educacional brasileiro no início dos anos de 1990. De acordo com Paraíso (2004, p. 285), os primeiros trabalhos apresentados, a partir das formulações pós-modernas, na reunião anual da Associação Nacional de Pós-Graduação e Pesquisa em Educação (ANPED) e o primeiro livro publicado com essa perspectiva no Brasil (SILVA, 1993) são de 1993. Nesse momento inicial, a diferenciação entre moderno e pós-moderno foi traduzida, para o campo educacional, pela distinção entre pedagogias críticas e pós-críticas. Apesar das tentativas iniciais 83 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Nessa direção, McLaren e Jaramillo (2007) reforçam a noção de uma pedagogia crítica revisitada capaz de lutar contra a integração da esfera pública nas práticas neoliberais e imperialistas do Estado e do capitalismo globalizado. Sob esse aspecto, a pedagogia crítica é anti-imperialista, antirracista, anti-homofóbica e sintonizada com outras reivindicações de grupos oprimidos (McLAREN, 2005). Sob influência da academia anglófona, Fischman e Sales (2010, p. 12-13) explicam: Sob influência da academia anglófona, Fischman e Sales (2010, p. 12-13) explicam: 84 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate Referimo-nos às pedagogias críticas como um conglomerado de perspectivas que tomam emprestados princípios e orientações dos ideários de John Dewey, da Escola de Frankfurt da Teoria Crítica, de Antônio Gramsci, de Paulo Freire, das perspectivas feministas, dos modelos antirracistas e até da educação popular e os aplicam à análise das instituições educativas. As pedagogias críticas têm presença importante nos cursos de formação de professores estadunidenses, embora gerem grandes resistências entre os professores nas escolas (Darder, 2002; Darder, Baltodano & Torres, 2003; Giroux, 1988, 1994, 2000, 2003; Giroux & McLaren, 1989; McLaren & Lankshear, 1993, 1994; McLaren & Fischman,1998; McLaren, 2005; McLaren & Kincheloe, 2007). A elasticidade do termo pedagogia crítica se vincula à equivalente maleabilidade e fluidez da expressão Teoria Crítica (Critical Theory) na academia anglófona (em especial, no Reino Unido e nos EUA), cujo sentido, por sinal, é bastante endógeno a esse ambiente e, segundo Jeanpierre (2010), não tem equivalência ou eco na Europa Continental. Na estranha e palatável classificação bibliográfica de Critical Theory, cabem filósofos, literatos, historiadores, etnólogos e sociológicos europeus ou americanos, como Adorno, Agamben, Althusser, Anderson, Balibar, Bataille, Baudrillard, Benjamin, Bey, Butler, Caillois, Camus, Deleuze, Derrida, Eagleton, Jameson, Rancière, Saïd, Sartre, Spivak, Virilio, Williams, Žižek, entre outros. Na Enciclopédia de Filosofia de Stanford, James Boham (2021) explica que o verbete Teoria Crítica possui um sentido estreito e amplo. Na acepção estrito, designa várias gerações de filósofos e teóricos sociais alemães da tradição marxista da Europa Ocidental conhecida como Escola de Frankfurt. Inspirada pela luta pela emancipação humana que, segundo Boham (2021) qualifica a teoria crítica no seu sentido mais estreito, muitas teorias críticas, articuladas a movimentos sociais, se desenvolveram a fim de explicar e transformar as circunstâncias que escravizam os seres humanos. Debate LOPES, 2013; RIBEIRO, 2016, 2017). A perspectiva é de que, apesar de heterogêneas, essas teorias educacionais podem se complementar: LOPES, 2013; RIBEIRO, 2016, 2017). A perspectiva é de que, apesar de heterogêneas, essas teorias educacionais podem se complementar: Apesar de ênfases distintas, tais vertentes, internamente também heterogêneas, podem operar sob diálogo em determinados pontos. Acreditamos que a temática do conhecimento pode servir a essa lógica que hibridiza perspectivas crítica e pós- crítica de currículo, que a acolhem como central a partir de ênfases distintas, mas que não se configuram como pólos antagônicos de análise (RIBEIRO, 2017, p. 577). Não deixa de ser paradoxal que “juntar” teorias críticas e pós-críticas foi a proposta inicial de Silva quando introduziu, no início dos anos de 1990, o debate pós-moderno na educação brasileira. Depois de ganharem identidade própria e se contraporem às teorias educacionais críticas no final da década de 1990, causa surpresa entrar em cena novamente essa aproximação “híbrida”. Estaríamos diante de um retorno histórico? De modo algum. A proposta de hibridização parece-nos mais uma das acrobacias da agenda pós na educação não apenas para lidar com seus limites e impasses de ordem teórica, mas também para sobreviver à interpelação da história hodierna. A nosso ver, a relação orgânica entre neoliberalismo e neoconservadorismo, assim como a recente pandemia mundial de Covid-19 têm tornado mais evidentes e profundas as aporias e os (des)caminhos dessa agenda. “[...] tanto no sentido amplo quanto no sentido estrito, uma teoria crítica fornece as bases descritivas e normativas para a investigação social destinada a diminuir a dominação e aumentar a liberdade em todas as suas formas” (BOHAM, 2021, s.p.). Emancipação humana contra opressão e dominação: “qualquer abordagem filosófica com objetivos práticos semelhantes poderia ser chamada de “teoria crítica”, incluindo feminismo, teoria racial crítica, e algumas formas de crítica pós-colonial” (BOHAM, 2021, s.p.). A absorção da proposta elástica de uma pedagogia crítica a partir do modelo anglófono tende a invalidar a designação de pós-crítica e a despedida de uma educação crítica. Mas não apenas isso. A expansão sobre o que considera pedagogia crítica possui, em geral, um enfoque plural vinculado ao antirrealismo (cf. BIESTA, 1998; LOPES, 2015), posição que mais se aproxima de uma estratégia malabarista de sustentar a sobrevida de argumentos da agenda pós nesse novo rearranjo de compromisso com a emancipação social e com grupos oprimidos. Fronteiriça a esse caminho, há no Brasil uma tendência que tem buscado aliar teorias educacionais críticas e as pós-críticas. No campo dos estudos curriculares, chamam-se híbridas as teorias que, sem abandonarem a noção pós-moderna de pluralidade cultural, conferem importância à centralidade do conhecimento, como defendida pelas teorias críticas (cf. MOREIRA, 2007, 2009, 2010; 85 erminal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate (para capturar consumidores para um determinado conteúdo e mantê-lo vinculado a esse conteúdo) foi usado em termos políticos: “O objetivo inicial foi comercial, e não o de criar instrumentos para a ascensão da extrema-direita, por exemplo. Mas a consequência residual, o efeito colateral, acabou sendo jogar as pessoas, cada vez mais, em bolhas” (BARBOSA, 2022). Cada pessoa parece habitar um mundo que só faz confirmar suas crenças, cada um vive em um mundo erigido por suas crenças: “Na pós-verdade, mais do que interpretar e significar o acontecimento, cria-se uma versão que o sujeito tende a interpretar como verdadeira ou não, independente de investigações científicas quaisquer” (SIEBERT; PEREIRA, 2020, p. 244). Esse clima de pós-verdade se entrelaçou com o peso das fake news (notícias intencionalmente falsas) e com outro fenômeno que tem se fortalecido nos últimos tempos: o negacionismo da ciência (terraplanistas, negacionistas do aquecimento global, antivacinas etc.). A conjugação entre descrédito das evidências em detrimento da verdade individual, a disseminação intencional de notícias falsas para fins de manipulação político-ideológica e o descrédito e negacionismo da ciência habitam a atmosfera de ascensão mundial da nova direita ou, parafraseando Adorno (2020), do novo radicalismo de direita. Seu traço neofascista precisa ser compreendido a partir do alerta ainda atual de Max Horkheimer (1939, p. 115): “Quem não quer falar do capitalismo deve-se calar-se também sobre o fascismo”. O fascismo não é sinal de desinformação ou um desvio da ordem democrático burguesa. Ele está entranhado à lógica do capital; ancora-se, portanto, na sua dinâmica social objetiva, mais precisamente nas suas crises orgânicas, momentos nos quais irrompe toda a irracionalidade desse modo de vida: a busca desvairada pela valorização do valor em detrimento da satisfação de necessidades humanas, a concentração de riqueza, a anarquia social etc. Como observa Andrade (2021, s. p.), a “normalidade” do capitalismo é “[...] a personificação da crise global do sistema expressando graus avançados de irracionalidade [...]”. Os rearranjos políticos e econômicos para o enfrentamento das crises não conseguem mais uma estabilização por longo tempo, o que gera situações cada vez mais caóticas. A hegemonia do capitalismo em sua feição neoliberal tem comprovado isso na medida em que se mostra incapaz de [...] conter as desastrosas consequências sociais, políticas e ambientais que só tendem a se agravar. A prática social-objetiva, o critério de verdade e o debate no plano do conhecimento Em 2016, o Dicionário Oxford elegeu o vocábulo “pós-verdade” como a palavra do ano na língua inglesa (WORD..., 2016). Até esse momento, o interesse prioritário da editora de dicionários da instituição britânica era por palavras da vida cultural e cotidiana da população anglófona. A escolha de pós-verdade representou uma mudança, pois o uso do verbete teve como solo o campo político, mais especificamente o contexto do referendo britânico sobre a União Europeia e as eleições presidenciais dos Estados Unidos, vencidas pelo republicano Donald Trump. Segundo o dicionário, pós-verdade relaciona-se ou denota “[...] circunstâncias nas quais fatos objetivos são menos influentes na formação da opinião pública do que os apelos à emoção e à crença pessoal” (POST-TRUTH, s.d.). Trata-se, portanto, de um neologismo criado para descrever como se modela a opinião pública, negligenciando fatos objetivos e apelando para o campo das emoções e crenças pessoais, dentro da lógica de que o que parece ser verdade é mais importante que a verdade. Assim, tanto no referendo sobre a separação do Reino Unido da União Europeia em 2016, como na eleição de Trump no mesmo ano, os eleitores tenderam a negligenciar aspectos objetivos da realidade e dos acontecimentos para fundamentar suas decisões. Esse comportamento coletivo não se descola do cenário econômico mundial que tem sido dominado por empresas “plataformizadas”, a partir da expansão do arcabouço maquínico- informacional-digital no mundo produtivo (ANTUNES, 2020). O sistema de algoritmos utilizados por essas firmas cria “bolhas” que isolam os usuários, fazendo-os receber somente informações que corroboram seu ponto de vista. O aperfeiçoamento dos algoritmos inicialmente em termos comerciais 86 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate Na costura neoliberalismo e neoconservadorismo, a democracia burguesa não hesita em afastar-se, sem pudor, de tonalidades liberais progressistas, em assumir traços totalitários e cultivar uma ambiência persecutória e neofascista. Essa tem sido a condição vivida no Brasil em especial na eleição presidencial de 2014, no golpe de Estado que tirou Dilma Rousseff da Presidência da República em 2016 e na ascensão vertiginosa da extrema direita no país, culminando com o governo de Jair Messias Bolsonaro como presidente em 2019. O momento atual nos permite perceber que o negacionismo do conhecimento científico ou a relativização desse saber está de mãos dadas com posições reacionárias e ultraconservadoras. Tal fato atravessou vários acontecimentos no país em sua história recente. A instalação pela presidenta Dilma Rousseff da Comissão da Verdade a partir de maio de 2012, para investigar violações de direitos humanos por parte do Estado ocorridas entre 1946 e 1988 no Brasil, foi confrontada várias vezes por argumentos relativistas. Em São Paulo, por exemplo, quando se discutiu a reabertura da Comissão Municipal da Verdade em março de 2013, o vereador Coronel Telhada (PSDB) votou contra e insistiu que cada um tem a sua verdade e, portanto, era necessário ouvir a violência sofrida pelos militares: “A verdade que está sendo apresentada é totalmente relativa. A violência que os militares sofreram na época nunca é levada em conta” (PREFEITURA..., 2014). O Escola sem Partido também se vale de tal relativização dos conhecimentos e da verdade. Nos deveres do professor, elaborados pelo movimento, encontramos: “4 –Ao tratar de questões políticas, socioculturais e econômicas, o professor apresentará aos alunos, de forma justa – isto é, com a mesma profundidade e seriedade –, as principais versões, teorias, opiniões e perspectivas concorrentes a respeito da matéria” (ESCOLA SEM PARTIDO, 2019, grifo nosso). No primeiro ano de governo, o presidente Bolsonaro também iniciou sua política anticiência com cortes de verbas para a ciência5 e de bolsas para a pós-graduação.6 O seu segundo Ministro da Educação Abraham Bragança de Vasconcellos Weintraub fez ataques constantes às instituições federais de ensino: interferiu na nomeação de reitores/as, acusou-as de ter plantações extensivas de maconha e anunciou cortes de verbas do orçamento, em especial das que estivessem promovendo “balbúrdia” (cf. WEINTRAUB..., 2019; KER, 2020). [...] Enquanto isso, parcelas significativas das massas em desespero e desesperançadas, assim como a classe média tendem abraçar o autoritarismo, o negacionismo, o fanatismo religioso, o anticomunismo que se expande através de movimentos políticos neofascistas, que emergem como filhos legítimos e intrínsecos da crise do capitalismo e do neoliberalismo (ANDRADE, 2021, s.p.). Nesse contexto, estabelece-se uma forte aliança entre interesses empresariais e setores neoconservadores: “[...] o neoconservadorismo é, portanto, perfeitamente compatível com o programa neoliberal de governança pela elite, desconfiança da democracia e manutenção das liberdades de mercado” (HARVEY, 2014, p. 92). Nas palavras de Harvey (2014, p. 92), “[...] o neoconservadorismo se assemelha a uma mera retirada do véu de autoritarismo com que o neoliberalismo se cobre”. 87 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. Em 2019, sua política anticiência também se manifestou na sua recusa de conduzir a vacinação em massa contra a Covid-19, fato que resultou em mais de 600 mil mortes em decorrência dessa doença no país. De certa maneira, confirmou-se o alerta dado anteriormente: quando os pressupostos pós são levados às últimas consequências, o fortalecimento não é das vozes minoritárias e invisibilizadas pela narrativa única, mas das posições conservadoras que se mantêm tão legítimas como quaisquer outras e sem poderem ser submetidas à crítica a partir da objetividade social. Como bem coloca Medeiros (2021), não se trata de culpar a agenda pós-moderna, em especial sua tendência de esquerda pela ascensão da extrema direita. Contudo, visto sob a perspectiva da totalidade dos fenômenos que marcam nossa época, também não é possível isentá-la de, em termos ideológicos, ser constituída por e constituir o capitalismo contemporâneo. Desse modo, 88 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Debate [...] não se deve deixar de apontar o que hoje podemos julgar como um suicídio ideológico: minar a capacidade da crítica científica de influenciar o pensamento cotidiano, a visão de mundo corrente. O fundamental, portanto, é deixar claro que todo proferimento ideológico tem significado prático, podendo alcançar um sentido e uma dimensão distintos do que o pretendido na formulação original. Não existem brincadeiras filosóficas, ao menos se as ideias que as fundamentam demonstram-se capazes de percorrer o tecido social. Se se ataca durante décadas a principal arma discursiva em favor da emancipação do gênero humano, a ciência, ela acaba de fato desprestigiada (MEDEIROS, 2021, p. 445). Registramos também que, ao abordar alguns dos rumos e movimentos da agenda pós, ficamos longe da intenção tanto de detalhar todas as suas sobrevidas ou ressurreições contínuas, como de anunciar mais um de seus fins. Como visto, as próprias confusões que muitas vezes foram (e têm sido) geradas no seio dessa agenda serviram de alimento e impulso para o seu desenvolvimento. Portanto, suas aporias e seus malabarismos podem se tornar não uma fragilidade, mas a força para a renovação de seu conservadorismo. Por fim, pontuamos que estamos diante de uma brecha histórica favorável à luta contra argumentações como as da agenda pós. Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. Nos últimos anos, em função do avanço de forças reacionárias em termos mundiais e da pandemia de Covid-19, a reação contra qualquer relativismo da verdade e depreciação da ciência tem sido assumida por vários segmentos sociais progressistas. Não por acaso várias manifestações de 2017 a 2021 ratificaram a defesa do conhecimento científico, de seus orçamentos públicos e de suas conquistas. Alguns cartazes dessas manifestações sinalizam isso em seus dizeres: “Pesquisa não é balbúrdia” “Esqueçam as princesas, eu quero ser astrofísica” “Sem ciência o Brasil não avança” “Sem ciência não há futuro” “Sem ciência não há consciência” “No começo de todo filme de desastre tem cientistas sendo ignorados” Debate Vivemos uma condição na qual a asserção pós-moderna de não existência de validade objetiva para a verdade e para o julgamento ético está enfraquecida. Além disso, a história (como prática social) tem se imposto e a atual defesa da ciência coloca sob suspeita a própria suspeição a que fora submetida pelas mãos do pós-moderno. Por essa razão, nosso momento traz bases objetivas e subjetivas para colocarmos o debate sobre a pesquisa científica e a prática educativa em outro patamar. Defender a ciência não implica abdicar da necessária crítica ao cientificismo positivista e a subserviência desse saber aos interesses do mercado. Essa provocação merece aprofundamentos até para articulá-la ao projeto de valorização da escola pública, do ensino e da transmissão e apropriação ativa de conhecimentos elaborados, como conhecimentos vivos acerca do fazer-se humanidade. Nesse aspecto, a Pedagogia histórico-crítica tem muito a oferecer. ANDERSON, Perry. As origens da pós-modernidade. Rio de Janeiro: Jorge Zahar, 1999. ANDERSON, Perry. As origens da pós-modernidade. Rio de Janeiro: Jorge Zahar, 1999. ANDRADE, Eliziário. A normalidade da irracionalidade. A terra é redonda... eppur si muove. 25 jun. 2021. Disponível em: https://aterraeredonda.com.br/a-normalidade-da-irracionalidade/#_edn. Acesso em: 21 ago. 2022. “No começo de todo filme de desastre tem cientistas sendo ignorados” “No começo de todo filme de desastre tem cientistas sendo ignorados” Chama atenção, nesse contexto, o pronunciamento de Vanuza Kaimbé, a primeira indígena a tomar a vacina no país. Sua abordagem da ciência afasta-se do viés totalitário e etnocêntrico que lhe atribui parcela da agenda pós. Segundo ela: A vacina salva vidas. É a única garantia da nossa existência. Desde 1500, nosso povo foi quase dizimado. Nós éramos 1.300 povos indígenas no Brasil, hoje nós somos apenas 305. Se nós não nos vacinarmos, nós vamos desaparecer. Eu estou aqui em defesa da minha vida, da minha família, do meu povo e da humanidade. Estou me sentindo hoje com esperança. Esperança de dias melhores, esperança de vida. A palavra, hoje, é essa. E é também gratidão. Gratidão à Ciência e aos educadores [...]. Quero dizer para os meus parentes: „vamos nos vacinar!‟. Vacinar é um ato de amor, de esperança. Eu sou um exemplo. Sou indígena, uma mulher de fé, sou uma pessoa que preserva os ensinamentos do meu povo, a minha ciência, a minha ancestralidade, minha crença. Mas sei que só a minha crença e a minha ancestralidade não são suficientes para combater este vírus, que é um inimigo feroz que destrói vidas em questão de minutos ou segundos (apud NUNES, 2021, s.p.). 89 m debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Referências: ADORNO, Theodor W. Aspectos do novo radicalismo de direita. São Paulo: Editora Unesp, 2020. ADORNO, Theodor W. Aspectos do novo radicalismo de direita. São Paulo: Editora Unesp, 2020. AHMAD, Aijaz. Postcolonial theory and the “post-” condition. Socialist register, London, v. 33, p. 353-381, 1997. AHMAD, Aijaz. Postcolonial theory and the “post-” condition. Socialist register, London, v. 33, p. 353-381, 1997. Debate ELLA FONTE, Sandra Soares. As fontes heideggerianas do pensamento pós-moderno. 2006. rientadora: Maria Célia Marcondes de Moraes. 233 f. Tese (doutorado) – Universidade Federal de DELLA FONTE, Sandra Soares. As fontes heideggerianas do pensamento pós-moderno. 2006. Orientadora: Maria Célia Marcondes de Moraes. 233 f. Tese (doutorado) – Universidade Federal de Santa Catarina, Centro de Ciências da Educação. Programa de Pós-Graduação em Educação, 2006. ( ) Santa Catarina, Centro de Ciências da Educação. Programa de Pós-Graduação em Educação, 2006 DOSSE, F. História do estruturalismo: o campo do signo 1945-1966. Campinas/ São Paulo: Editora da UNICAMP/ Ensaio, 1993. DUAYER, Mário. Economia depois do relativismo: crítica ontológica ou ceticismo instrumental? Niterói: UFF, 2003. 14p. (Mimeo.). EAGLETON, Terry. After theory. London/ New York: Penguin Books/ Basic Books, 2003. EAGLETON, Terry. As ilusões do pós-modernismo. Rio de Janeiro: Jorge Zahar, 1998. ESCOLA SEM PARTIDO. Deveres do professor. 2019. Disponível em: https://www. escolasempartido.org/blog/deveres-doprofessor/. Acesso em: 4 jul. 2019. FALAVIGNA, Mauricio. Ciência e Pesquisa: Governo Bolsonaro derrubou investimentos em quase 75% em relação a 2015. Reconta aí, 4 fev. 2022. Disponível em: https://recontaai.com.br/ciencia-e- pesquisa-governo-bolsonaro-derrubou-investimentos-em-quase-75-em-relacao-2015. Acesso em: 7 jun. pesquisa-governo-bolsonaro-derrubou-investimentos-em-quase-75-em-relacao-2015. Acesso em: 7 jun. 2022. FISCHMAN, Gustavo; SALES, Sandra Regina. Formação de professores e pedagogias críticas - É possível ir além das narrativas redentoras? Revista Brasileira de Educação, Rio de Janeiro, v. 15, n. 43, p. 7-20, jan./abr. 2010. FOUCAULT, Michel. Qu‟est-ce que les Lumières? In: FOUCAULT, Michel. Dits et écrits (1954- 1988). Paris: Editions Gallimard, 1994. v. IV. p. 679-688. HABERMAS, Jürgen. Que significa socialismo hoje? Novos Estudos Cebrap, São Paulo, n. 30, p. 43- 61, jul. 1991. HABERMAS, J. Modernity – an incomplete project. In: DOCHERTY, Thomas (org.). Postmodernism: a reader. London: Harvester Wheathsheaf, 1993. p. 98-109. HARVEY, David. O neoliberalismo: história e implicações. São Paulo: Loyola, 2014. HOBSBAWM, Eric. Ecos da Marselhesa: dois séculos revêem a Revolução Francesa. São Paulo Companhia das Letras, 1996. HORKHEIMER, Max. Die Juden und Europa. Studies in Philosophy and Social Science, n. 8, p. 115-137, 1939. JAMESON, F. Foreword. In: LYOTARD, J. F. The postmodern condition: a report on knowledge. Manchester: Manchester University Press, 2004. p. VII-XXI. JEANPIERRE, Laurent. A invenção e as reinvenções transatlânticas da “Critical Theory”. História: Questões & Debates, Curitiba, n. 53, p. 171-202, jul./dez. 2010. KELLER, J. After attack, postmodernism loses its glib grip. The Chicago Tribune, 27 de setembro de 2001. 3p. Disponível em: http://squawk.ca/Ibo-talk/0109/3024.html. Acesso em: 24 jun. 2004. KER, João. NTUNES, Ricardo (org.). Uberização, trabalho digital e indústria 4.0. São Paulo: Boiempo, 202 ANTUNES, Ricardo (org.). Uberização, trabalho digital e indústria 4.0. São Paulo: Boiempo, 2020. APPLE, Michael W. Podem as teorias críticas sustar as políticas de direita? Cadernos de Pesquisa, São Paulo, n. 116, p. 107-142, jul. 2002. BIESTA, Gert. Say you want a revolution…Suggestions for the impossible future of critical Pedagogy. Educational Theory, v. 49, n. 4, p. 499-510, 1998. BOHMAN, James. Critical Theory. In: ZALTA, Edward N. (org.). The Stanford Encyclopedia of Philosophy, Stanford 2021. Disponível em: <https://plato.stanford.edu/archives/spr2021/entries/critical-theory/>. Acesso em: 10 fev. 2022. CALLINICOS, A. Against postmodernism: a Marxist critique. Cambridge: Polity Press, 2002. 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ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. IS Balbúrdia, plantação de maconha e bloqueio de recursos: os ataques de Weintraub às universidades. O Estado de São Paulo, São Paulo, 19 fev. 2020. Disponível em: // d d b / / b b d d b d p https://educacao.estadao.com.br/noticias/geral,balburdia-plantacao-de-maconha-e-bloqueio-de- recursos-os-ataques-de-weintraub-as-universidades 70003203018 Acesso em: 1 jun 2022 https://educacao.estadao.com.br/noticias/geral,balburdia-plantacao-de-maconha-e-bloqueio-de- recursos-os-ataques-de-weintraub-as-universidades,70003203018. Acesso em: 1 jun. 2022. p // / /g , p q recursos-os-ataques-de-weintraub-as-universidades,70003203018. Acesso em: 1 jun. 2022. LATOUR, B. Jamais fomos modernos: ensaio de antropologia simétrica. Rio de Janeiro: Editora 34, 1994. LOPES, Alice Casimiro. Teorias pós-críticas, política e currículo. Educação, Sociedade & Culturas, Porto, n. 39, 2013. LOPES, Alice Casimiro. 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Disponível em: https://www.andes.org.br/conteudos/noticia/numero-de-bolsas-para- pesquisas-cientificas-cai-17-5-na-gestao-de-jair-bolsonaro1. Acesso em: 7 jun. 2022. UNES, Mônica. Debate IBEIRO, Márden de Pádua. Currículo e conhecimento sob diferentes perspectivas teóricas. urrículo sem Fronteiras, v. 17, n. 3, p. 574-599, set./dez. 2017. Disponível em: RIBEIRO, Márden de Pádua. Currículo e conhecimento sob diferentes perspectivas teóricas. Currículo sem Fronteiras, v. 17, n. 3, p. 574-599, set./dez. 2017. Disponível em: http://www.curriculosemfronteiras.org/vol17iss3articles/ribeiro.pdf. Acesso em 12 out. 2020. , , , p , p http://www.curriculosemfronteiras.org/vol17iss3articles/ribeiro.pdf. Acesso em 12 out. 2020. RIBEIRO, Márden de Pádua. Teorias críticas e pós-críticas: pelo encontro em detrimento do radicalismo. Movimento Revista de Educação, Rio de Janeiro, v. 3, n. 5, p. 284-317, 2016. Disponível em: https://doi.org/10.22409/mov.v0i5.32619. Acesso em: 13 out. 2020. RORTY, R. 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São Paulo: Cortez, 2010. SANTOS, S. L. A logogização do infantil: uma prática escolar. In: REUNIÃO ANUAL DA ASSOCIAÇÃO NACIONAL DE PÓS-GRADUAÇÃO E PESQUISA EM EDUCAÇÃO, 23., 2000, Caxambu. Anais... Caxambu: ANPED, 2000. GT 12 - Currículo. SAVIANI, Dermeval. História das ideias pedagógicas no Brasil. Campinas: Autores Associados, 2007. SCHURSTER, K.; LEITE, Alana de Moraes. Disputas Historiográficas acerca do Nazismo e do Holocausto. Locus: Revista de História, [S. l.], v. 26, n. 2, p. 386-406, 2021. SIEBERT, Silvânia; PEREIRA, Israel Vieira. A pós-verdade como acontecimento discursivo. Linguagem em (Dis)curso – LemD, Tubarão, v. 20, n. 2, p. 239-249, maio/ago. 2020. SILVA, Tomaz Tadeu da. Documentos de identidade: uma introdução às teorias do currículo. 2. ed. Belo Horizonte: Autêntica, 2002. SILVA, Tomaz Tadeu da. Identidades terminais: as transformações na política da pedagogia e na pedagogia da política. Petrópolis: Vozes, 1996. SILVA, Tomaz Tadeu da (org.). “Sinto esperança e gratidão”, diz Vanuza Costa Santos, da etnia Kaimbé, a primeira dí i d C id 19 B il C ã l 18 j 2021 Di í l NUNES, Mônica. “Sinto esperança e gratidão”, diz Vanuza Costa Santos, da etnia Kaimbé, a primeira indígena vacinada contra Covid-19 no Brasil. Conexão planeta, 18 jan. 2021. Disponível em: https://conexaoplaneta.com.br/blog/sinto-esperanca-e-gratidao-diz-vanuzia-costa-santos-da-etnia- kaimbe-a-primeira-indigena-vacinada-contra-covid-19-no-brasil/. Acesso em: 20 mar. 2022. NES, Mônica. Sinto esperança e gratidão , diz Vanuza Costa Santos, da etnia Kaimbé, a primeira ena vacinada contra Covid-19 no Brasil. Conexão planeta, 18 jan. 2021. Disponível em: //conexaoplaneta.com.br/blog/sinto-esperanca-e-gratidao-diz-vanuzia-costa-santos-da-etnia- b i i i di i d id 19 b il/ A 20 2022 p p g p g imbe-a-primeira-indigena-vacinada-contra-covid-19-no-brasil/. Acesso em: 20 mar. 2022. PARAÍSO, Marlucy Alves. Pesquisas pós-críticas em educação no Brasil: esboço de um mapa. Cadernos de Pesquisa, São Paulo, v. 34, n. 122, p. 238-303, maio/ago. 2004. POST-TRUTH. Oxford Dictionary. Oxford: Oxford Lexico. s.d. Disponível em: https://www.lexico.com/en/definition/post-truth. Acesso em: 20 maio 2022. PREFEITURA de São Paulo terá comissão da verdade. Sindicato dos Bancários, São Paulo, 28 maio 2014. Disponível em: https://spbancarios.com.br/05/2014/prefeitura-de-sao-paulo-tera-comissao-da- verdade. Acesso em: 12 abr. 2022. 92 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. Notas 1 Doutora em Educação e em Filosofia. Professora do Programa de Pós-Graduação em Educação da Universidade Federal do Espírito Santo (Ufes). Membro do Grupo de pesquisa Estudos Marxistas em Educação. Currículo Lattes: http://lattes.cnpq.br/9396743098041438. Orcid: https://orcid.org/0000-0002-9514-7202. E- mail: sdellafonte@gmail.com. 1 Doutora em Educação e em Filosofia. Professora do Programa de Pós-Graduação em Educação da Universidade Federal do Espírito Santo (Ufes). Membro do Grupo de pesquisa Estudos Marxistas em Educação. Currículo Lattes: http://lattes.cnpq.br/9396743098041438. Orcid: https://orcid.org/0000-0002-9514-7202. E- mail: sdellafonte@gmail.com. 1 Doutora em Educação e em Filosofia. Professora do Programa de Pós-Graduação em Educação da Universidade Federal do Espírito Santo (Ufes). Membro do Grupo de pesquisa Estudos Marxistas em Educação. Currículo Lattes: http://lattes.cnpq.br/9396743098041438. Orcid: https://orcid.org/0000-0002-9514-7202. E- mail: sdellafonte@gmail.com. 2 Esse é um movimento complexo, pois, em alguns casos, coloca em dissonância intencionalidades políticas individuais e os desdobramentos teóricos e históricos das argumentações pós. Vide, por exemplo, as relevantes críticas de autores/as que se situam na agenda pós à Escola sem partido e à Base Nacional Comum Curricular (cf. MACEDO, 2018). 2 Esse é um movimento complexo, pois, em alguns casos, coloca em dissonância intencionalidades políticas individuais e os desdobramentos teóricos e históricos das argumentações pós. Vide, por exemplo, as relevantes críticas de autores/as que se situam na agenda pós à Escola sem partido e à Base Nacional Comum Curricular (cf. MACEDO, 2018). 3 A expressão virada ou giro linguístico foi utilizada, em 1966, quando Richard Rorty reuniu em um volume um número significativo de textos importantes a respeito de “filosofia linguística”, com o título The linguistic turn. A partir daí, a expressão ganhou popularidade, indicando o movimento ocorrido na filosofia no século XX centrado na linguagem e não na consciência. Pensadores da linguist turn afirmam que o que sustenta a legitimidade dos discursos é também um discurso a ser legitimado. Se o acesso ao mundo depende da mediação linguística e sendo esta, por sua vez, contextual e sujeita a interesses, a crítica seria, na verdade, mais um discurso vinculado a determinado contexto que carreia os interesses de seus proponentes. 4 Trata-se do experimento literário no qual o físico Alan Sokal intencionalmente elaborou um texto permeado de nonsenses, com o intuito de verificar se tal artigo seria aceito para publicação. O artigo foi publicado em uma revista com reputação de esquerda (SOKAL, 1996a). Debate WEINTRAUB diz que faculdades plantam maconha. Isto é, São Paulo, Seção Dinheiro, 23/11/2019. Disponível em: https://www.istoedinheiro.com.br/weintraub-diz-que-faculdades-plantam-maconha/. Acesso em: 1 jun. 2022. WEINTRAUB diz que faculdades plantam maconha. Isto é, São Paulo, Seção Dinheiro, 23/11/2019. Disponível em: https://www.istoedinheiro.com.br/weintraub-diz-que-faculdades-plantam-maconha/. Acesso em: 1 jun. 2022. WOOD, Ellen Meiksins O que é a agenda “pós-moderna”? In: WOOD, Ellen Meidsins; FOSTER, John Bellamy (org.). Em defesa da história: marxismo e pós-modernismo. Rio de Janeiro: Jorge Zahar Editor, 1999. p. 7-22. WOOD, Ellen Meiksins. What is the “postmodern” agenda? An introduction. Monthly Review, v. 47, n. 3, p. 1-12, jul./ago. 1995. WORD of the year 2016. Oxford languages. Oxford: Oxford University Press, 2016. Disponível em: https://languages.oup.com/word-of-the-year/2016/. Acesso em: 20 maio 2022. Teoria educacional crítica em tempos pós-modernos. Porto Alegre: Artes Médicas, 1993. SOKAL, A. D. Transgressing the boundaries: an afterword. Dissent, vol. 43, n. 4, p. 93-99, 1996c. 15p. SOKAL, A. D. A physicist experiments with Cultural Studies. Lingua Franca, p. 62-64. maio/jun. 1996b. 7p. SOKAL, A. D. Transgressing the boundaries: toward a transformative hermeneutics of quantum gravity, Social text, n. 46-47, Science Wars, p. 217-252, 1996a. 93 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604 Recebido em: 15 de out. de 2022 Aprovado em: 10 de nov. de 2022 Notas No mesmo ano, o autor revelou a paródia (SOKAL, 1996b) e explicou que o motivo de sua sátira foi duplo: “[...] combater um discurso pós-modernista/pós- estruturalista/construcionista social atualmente em moda e, de forma mais geral, uma inclinação para o subjetivismo que é, a meu ver, inimigo dos valores e do futuro da esquerda” (SOKAL, 1996c, p. 3) e afirmar que “[...] há um mundo real; suas propriedades não são apenas construções sociais; fatos e evidências importam” (SOKAL, 1996b, p. 4). 5 Em 2022, o valor em orçamento dedicado à Ciência e Tecnologia é 73,4% menor do que o de 2015 (FALAVIGNA, 2022). 6 Na gestão de Jair Bolsonaro até 2022, o número de bolsas para pesquisas científicas sofreu uma diminuição: queda de 17,5% no número de bolsistas contemplados pelo Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e de 16,2% pela Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) (NÚMERO..., 2022). Recebido em: 15 de out. de 2022 Aprovado em: 10 de nov. de 2022 94 Germinal: marxismo e educação em debate, Salvador, v.14, n.3, p. 73-94, dez. 2022. ISSN: 2175-5604
https://openalex.org/W615794056	https://www.biodiversitylibrary.org/itempdf/117766	English	null	Breaking and riding, with military commentaries	null	1,900	public-domain	124,102	BREAKING AND RIDIN BREAKING AND RIDIN BREAKING AND RIDIN by JAMES FILLIS This book was digitized by Microsoft Corporation in cooperation with Cornell University Libraries, 2007. You may use and print this copy in limited quantity for your personal purposes, but may not distribute or provide access fo it (or modified or partial versions of it) for revenue-generating or other commercial purposes. Digitized by Microsoft® win 3 192 win 3 192 Digitized by Microsoft® BREAKING AND RIDING Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® d by Microsoft® tize igi d by Microsoft® tize igi Digitized by Microsoft® THE CHAPEL fa RIVER iy PRESS Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® PREFACE. IN this book, I do not presume to discuss scientific subjects. I am simply a horseman who has been among horses for sixty years, who knows them, loves them, and is capable of reasoning about them. The fundamental p-inciple of the studies which I submit to the public is that it is necessary for a horse to be correctly balanced and light in forward movements and propulsion, in order that the rider may obtain the most powerful effects with the least exertion. My method of equitation consists in distribution of weight by the height of the neck bent at the poll and not at the withers; propulsion by means of the hocks being brought under the body; and lightness by the loosening of the lower jaw. When we know this, we know everything, and we know nothing. We know everything, because these principles are of universal application; and we know nothing, because they have to be applied practically. Practice cannot be taught in a book; but I will try to set forth principles. Probably, I would not have had the bold- ness to do this, had not one of my pupils entreated me to Digitized by Microsoft® PREFACE. viii write this book; because he had been greatly struck with the aptness of the explanation which I gave him respecting the details of the breaking lessons. In judging this work, I trust that my readers will give it the attention it deserves, as the result of sixty years’ serious study and hard practical work. I crave the indulgence of the public, and the impartiality of my reviewers. JAMES FILLIS. Digitized by Microsoft® Digitized by Microsoft® TRANSLATOR’S PREFACE. Mr. FILLIS, at whose request [ have had the pleasure of translating his Principes de Dressage et @Equitation, is acknowledged throughout Europe as the greatest high school rider of all time. In fact, he has had the honour of giving private riding exhibitions before The Emperor and Empress of Germany, The Tsar, President Carnot, The Queen of Belgium, The King and Crown Prince of Denmark, and the Emperor of Austria, all of whom have accorded him special marks of their appreciation of his unrivalled ability. Having fortunately had many opportunities of seeing Mr. Fillis ride both in Germany and at the St. Petersburg Cavalry Riding School, where he is Ecuyer en chef, 1 can fully endorse the correctness of the good opinion held about him by his most enthusiastic admirers. A school rider obtains control and guidance by the com- bined action of hands and legs; but an ordinary horseman depends almost entirely on the reins for collecting and directing his mount, and consequently his power over the animal is insufficient for military and polo requirements. With extremely few exceptions, school riders abuse their Digitized by Microsoft® TRANSLATOR’S PREFACE. x power, and sacrifice freedom of movement to exaggerated control, with the result, in many cases, of getting their animals behind their bits and straining their hocks. Such men ride in a mechanical manner, which is inelegant in a school or circus, and is entirely unsuited for work in the open. Mr. Fillis on the contrary, acting on his motto “ex avant” (forward), has succeeded in showing how a horse can be made clever in his movements, without in any way diminishing his usefulness on the road or over a country. I therefore trust that all English-speaking horsemen, and _ especially cavalrymen and polo players, will profit by the valuable instruction which he now puts before them. power, and sacrifice freedom of movement to exaggerated control, with the result, in many cases, of getting their animals behind their bits and straining their hocks. Such men ride in a mechanical manner, which is inelegant in a school or circus, and is entirely unsuited for work in the open. Mr. Fillis on the contrary, acting on his motto “ex avant” (forward), has succeeded in showing how a horse can be made clever in his movements, without in any way diminishing his usefulness on the road or over a country. CONTE. ts. PAGE PREFACE. . : 2 . Vil TRANSLATOR’S Paes ‘ : ’ ix PREFACE. . TRANSLATOR’S Paes PREFACE. . TRANSLATOR’S Paes TRANSLATOR’S PREFACE. I therefore trust that all English-speaking horsemen, and _ especially cavalrymen and polo players, will profit by the valuable instruction which he now puts before them. M. H. HAYES. Digitized by Microsoft® CHAPTER I. ORDINARY RIDING. Lunging « Horse—Close work, advancing—Collecting a Horse and direct Flexion—Objects to be obtained by direct Flexion—Making a Horse quiet to mount—How to hold the Reins—Teaching a Horse by the Whip to obey legs and spurs—Horse mounted, first Defences, means for overcoming them—The Walk—To halt and stand still—Changes of Direction—Lateral flexions—Objects of Lateral flexions—Rotation of the Croup and Shoulders—School Walk—-The rein back—The ramener, collection, rassembler, and equestrian tact—Side steps and two tracks—The trot—The canter —Voltes and demi voltes at the canter—Changes of leg—The Hack . Z : 3 : 32 to 183 CHAPTER i HORSES WITH VICES. Nervous Horses—Horses which throw their heads about—Horses which runaway. : ‘ i gj F , : . 184 to 194 CHAPTER 1, HORSE AND MAN. The Horse—Feeding—Intelligence of the Horse—Influence of a Man’s expression of face on a Horse—Influence of the Human Voice ona Horse—Making much of a Horse—Punishment—Bitting—Martin- gale—Saddle—Stirrups—Cutting ieee lies s Seat— Lady's Seat . : ; j . I to 31 The Horse—Feeding—Intelligence of the Horse—Influence of a Man’s expression of face on a Horse—Influence of the Human Voice ona Horse—Making much of a Horse—Punishment—Bitting—Martin- gale—Saddle—Stirrups—Cutting ieee lies s Seat— Lady's Seat . : ; j . I to 31 CHAPTER. VI. CHAPTER Vit. THE ARMY HORSE ; 3 2N3°to 235 CHAPTER i HORSES WITH VICES. HORSES WITH VICES. Nervous Horses—Horses which throw their heads about—Horses which runaway. : ‘ i gj F , : . 184 to 194 Digitized by Microsoft® xii CONTENTS. TAGE CHAPTER IV. JUMPING 3 « 195 to 205 HIGH-SCHOOL RIDING The Spanish walk—Reversed pirouettes on three legs—Reversed and ordinary pirouettes with the feet crossed—Reining back without reins—Rocking the forehand—Rocking the haunches—Spanish trot —Shoulder-in at the canter—Pirouettes at the canter—Changes of leg’at each stride—Changes of leg without gaining ground—Piaffers and passages—Differences between the fassage and Spanish trot— Serpentine at the trot—Canter on three legs—Passage to the rear— See-saw piaffer—Canter without gaining ground and canter to the rear—New school movements—School horse for ladies . 236 to 331 The Spanish walk—Reversed pirouettes on three legs—Reversed and ordinary pirouettes with the feet crossed—Reining back without reins—Rocking the forehand—Rocking the haunches—Spanish trot —Shoulder-in at the canter—Pirouettes at the canter—Changes of leg’at each stride—Changes of leg without gaining ground—Piaffers and passages—Differences between the fassage and Spanish trot— Serpentine at the trot—Canter on three legs—Passage to the rear— See-saw piaffer—Canter without gaining ground and canter to the rear—New school movements—School horse for ladies . 236 to 331 CHAPTER Idx. COMMENTARIES ON BAUCHER : . : : + 332 to 344 TAGE CHAPTER IV. JUMPING 3 « 195 to 205 CHAPTER. ¥. FLAT RACING : . 206 to 209 CHAPTER. ¥. FLAT RACING : CHAPTER. ¥. FLAT RACING : . 206 to 209 CHAPTER. VI. THE HUNTER : . 210 to 212 CHAPTER. VI. . 210 to 212 CHAPTER. VI. THE HUNTER : CHAPTER AL ICERS 3 ‘ ‘ ‘ CHAPTER AL ELGIAN OFFICERS 3 ‘ ‘ ‘ - 348 to 356 Digitized by Microsoft® LisT OF ILLUSTRATIONS: FIG. . Leading a horse forward . . Jaw contracted . Jaw and bit free . Going forward when mobilising the lower jaw, so as to prevent the horse getting behind his bit . Muzzle on a line with upper part of shoulder . Correct preparation for direct flexion Preparation for Baucher’s direct flexion . Incorrect and frequently employed flexion . Lowering the head—a faulty practice a5 ” a . Direct flexion when going forward . . Baucher’s flexion when mounted . Correct flexion when mounted . Exaggerated example of Baucher’s incorrect flexion . . Proper way to mount . Equal tension on all four reins Action of the curb . Action of the snaffle . Action of the off curb rein Action of the near curb rein PAGE 4! 45 47 49 51 55 55 LW 57 59 59 63 64 65 67 71 71 71 73 73 Digitized by Microsoft® LIST OF ILLUSTRATIONS. FIG. 21. Separating the curb and snaffle reins . Passing the right hand between the off curb rein and oft snaffle rein . Closing the hand on the off curb and off snaffle rein . Separating the reins . Doubler . Volte . Demi-volte 28. Diagonal change of hand . Reversed change of hand . Counter-change of hand . . Preparation for lateral flexion; jaw contracted . . Lateral flexion ; jaw relaxed . Correct lateral flexion . Incorrect lateral flexion . Correct lateral flexion when mounted . Baucher’s lateral flexion when mounted . Incorrect lateral flexion . Lateral effects in rotation of croup and shoulders . Direct effects ¥5 ” ” . Diagonal ,, 53 ” » . On “ two tracks ” at the school walk, from right to left ” ” 0o ge 55 left to right . First time of the canter; near hind support . Second time of the canter; left diagonal in support . Third time of the canter; off fore in support . Clearing a fence . Horse raising his forehand a good deal when jumping croup when clearing a fence . ” ” PAGE 73 75 75 75 go gI g2 93 94 95 97 99 101 103 105 106 107 108 1cg 110 I4t 143 160 161 162 199 200 201 LIST OF ILLUSTRATIONS. LIST OF ILLUSTRATIONS. XV FIG. PAGE 49. Horse and rider being drowned. Fi ‘ : of 231 50. Digitized by Microsoft® HORSE AND MAN The Horse—Feeding—Intelligence of the Horse—Influence of a Man’s expres- sion of face on a Horse—Influence of the Human Voice on a Horse— Making much of a Horse—Punishment —Bitting—Martingale—Saddle— Stirrups—Cutting Whip—Spurs —Man’s Seat—Lady’s Seat. LisT OF ILLUSTRATIONS: Rider making his horse swim according to Mr. James Fillis’ method. : ; ‘ : : : . 232 51. Spanish trot : ; ; : ; : 3 253 52: ” ” . ; e ; ; : 2 255 53. Shoulder-in at the canter ; ‘ : : : 263 545 oy » ” 265 555 ” i ” 267 56. Passage ; right diagonal in the air . F F 3 s a7 57- 3 1% Jett ¥e Pa : ; : ; . 279 58. $s on ‘‘ two tracks” Z 4 2 : . 283 59- 5 9 55 : ‘ ‘ . - . 285 6o. Piaffer . : : : : : : . : . 289 Or. o> ‘ : 2 é i 5 : : . 291 62. Teaching the piaffer by means of a cutting whip. . 299 63. Cantering to the left on three legs . a : ‘ » 307 64. 5 » tight . : é ‘ ; . 309 65. Half-rear : : . i ‘ ‘ : » 3ir 66. See-saw piaffer ; : : : : ; = BFS 67. 5 55 . i Z : - : : ae) 9) 68. Cantering tothe rear. 3 ‘ : : : a 323 69. i ,, ‘ight on three legs to the rear ; - 327 70. 4s 7 7 n ¥) . - 329 Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® THE HORSE. ALTHOUGH I break in only thorough-breds for my own use ; I in no way assume that three-quarter or half-breds cannot be good riding horses. I do not care for very big animals, and prefer those about 15.2, or, say from 15.1 to 15.3. When examining a horse, \| at first take a general view of him at a distance of a few yards. If the first impression is pleasing, I go over his various points in detail, with a fairly liberal spirit, as regards trifling faults; but if it is not favourable, I become all the more critical. In any case, it is hopeless to expect perfection. In this first general examination, we should be particularly careful to see how he moves at the walk, trot and canter, both when led and ridden. ng still ; but Some horses appear badly shaped when standing still ; but become good-looking, light and active as soon as they begin to move. Others, which seem almost perfect in repose, are heavy and awkward in their paces. I prefer the former to the latter, because they can utilise what they have got. I Digitized by Microsoft® HORSE AND LS) HORSE AND MAN. LS) I like a handsome head, long and light neck, prominent withers, short and strong back and loins, long croup, long and oblique shoulders, close coupling between the point of the hip and the last rib, hocks well let down, short canon bones, long fore arms, and the pasterns fairly long. These desirable points are rarely found in one animal. A horse should be close to the ground, which he will be when the distance from the brisket to the ground will be equal to that from the withers to the brisket. A horse which is high off the ground is generally clumsy in his movements, and liable to stumble. I would have nothing to do with a horse whose fetlock and pastern joints are stiff, because he would be deficient in elas- ticity, would drag his feet, and would consequently be liable to trip. I take particular care to see that the heels are not con- tracted. In order to prevent my horses acquiring this defect, I do not have them shod when they work on soft ground, in which case the heels remain well apart, on account of the frog being subjected to pressure. Digitized by Microsoft® THE HORSE. I always have my horses shod with tips, the ends of which are imbedded into the wall at the heels; and I thus prevent them having their heels con- tracted and their feet becoming diseased. Horses are like men; having obtained physical perfection in them, we further require good temper and courage. The highest mental qualification of a horse is to be a free- goer; because such an animal, contrary to what is often thought, is neither irritable nor sulky. I will again refer to this point. At present \| confine myself to stating that such a horse is valuable, even if his make and shape are only moderate. If an animal has not the essential requirement of being always ready to go forward, he is useless, although he may be a perfect picture to look at. I like to begin breaking a horse when he is two off and not more than three years old, and to buy if possible in or near Digitized by Microsoft® THE HORSE. 3 September, at which time the animal would be about two and a-half years old. At that age he would have done little or no work, and would consequently be sound. Besides, it is easy to get such horses, because there are a large number of young thorough-breds which are capable of becoming mar- vellously good horses for riding-school purposes and other work, although they might be of no use on a race-course. Also, for many other reasons, we can get a large choice of thorough-breds at that age. I never buy mares, because they often become peevish, especially when touched with the spurs. I always have my horses “added to the list,” because thorough-bred entires are greatly inclined to rush at every animal they meet, which is a habit that is not pleasant for their riders, and they are always ready to get on their hind legs. Besides, many of the horses which I break are required for ladies, who should never be allowed to ride a rearer. These objections do not generally apply to Arab and Trakene (German) entires, which live with mares and pay little or no attention to them. In course of time the thorough-bred entire gets heavy in front and be- comes poor behind. A riding horse should, on the contrary, have his hind quarters well developed and his forehand light. THE HORSE. Thorough-bred stallions which are used for stud purposes preserve their proper proportions, although they get very fat. Everyone knows that a gelding is much more quiet than an entire. After the hot weather, I send my young horses to be cas- trated at the Veterinary College of Alfort, where they remain a fortnight, and afterwards I turn them out to grass for three months and a-half at a place where they are properly looked after. I do not put even a saddle on their backs during these four months, after which I begin their education in the quietest manner possible. When \| have taught them to go collectedly when walking, 1* 1* Digitized by Microsoft® HORSE AND MAN. 4 trotting, cantering, turning, reining back, and moving from one side to the other for a few steps, I begin to take them out- side, and they will then need only a few days to make them pleasant to ride in the open. In this way, I first of all make them into hacks. During the following two or three months I repeat, in the open air, the work which my horses have learned in the school, so as to make them light and supple in their natural paces. Thus, from September to the end of December, nothing but care and repose; from January to March, breaking in the school; from April to June, confirma- tion, outside, of the work learned during the preceding months. In July I send my horses for their holiday to grass, leave them loose in the fields, and give them oats. In August I resume work in the open, and as the animals have rested and got strong, I begin at the same time high school riding. My horses being handy, light and well balanced, their progress is rapid, and I generally finish their school training towards the end of December, with a limit of two or three months one way or the other, according to the difficulties which I have to overcome and the extent of the instruction. I then give them another holiday of a fortnight and immediately after that I take them out hunting if pos- sible. I do not consider their education complete until I have tested them in all these ways. THE HORSE. In this manner I obtain an excellent hack for spring and summer, a hardy hunter for autumn, and a pleasant school horse for winter. Digitized by Microsoft® INTELLIGENCE OF THE HORSE. 5 which is their time of rest. Not being disturbed, they eat slowly and thoroughly chew their food, which on that account becomes properly digested. I give one-and-a-half quarterns in the morning, three-and-a- half at noon, and five in the evening. The morning oats is given at least two hours before work, so that the stomach of the animal may be empty at that time. For the same object I rack him up, so that he may not eat his bedding. Half an hour after work I give each horse 2 or 3 lbs. of hay ; at noon three-quarters of a bucket of water; and after that, oats. At four o’clock in the afternoon, each horse gets a bundle of straw as bedding; at five o'clock, 2 or 3 lbs. of hay; and finally, at seven o’clock in the evening, the same quantity. of water as at noon, and the evening ration of oats, instead of which I give, twice a week, a bran mash. FEEDING. I feed my horses liberally, especially as regards oats, of which I give them 10 quarterns a day. This makes them lively, but not more so than I wish. I give them only a small quantity of oats in the morning, so as not to overload their stomachs ; but I allow them all they can eat in the evening, Digitized by Microsoft® INTELLIGENCE OF THE HORSE. INTELLIGENCE OF THE HORSE. The great difficulty in breaking is to make the horse under- stand what we want him to do, which is no easy matter, because a horse, contrary to what many think, has only a small supply of intelligence. His only well-developed mental quality is his memory, which is particularly acute, and should therefore be specially utilised. A horse is incapable of affection for man: he possesses only A horse is incapable of affection for man: he possesses only habits, which he often acquires far too easily, and frequently sticks to them with too much persistence, a fact we should always bear in mind. On this subject I have made hundreds of experiments. For instance, one of my friends had a horse which went to him when he called him, neighed when he entered the stable, etc. He averred that this animal was par- ticularly attached to him, and that the horse would pine away if he left him. Having learned all about the habits of the horse from the owner, I begged him to lend me the animal, which I took to my stable, where I treated him exactly as he Digitized by Microsoft® Digitized by Microsoft® HORSE AND MAN. 6 had been treated in his own stable. On the following day I worked him during periods of time similar to those his owner had adopted; I gave him carrots according to established custom ; I imitated the voice of his master; and brought him his food at the accustomed times. On the following day I re-assumed my natural tone of voice, and in forty-eight hours the animal made similar manifestations of affection to me as he had done to his master, whose loss he did not appear to feel in the slightest degree. had been treated in his own stable. On the following day I worked him during periods of time similar to those his owner had adopted; I gave him carrots according to established custom ; I imitated the voice of his master; and brought him his food at the accustomed times. On the following day I re-assumed my natural tone of voice, and in forty-eight hours the animal made similar manifestations of affection to me as he had done to his master, whose loss he did not appear to feel in the slightest degree. INTELLIGENCE OF THE HORSE. After the morning lesson I give a large quantity of carrots to my horses, who neigh the moment I speak to them on entering the stable. If a stranger accompanies me on these occasions, he will always tell me that the animals recognise me and love me, which is a mistake ; ,because if another person takes my place in distributing the carrots at the usual hour, the horses will not notice my absence. This can easily be proved by the fact that if I go into the stable a few minutes after they have finished eating, they will not take the slightest notice of my arrival. I could give a hundred other instances of the in- difference of horses towards those who tend them or ride them. This is not a matter of regret; for if horses were differently constituted, they would be willing to obey only one master. INFLUENCE OF A MAN’S EXPRESSION OF FACE ON A HORSE. Despite many arguments to the contrary, I am thoroughly convinced that the human expression of face has no influence on a horse, who pays no attention to one’s look, whether it is hard, angry, soft or caressing. I have proved this fact by many experiments on young and old animals; and I can certify that if a person who is looking at a horse works only the muscles of his face, without moving his body or limbs, the animal will not take the slightest notice of the changes of expression. Digitized by Microsoft® INFLUENCE OF THE HUMAN VOICE. 7 I have scores of times tried the effect of giving a horse an angry look and of smiling on him without any result whatsoever. If you make the most horrible grimaces at your horses or stick your tongue out at them, you will find that they will take absolutely no notice of such a proceeding, but they will be quick to perceive any movement of the body, and especially of the hand. INFLUENCE OF THE HUMAN VOICE ON A HORSE. The human voice has a great influence on a horse, but of course it is only the tone which he remembers. The sweetest words, repeated in a short, high tone, will frighten him, and the most horrible threats, uttered in a soft voice, will soothe him. The voice is the most useful help for breaking a horse “at liberty,” in which case he is turned loose in a riding school, circus, or other suitable enclosure. Thus, to teach a horse at liberty to move forward at a walk, trot or canter, one says: “walk,” in a comparatively weak voice; “trot,” in a higher voice; and “canter!” in a tone of command. You may say “canter” in a soft voice, and the horse will remain at the walk; but if you say “walk!” in a high tone, the animal will immediately strike off into the canter. The voice is also of great use when breaking a horse which one is riding. For instance, if my mount kicks, rears, or plays up in any other way, I correct him with whip or spurs, and speak to him, at the same time, in a tone of reproof, and he thus soon becomes attentive to my voice. If he makes a mistake, or tries on a “defence,” it will generally be enough for me to raise my voice, which, by reminding him of the previous punishment, will make him quiet. By acting in this manner, I save him from fresh punishment. The voice can be used not only for correction, but also to encourage and quieten a horse, in which case a pat on the neck of the horse will help to aid its good effect. Digitized by Microsoft® Digitized by Microsoft® HORSE AND MAN. 8 Its action is all the more useful because it can be employed in all cases with a varied choice of tone. A rider cannot, on the contrary, always bring his hands and legs into play. Let us suppose that a hot-headed, impetuous horse which one is riding, gets startled and becomes maddened in a place where there are several carriages, and that one cannot get him out and steady him by means of the reins and legs. The voice, however, will calm him down, if he has been trained to obey it. INFLUENCE OF THE HUMAN VOICE ON A HORSE. The effect of the voice has often been a great help to me, and has brought me out of many a difficulty. I like impetuous horses, and I rarely use any others. I make them sufficiently quiet to carry ladies; but I never employ them for this purpose until I am certain that my voice will soothe them when they are excited. I have avoided accidents by always acting in this manner. MAKING MUCH OF A HORSE. The good effect of “making much” of a horse should not be neglected, as we shall see further on. The education of the horse is based on the principles of reward and punish- ment. Making much of a horse gives him confidence, by placing the rider in direct contact with him otherwise than by impulsion. All horses, even the most impatient, accept a pat on the neck, which is therefore the place the rider should caress. The pats on the neck should be given forcibly enough to attract the attention of the horse, but without hurting the animal. We should avoid touching him too lightly, which might only have the effect of tickling him. A pat on the neck should be given at an appropriate time, that is to say, zmmediately after an act of obedience on the part of the horse, in the same manner as punishment should Digitized by Microsoft® PUNISHMENT. 9 promptly follow disobedience. The moment he yields, give him his proper reward by patting him on the neck and slackening the reins, which will greatly facilitate the process of breaking. To obtain its full effect, a pat on the neck should be accompanied by the voice. A combination of these two soothing means will produce the best possible effect and, as a rule, will accomplish its object. PUNISHMENT. The education of a horse, as I have already said, depends entirely on the manner in which the rider applies the principles of reward and punishment; the appropriate appli- cation of the latter being even more essential to success than that of the former. Above all things, the rider of a difficult horse should never lose his temper. When a horse deserves punishment, he should get it with an amcunt of severity which might be regarded as the outcome of anger, but which should be proportionate to the offence. In fact, we should treat horses as we do children. We all know that nothing is worse than to punish a child when we are in a rage. A horse can in no case understand the feeling which prompts a man to punish him, and he will remember only the pain he has suffered and the occasion on which it was inflicted. His intelligence enables him to connect his action with the punishment it provoked ; but it does not allow him to go further than that. On this account, if punishment is not administered at the precise moment the fault 1s com- mitted, it will lose all its good effect, and will be an element of confusion in the memory of the animal. For instance, if a horse which kicks receives punishment when his hind legs are off the ground, he will remember that he got hurt for kicking. If, on the contrary, the punishment is received after his hind legs have come down, he will be unable to connect in his mind the ideas of these two acts; in fact, the Digitized by Microsoft® HORSE AND MAN. 10 probability is that he will try, by a fresh kick, to get rid of the person who is hitting him. I have said that every deliberate act of disobedience com- mitted by a horse should be punished; but I do not hesitate to add that it is better not to punish him than to do it too late. Both are bad, but it is necessary to choose the lesser of the two evils. It is also important to find out what is the motive which has caused a horse either to wilfully disobey or to act on the defensive; in other words, to find out whether he is vicious or in pain. PUNISHMENT. Thus, if a horse kicks because his loins or hocks are painful, we would not be justified in punishing him, and should do everything to relieve his suffering. But if the kicking is a vice, we ought to severely correct him the moment it is practised, and, to prevent it, we should forcibly raise his head and neck in order to put the weight on his hind quarters. BITTING. I have nothing particular to say about the snaffle, except that it ought to be rather thick, so as to reduce its severity, and that it should be placed at about an equal distance from the bit and the corners of the mouth. The form of a curb bit and the position it is placed in the mouth are points of great importance. It is impossible to decide at first glance what kind of curb will suit a young horse best, and what position it should occupy in his mouth. Baucher says that he would use the same kind of bit for all kinds of horses, which statement is the consequence of his pet theory that all horses have the same kind of mouth. I will discuss this theory further on, and I will here content myself with saying that even the most inexperienced horseman will state that horses go better in one bit than in another, and that certain animals will go kindly in a snaffle, but will resent Digitized by Microsoft® BITTING. II the use of a rather severe curb. This is a generally accepted fact which has been amply proved. We can find the best curb for a horse only by trial. But there are always certain general principles to help us in making our experiments, which we may sum up as follows: The curb which is used at the beginning of the breaking should have a thick mouth-piece, low port, and short cheek- pieces, so that it may be easy to the mouth. Its width should be proportionate to that of the mouth of the horse. If it is too narrow, the lips will be compressed by the cheek- pieces. If it is too wide, the horse, either in play or to relieve the bars of his mouth from pressure, will bring it too much over on one side of his mouth, so that a part of the port will rest on one of the bars, and consequently the mouth-piece will exert an uneven pressure, which will almost always cause the horse to carry his head sideways. In order that the curb may fit properly, it should be wide enough for the cheeks to keep clear of the lips on each side. BITTING. The mouth-piece should have an even feeling on both sides of the mouth, and should be mid-way between the tushes and the corners of the lips, and consequently it will be lower in the mouth than the snaffle in the case of a double bridle. Later on I shall point out some exceptions to this rule. The lower ends of the cheek-pieces, yielding to the pull of the reins, cause the upper ends to revolve forward, and thus to produce pressure on the bars of the mouth. The curb-chain, which prevents the upper ends of the cheeks from revolving forward, increases the pressure on the bars proportionately to its shortness. Hence, the length of the curb-chain should be proportionate to the lightness of the horse’s mouth, which we are unable to estimate in the case of a perfectly “ green” horse. With such an animal, we should at first leave the curb-chain very loose, as there will always be time to take it up. It would not, on the contrary, be right to say that there Digitized by Microsoft® HORSE AND MAN. 12 is always time to slacken a tight curb-chain, which causes pain that will continue to be felt by the horse after the curb- chain has been let out. If, however, we begin with a loose curb-chain, and gradually shorten it to the desired extent, we shall avoid hurting the bars of the mouth and irritating the horse to resistance, and we gain time. But if, at the begin- ning of the work, we hurt or even irritate the bars by undue pressure, we shall fail to obtain any of the required indica- tions. By artificially increasing the sensitiveness of the bars, we fail to estimate it correctly, and work on a wrong method from the beginning. The bruising or even the mere irritation of the bars does not disappear as soon as the work is finished and the bridle taken off, but continues for a more or less long period. Consequently, when the next lesson is given, the bars are congested and painful. Digitized by Microsoft® BITTING. The rider will then be apt to form an opinion of the effects he produces on the mouth of the horse without taking into consideration that it is in an abnormal condition, and he will increase the evil which has been done, and he will become more and more unable to correctly estimate what is the natural condition of the mouth. In a word, he will do the very things he ought not to do. From the foregoing observations we can see that, at the beginning of the breaking, the curb-chain should he ioose. In fact, it is better to take it off. The knowledge of the mouth of a “green” horse is an important and delicate manner. In order to gently feel the mouth without spoiling it, we must begin with great lightness of hand, and gradually increase the pressure up to the point of making the horse feel it, which sensitiveness varies in degree according to the animal. If a horse yields to the light pressure of a curb which has no curb-chain, of what use is the curb-chain, and what is the good of seeking for a more powerful means of restraint? I have thoroughly broken horses, not only for the manége, Digitized by Microsoft® MARTINGALE. 13 but also for outdoor work, without using a curb-chain, which may remain hooked up on one of the curb-hooks, so that it may be instantly used in case of need. As a rule, it should not be employed unless the rider finds that he needs its help. When he uses it, he should never put more tension on it than is actually required; the proper maximum being when the curb-chain is tightened up, so that it makes an angle of 45° with the lower jaw. As the tension of the curb-chain should be proportionate of the sensitiveness of the bars, so should the pressure caused by the pull of the reins be proportionate to the resistance. If this resistance is slight, the effort to overcome it should be light, and the point of its application should be high up on the jaw. If the resistance is great, the effort should be energetic, and it should be applied low down. BITTING. Therefore, without greatly altering the middle position which the mouth-piece should occupy between the tushes and the corners of the lips, we may raise or lower the mouth-piece so that the horse may yield his jaw by, respectively, a light feeling of the reins, or by a strong pull on them. In other words, the softer the mouth is, the higher should be the mouth-piece ; and the harder, the lower should it be placed. In no case, however, should it press on, or even touch, the corners of the lips or the tushes. We learn from the foregoing remarks that the proper tension of the curb-chain and the right position of the mouth- piece in the mouth of a young horse can be found only from experience, which should be gained from very slight effects that can be gradually increased in severity as the case may demand. Digitized by Microsoft® Digitized by Microsoft® MARTINGALE. The martingale prevents a horse from star-gazing and throwing up his head, and helps the rider to guide him. ifa Digitized by Microsoft® HORSE AND MAN. 14 horse is well broken he will not require its use. I would recommend its employment only when the teacher has not time or knowledge to properly break in a horse. It would be well to put it on a horse which we are going to ride for the first time, whether for hacking or for hunting, if we had reason to think that he threw his head about or carried it too high. There are three kinds of martingales, namely, the standing or fixed martingale buckled on to the nose-band, the running martingale, and the standing martingale buckled on to the rings of the snaffle. The first is the only one I would recommend, because it produces its effect on the animal's nose and not on his mouth, and, being unconnected with the rider’s hands, its use is consequently free from danger. It ought to be long enough to allow the horse to carry his head high, and short enough to prevent him bringing his nose in a horizontal position, in which case the curb, by revolving from below upwards, would produce no effect on the mouth. If the martingale is too short, it will interfere with the move- ments of the horse, and can then become dangerous, especially if he does not go freely forward ; because when it is fixed in this manner, he will draw his chin in towards his breast so as to escape from its action. The running martingale has two rings, through which pass the reins of the snaffle, and occasionally those of the curb, which is a very dangerous arrangement. It is therefore con- nected with the hands of the rider and the mouth of the horse. It may be of great service to experienced riders, but on account of the strong effect which can be produced with it, it is particularly dangerous for other people. The martingale which buckles on to the rings of the snaffle, being unyielding, is always dangerous, particularly if the horse runs back; because, in this case, it will continue to pull on his mouth. If he rears, which he will have a ten- SADDLE. SADDLE. I would not advise the use of a new saddle, which rarely gives satisfaction to the rider. New leather is hard and stiff, and consequently the seat of the saddle will not be com- fortable. It is best to try several second-hand saddles, one of which is certain to give satisfaction. The seat of a saddle ought to be fairly straight. If it is too high at the pommel the rider will be thrown back, and if it is too high at the cantle he will be thrown for- ward. I am also of opinion that a saddle should have very little stuffing, so that the rider may get close to his horse. The length of the flaps should be proportionate to that of the thighs of the rider. If they are too short, the rider might hurt the calves of his legs. If too long, he will not be able to feel the sides of the horse with his legs. The rider can please himself as regards plain flaps or knee rolls, which are a matter of habit and firmness of seat. I believe I was the first to use a plain flap saddle for high school riding. I always begin with a pupil by putting him on a French saddle in preference to an English one, as he is able to get better into it, and is consequently more at his ease. After the beginner acquires a firm seat in a French saddle, I put him in an English saddle covered with doeskin, and when he is able to trot, canter and turn without rolling about, I give him a plain English saddle to ride on. MARTINGALE. 1S dency to do, in order to escape from the pressure of the mouth-piece, this martingale will be liable to make him fall backwards. Digitized by Microsoft® Digitized by Microsoft® STIRRUPS. I never allow a pupil to use stirrups until he has acquired a good seat at all paces, and in this respect I follow the Digitized by Microsoft® HORSE AND MAN. 10 example of all the old riding masters, French and foreign. We must admit that formerly riders had quite a different seat to those of the present time. Stiffness has replaced the pliability, ease and grace of the olden time, because pupils have fallen into bad habits from the commencement of their instruction, either from want of comprehension or on account of being badly taught. Not only the premature, but also the excessive use of the stirrups has other faults. I am of opinion that the great majority of dangerous falls are caused by their abuse. For example, Mr. X is run away with by his horse in the Forest of Saint-Germain; he sits well down into the saddle, succeeds in stopping the animal, and starts into a trot on his stirrups; unfortunately, a stirrup leather breaks at this moment, and Mr. X falls on his head and is killed. Every horseman knows that the breaking of a stirrup leather can cause a rider to fall on his head, only because the man had put all his weight on the stirrups, and was consequently more or less out of the saddle. If he was not bearing his weight on the stirrups he might, no doubt, roll off, but the severity of the fall would be more or less broken by the fact that the knees would be gripping the flaps of the saddle. I would go further and say that if a rider did not depend on his stirrups, he would hardly ever fall off on account of a stirrup leather breaking. Let us take another example. Mr. Z , at Toulouse, rides out of his stable at a walk; his horse falls, and Mr. Z is thrown forward on to his head and is killed. We need no knowledge of riding to understand that if he had been sitting in his saddle he could not have been thrown forward with such violence. I hasten to add that I recall these two well-remembered accidents, not in a spirit of criticism, but in order to explain the sad consequences which may arise from the abuse of the stirrups, and in the hope Digitized by Microsoft® STIRRUBPS. SPURS. The box spur is the only spur of which I approve, because it remains fixed in its place, and it can therefore be applied with precision. All the other kinds are liable to become displaced, either up or down, so that one is never certain of touching the horse on the spot one wishes. We ought to use dummy spurs (spurs with round heads), if We ought to use dummy spurs (spurs with round heads), if they are sufficient for our purpose, and we can replace them by spurs with very blunt rowels, the sharpness of which should be increased only when necessary as, for example, when the horse does not answer to their touch. Without trying them, it is difficult to find out the proper length for the neck of the spurs, which should be short, if the legs of the rider are short ; because, in this case, his heels will always remain close to the animal’s sides. -If the rider has long legs, he will be obliged to raise his heels when he wants to use the spurs, which should have proportionately long necks, so that he may have to shorten his legs as little as possible. CUTTING WHIP. I use the cutting whip only in work on foot, in order to teach the horse to go forward, and to give way to the spur, but I discard it the moment I mount. True horsemen require legs and hands; incompetent riders need a whip. STIRRUPS. in getting rid of them; because it is extremely difficult, if not impossible, to leave off a bad habit. in getting rid of them; because it is extremely difficult, if not impossible, to leave off a bad habit. Digitized by Microsoft® STIRRUPS. 17 that my modest advice may render such accidents rare in the future. Standing on the stirrups is like standing on a spring-board, in which case, if any violent movement is produced by the horse, the rider is liable to be shot forward as if from a catapult, and always on to his head. Even when the horse plunges forward, the rider will be rarely unseated if he is sitting well into the saddle, with the weight resting on his seat. Then the worst thing that can happen to him is to be shifted on to the horse’s neck, in which case he will fall softly; but if he is bearing his weight on the stirrups, with his legs straight, nothing will be easier for the horse than to send him between his ears. When the rider adopts this faulty position, his body is carried forward, and the horse is able to plunge as he likes, on account of the weight being taken off his loins. The rider who stands on his stirrups is in the position of a gymnast standing on the hands of a comrade, whose busi- ness is to give him the necessary propulsion for making a dangerous leap. In order for him to be projected very far, it is necessary for him to keep his body and legs straight, but if he bends his knees he cannot go far. The stirrups play a véZe similar to that of the hands of the gymnast who is ready to give the propulsion. Hence, if the rider bends his knees he will remain in the saddle. We should bear in mind that if the pupil, before his legs have got well down, is allowed to use the stirrups, he will not be able to keep his feet in them. He will then make all sorts of contortions in order to retain them, in which case, not only the muscles of the legs, but also those of the body and face, will become contracted, and the neck and the shoulders will assume a characteristic form of stiffness. Notwithstanding all the care which the teacher may take later on to correct these faults, the pupil very rarely succeeds 2 2 Digitized by Microsoft® HORSE AND MAN. 18 in getting rid of them; because it is extremely difficult, if not impossible, to leave off a bad habit. MAN’S SEAT. As a rule, every one can acquire a good and strong seat, but every one cannot become a fine and graceful horseman. A pupil who has been taught well from the beginning, has done his best to learn, has trotted without stirrups for several Digitized by Microsoft® MAN’S SEAT. 19 months, has ridden horses that were lively and difficult, with- out being dangerous, will of necessity acquire a firm seat. He will hold his head in a free and unconstrained position, so that he can move it with ease in every direction. He will keep it high in all ordinary paces and when jumping, and will lower it a little, with the chin towards the breast, when going fast. Huis eyes should preserve their mobility, and should not become fixed in any one direction, so that they may take notice of all the inequalities of ground, fences and other diffi- culties which the horse may meet. He should keep his neck supple, and his shoulders down and without contracting them, as is often done. His arms should hang naturally down to the elbows, which should be kept close to the sides, and should on no account be turned outwards; for the hands cannot be kept light if the elbows are in this position. Lightness of hand is absolutely necessary for using the reins with precision and without jerking them. With the elbows firmly fixed to the sides, it is easy to prevent the hands from moving about. A rider who has a good seat will keep his elbows at the height of his waist, the insides of the wrists facing each other and the fingers turned inwards. He should avoid rounding the wrists, which would tend to bring the elbows away from the sides, and would prevent him having a straight line of communication with the horse’s mouth. The action of the bit on the mouth of the horse should be produced only by the tightening and slackening of the fingers on the reins. A rider who works his hands a good deal, does not know how to use them ; but a man with good hands produces the necessary effect by a simple movement of the wrist and fingers. The rider ought to keep his body upright, but without any stiffness, which prevents the independent action of the “ aids ” (hands and legs). MAN’S SEAT. Stiffness in one part produces muscular contraction of the whole body, which is a condition entirely 2* Digitized by Microsoft® Digitized by Microsoft® HORSE AND MAN. 20 opposed to good horsemanship. He should in no case hollow out the back, because that will produce stiffness which is the thing we should avoid. The loins (not the shoulders), on the contrary, should be slightly rounded, so as to preserve their elasticity. The chest should be kept in a natural position, without being pressed out in any way, and the muscles of the pelvis relaxed, which is the only means of obtaining ease. The weight of the upper part of the body should be carried by the buttocks, which are its only proper support. The legs should be stretched well down, the thighs flat, the knees close to the flaps of the saddle, and the toes turned slightly out- wards, so that the horse may feel the leg before he is touched with the spur. When the toes are turned too much inwards, the calves of the legs will be forced outwards, and then the rider will be able to use the spurs only by prods. The fact of the knees forming a kind of fixed pivot will give great mobility to the lower portion of the legs, which should fall naturally from the knees, and should not be kept close to the animal’s sides. In order for the rider to be at his ease, he should sit well down in the saddle, without hanging on by his hands or gripping by his knees, which should grip only when occasion demands. By always gripping with the knees, the rider is made to assume a cramped position on horseback. On the contrary, the seat should be maintained by balance and not by grip. When we use the leg, we should use it from the knee to the heel. Fatigue of the muscles above the knee is caused by stiffness and is a sign that the seat is bad. The rider will also appear cramped if his knees are too high, if too low he will rest not on his buttocks, but on the inner part of his thighs, which fact will increase the firm- ness of his seat, because the entire length of the legs will be applied to the horse. Digitized by Microsoft® MAN’S SEAT. This seat can be used with advantage by cavalry when charging, oniy at the moment of meeting Digitized by Microsoft® MAN’S SEAT. 21 the enemy, because it helps them to avoid being displaced by the shock. With this seat, it is difficult to keep close to the horse when he changes from the canter to the trot. In con- clusion, I may point out that a rider who has a good seat sits on a saddle in the same manner as he would do on a chair. When the rider uses stirrups, his toes ought to be higher than his heels. Without stirrups, the feet ought to fall naturally, and consequently the toes will be lower than the heels. We may see that with the feet out of the stirrups, it is impossible to keep the toes up without contracting (stiffening) the muscles of the legs. In Germany, pupils are taught to keep the toes higher than the heels, when riding without stirrups, which practice gives German horsemen their characteristic stiffness. I am aware that Germans are naturally stiff. Even a Frenchman made to ride in that manner would become stiff. The length of the stirrups should be proportionate to that of the legs. The traditional plan of measuring the length of the stirrups by that of the arm, affords a useful approxima- tion which one has always to rectify when mounted. To do this, it is necessary to take the feet out of the stirrups and to leave the legs hanging down. Then the stirrup leathers will be the correct length when the “tread” of the iron comes just below the ankle joint. It is generally recommended to keep the foot in contact with the internal side of the stirrup iron. I place my foot at an equal distance from both sides of the iron. We can give great suppleness to the ankle joints by working them in various directions, and can then let go or pick up the irons very easily. In the open I ride with the leathers one hole shorter than in the school, and then obtain a better support in quick paces, especially when rising at the trot. MAN’S SEAT. In the manége one requires to have the legs lower down than in the open, so as to get the legs round the horse, and it is also necessary to be entirely on the buttocks, in Digitized by Microsoft® HORSE AND MAN. 22 order to catch each movement of the horse. We all know that this feeling can be acquired only by long training. order to catch each movement of the horse. We all know that this feeling can be acquired only by long training. Many physical qualities are necessary for obtaining a strong and graceful seat. For instance, it is evident that a stout, short man is less likely to ride well than one who is sufficiently tall and slight. I say “sufficiently tall,” because it is a common error to think that one must be tall in order to ride well. On the contrary, the taller the horseman, the more difficulties will he have in riding. First of all, the longer the body, the more easily can it be displaced, on account of the height of the centre of gravity, and the harder will it be for the equilibrium to be restored. Also, long legs cannot adapt themselves to the sides of the horse so well as those of medium length, because the feet are below the sides of the horse, and if the rider wishes to use the spurs he is obliged to bend his knees, in order to shorten his legs, which action is ugly, and weakens the seat. I recognise the fact that every man can acquire a strong seat if he works hard. The remarks I lately made about German stiffness also apply to the English. Nations of Teutonic origin have justly earned the reputation of being the best horsemen, which they have become by their great perseverance and love of work. The Latin races, being of middle height, are better fitted, by their suppleness and agility, to work in harmony with a horse, and if they were industrious they would certainly be the finest horsemen in the world. But, unfortunately, they are content with being nearly the best. It goes without saying that I am speaking generally, and that there are good and bad horsemen in every country. Digitized by Microsoft® MAN’S SEAT. 24 This faulty practice is avoided when the snaffle reins are held one in each hand. We should bear in mind that it is easier to put a beginner into a good position than to rectify a bad seat later on. T seat later on. To sum up, I may say that the chief good point about a rider is firmness of seat, which is obtained by a correct position and practice. The rider should have, not merely blind pluck, but self-confidence and coolness, without which he will not be able to utilise the useful things he has learned. It is not necessary to know all about horses in order to ride well. I prefer an unscientific man who can stick on any rideable horse, to one who is strong in theory but weak in practice. The purely theoretical man almost always succeeds in making restive horses which he pretends to break in. He may have enough firmness of seat to give the horse an indication to move, but not sufficient to enable him to enforce his orders when the animal “ plays up.” To sum up, I may say that the chief good point about a rider is firmness of seat, which is obtained by a correct position and practice. The rider should have, not merely blind pluck, but self-confidence and coolness, without which he will not be able to utilise the useful things he has learned. It is not necessary to know all about horses in order to ride well. I prefer an unscientific man who can stick on any rideable horse, to one who is strong in theory but weak in practice. The purely theoretical man almost always succeeds in making restive horses which he pretends to break in. He may have enough firmness of seat to give the horse an indication to move, but not sufficient to enable him to enforce his orders when the animal “ plays up.” Nothing is worse than to provoke a horse to resistance if the rider has not the pluck to make him give in. MAN’S SEAT. The ease, firmness of seat and confidence of the rider generally depend on the first lesson which he has received ; and, as I have said, a good seat is acquired only by trotting without stirrups for a long time Digitized by Microsoft® MAN'S SEAT. 23 The only horses beginners should ride are those which have easy paces and very good tempers. One cannot take too much pains to give confidence to a novice. It is only the confidence which he gets in his first lessons that will enable him later on to ride with entire absence of stiffness. A man whose movements on horse-back are 8tiff, may remain on the outside of his mount, but does not ride him. Stiffness of arms, legs and body makes fine horsemanship impossible. How can contracted or stiff limbs keep in touch with the horse while they are occupied in holding the body on the saddle? When they become supple, and when the body maintains its position in the saddle by balance, they will acquire a delicate feeling with the horse, which they had not at first. Flexibility of the limbs and a good seat are indispensable conditions for attaining “ equestrian tact.” Being merely stuck on a saddle is not riding. Generally the pupil gradually gets out of this faulty position only by acquiring confidence in his seat. I need hardly say that a man may be very brave and yet have no confidence in the saddle. Slight horses are best for the first lessons, especially in the case of boys and men who have short legs. A too great separation of the legs might have grave results, and it fatigues the groins without benefiting the rider in any way. I have seen malformations of the hips caused by this practice. Later on, the rider becomes accustomed to this form of gymnastics, and he will be able to ride a horse of any shape without discomfort. I think that beginners should not ride with a double bridle, and that it is better for them to use an ordinary snaffle, with a rein in each hand. If they are at first given a double bridle, the body will probably follow the movements of the hands, because, at the beginning, they almost always carry the hands and alse the arms either to one side or the other. Digitized by Microsoft® HORSE AND MAN. Digitized by Microsoft® LADY'S SEAT. Except as regards the legs, a lady should sit on a saddle exactly like a man. For some time there has been talk of ladies riding astride, which practice would deprive her of all feminine grace, and would afford no useful result. The great want in a man’s seat is firmness, which would be still more difficult for a woman to acquire if she rode in a cross-saddle, because her thighs are rounder and weaker than those of a man. Discussion of this subject is therefore useless. Ladies who ride astride get such bad falls that they soon give up this practice. At all paces the shoulders should be parallel to the ears of the horse, which is possible only when the hips occupy a similar position. Therefore the position of the lady depends Digitized by Microsoft® LADY’S SEAT. 25 entirely on that of the hips. The two legs being on the left, the right leg is hooked round the upper crutch, and is more advanced and higher than the left leg, which presses against the leaping-head, a little above the knee, and the foot rests in the stirrup. It has been proposed that ladies should sit on the right side of the saddle. English and American journals have dwelt on the bad effects of girls on only one side of the horse, and they have tried to make out that this practice causes curvature of the spine. As I judge only by practical observations, I can- not say how it would affect children of five or six years old; but as I have often taught beginners of twelve or thirteen years old, I can certify most positively that girls of that age have nothing to fear on that score. I am well aware that in England and America young girls are given their first lesson in riding by coachmen and grooms, who are apt to teach wrongly and to pass over, and even accentuate faults. The same remark applies to many riding masters who have studied both theory and practice, and who succeed, only after a long course of teaching, in correcting small mistakes, which are apt to become greater. I maintain that if a lady is taught by a good master, her figure, instead of being spoiled by riding exclusively on the left side of the saddle, will increase in grace and suppleness. LADY'S SEAT. If, on the contrary, the left shoulder is not so far advanced as the right one, her equilibrium will be unstable and she will be in danger of falling off, which kind of fall should be provided against, because it is dangerous; for the lady will fall on her head, supposing that she gets clear of the crutches and stirrup. If, in falling, her foot catches in the stirrup, or if her skirt becomes hooked on the crutches, she will become dragged without having any means of freeing herself. A lady is rarely thrown to the left, because she is supported on that side by the crutches, and, if need be, by the stirrup. All the danger of a fall is therefore on the right, and it increases in proportion as the left shoulder is drawn back. It is easy to understand that if a horse makes an abrupt It is easy to understand that if a horse makes an abrupt movement, or makes a shy from the right to the left, the upper part of the body will be forcibly thrown to the nght, a displacement which can be easily prevented by the lady holding herself in a correct position, that is to say, if her shoulders are placed as I have just indicated. If, on the contrary, the left shoulder is not so far advanced as the right one, her equilibrium will be unstable and she will be in danger of falling off, which kind of fall should be provided against, because it is dangerous; for the lady will fall on her head, supposing that she gets clear of the crutches and stirrup. If, in falling, her foot catches in the stirrup, or if her skirt becomes hooked on the crutches, she will become dragged without having any means of freeing herself. That which gives firmness of seat to the horsewoman also endows her with elegance. Consequently, she need not sacri- fice either of these advantages for the other. It is necessary: Firstly, that the knees are brought as closely together as possible, the right leg being firmly hooked round the upper crutch, with its pressure acting from front to rear. The left leg, on the contrary, by reason of the fulcrum afforded by the stirrup, exerts its pressure from rear to front. Digitized by Microsoft® LADY'S SEAT. We may therefore continue to make ladies ride only on the left side. Placing her on the right side would be accompanied by the serious inconvenience of making her hold the whip in her left hand, which she cannot use as easily as her right. Her whip hand acts the part of a horseman’s leg. The position of a lady on the left side of a saddle causes almost all the weight of her body to be carried to the right side ; consequently, the left hip being freer from weight, is apt to be stuck out behind the right hip, which is a fault that ought to be avoided. The weight of the body ought to Digitized by Microsoft® Digitized by Microsoft® HORSE AND MAN. 26 be equally distributed on both sides. Like a man in his saddle, she ought to sit in her saddle exactly as she sits in a chair, namely, with the hips and shoulders parallel to the ears of the horse. This is a question not only of correct attitude, but also of strength of seat, which is the main thing. be equally distributed on both sides. Like a man in his saddle, she ought to sit in her saddle exactly as she sits in a chair, namely, with the hips and shoulders parallel to the ears of the horse. This is a question not only of correct attitude, but also of strength of seat, which is the main thing. A lady is rarely thrown to the left, because she is supported on that side by the crutches, and, if need be, by the stirrup. All the danger of a fall is therefore on the right, and it increases in proportion as the left shoulder is drawn back. It is easy to understand that if a horse makes an abrupt movement, or makes a shy from the right to the left, the upper part of the body will be forcibly thrown to the nght, a displacement which can be easily prevented by the lady holding herself in a correct position, that is to say, if her shoulders are placed as I have just indicated. LADY'S SEAT. Secondly, the left shoulder should be carried well forward, and the body slightly bent forward, so as to make it more supple. If the left shoulder is drawn back when the horse is at a Digitized by Microsoft® LADY’S SEAT. 27 walk, and consequently when all the weight is on the saddle, the position of the lady is bad and particularly ungraceful. When rising at the trot, her seat is still worse; because the left shoulder is jerked forward when she rises, and goes back when she comes down on the saddle. This is the well-known ungraceful cork-screw movement. When When the lady sits evenly on both sides of the saddle and has a good position at the walk, her hips and shoulders will easily remain well placed at the trot. She should keep her body well under her when rising at the trot. The upper part of the body makes no effort, but allows itself to be raised by the movement of the horse. The foot rests in the stirrup iron without stiffness, and the ankle and knees act only as hinges. The slightest muscular con- traction, or the least effort made by the ankle, knees or loins will give the rider a stiff and ungraceful appearance, and will cause fatigue. If the lady will carry out these rules, she will ride in two-time, namely, one on the saddle and one in the air. Otherwise she will come down too quickly, and will mark two-times on the saddle, which will give her a useless and fatiguing shock. I will refer to this fact at greater length when discussing the position of a horseman at the trot (p. 146). A horsewoman should have great pliability of body, which she will acquire by practice in riding and other preliminary exercises, of which dancing is the best. It also depends on certain small details of dress, about which I may give the following advice. A lady on horseback is apt to cut herself. The slightest crease in her clothes may cause an abrasion. For a long ride, and still more for hunting, she should wear a short chemisette, made of very fine material, and fitting close to the body. The collar and cuffs ought to be strongly connected to this chemisette, and not fixed to it merely by pins, which are Digitized by Microsoft® HORSE AND MAN. Digitized by Microsoft® LADY'S SEAT. 29 the mane at the withers does not become pressed upon by the pommel of the saddle, the irritation from which often causes the animal to “play up.” the mane at the withers does not become pressed upon by the pommel of the saddle, the irritation from which often causes the animal to “play up.” A few words about putting a lady into a saddle may be useful, not only for horsewomen, but also for men who have the somewhat parlous honour of touching a lady’s foot. I regret to say that when being “ put up” a lady generally does the very opposite of what she ought to do. She places the left foot in the hands of her male attendant, and jumps from the right foot on to the left foot, while carrying the body for- ward, with the result that all the weight falls suddenly on the hands of the man, who is inevitably pushed backwards and away from the shoulder of the animal. On the contrary, when her left foot is on his hands, she ought to use the right leg only to make a slight spring which will enable her, by straightening her left knee, to hold the body upright and a little inclined to the rear. This is a very simple movement, and is exactly similar to that which one makes when getting up a rather high step of a staircase. The lady should not try to jump up, but should limit her spring to straightening the left knee, while keeping the upper part of the body well bent forward; and she should use her arms, her left hand resting on the shoulder of her attendant and her right hand on the upper crutch. In acting thus, she will mount quite straight by the impulse given by the hands, and will come naturally on the saddle, and will carry the seat a little to the rear. She should not try to place herself on the horse, which is the busi- ness of her attendant, but should merely occupy herself with sitting down. If a lady attempts to jump into the saddle, she will generally strike it and fall on the man. I cannot help adding that the old practice of giving the left foot is bad, and am unable to explain its origin or the reason of its continuance. LADY'S SEAT. 28 liable to fall out or hurt the wearer. I strongly advise that she should wear socks instead of stockings; because a garter is always inconvenient and may cause serious wounds. The socks should be furnished with a close-fitting collar of some soft and elastic material, such as knitting or jersey, lined with silk, or, still better, very fine doeskin. The trousers should be strapped with india-rubber and should fit rather closely, so that they may not wrinkle. The boots should have elastic sides and not buttons, which might cause wounds. I prefer ordinary boots to long boots, which are too hard, and are consequently apt to cut the wearer under the knee, and to prevent her feeling the horse with her leg. The corset should be very short and low. A long busk is not only inconvenient, but is also dangerous. I would not have touched on these details but for the fact that the dress of the horsewoman is closely connected with her strength of seat and ease in the saddle. I have seen so many ladies returning from a ride in pain, and condemned to spend many days in a long char, that I am certain the points to which I have drawn attention are important. The head-dress of the lady should be firmly arranged, so that it may not occupy her attention, in which case she will think too little of her horse. Then, if she loses her hat, she will probably lose her head. The choice of a saddle is of great importance, both for the rider and for the horse. Its seat should be quite level, so that the knees of the rider may not be higher than her seat, and it should have but little stuffing, so that it may fit close to the horse, and may not become shifted. The smallest displace- ment of the saddle may seriously injure the animal's withers. If the cantle of the saddle is too short, it will certainly hurt the rider, and if too long, it will hurt the horse’s loins. A lady’s horse should have high withers, so as to prevent the saddle turning round. Great care should be taken that LADY’S SEAT. LADY'S SEAT. In fact, to be put on the saddle with the left foot on the hands of the attendant, the lady, when she is Digitized by Microsoft® HORSE AND MAN. 30 raised up, ought to carry her seat from front to rear, and from left to right, while the man at the same time makes a movement from rear to front and from right to left. Here we have a double displacement. If, on the contrary, the lady gives her right foot, which is nearer to the horse than the left, she need make only a very slight spring with her left foot and to straighten the right knee, to come naturally on the middle line of the saddle, and to sit down without the slightest displacement. I do not claim the merit of having discovered this simple method, which has long been practised by many of the best horsewomen. I have had the honour of putting into the saddle sovereign ladies who always mounted in this manner. Ladies, without having previously made up your mind, please try for a week this method of being put up on the saddle, and I am certain you will adopt it. As soon as the lady is in the saddle she ought to place her right leg over the other crutch without waiting to settle her skirt, so that she may avoid falling off in the event of the horse starting to one side. I may add that the hands of the man ought not to quit the foot of the lady until her right leg is in its right place. To dismount, the lady takes her foot out of the stirrup and gives her left wrist to the attendant. She thereupon removes her right leg from the upper crutch, gives him the right wrist, and lets herself slip down to the ground, while supporting herself a little with her arms. She ought to alight on her toes, and should bend her knees, so as to avoid any concussion, which, without this precaution, she is apt to receive after a long ride, on account of her legs being stiff and numbed. I repeat that the lady ought to give her wrists, and instead of jumping ought to shde down. It often happens that the lady throws herself from the saddle and the cavalier takes hold of her by the waist. Digitized by Microsoft® ORDINARY RIDING. Lunging a Horse—Close work, advancing—Collecting a Horse and direct Flexion —Objects to be obtained by direct Flexion—Making a Horse quiet to mount —How to hold the Reins—Teaching a Horse by the Whip to obey legs and spurs—Horse mounted, first Defences, means for overcoming them—The Walk—To halt and stand still—Changes of Direction—Lateral flexions— Objects of Lateral flexions—Rotation of the Croup and Shoulders —School Walk—The rein back—The ramener, collection, rassembler, and equestrian tact—Side steps and two tracks—The trot-—The canter—Voltes and demi voltes at the canter—Changes of leg—The Hack. Digitized by Microsoft® LADY'S SEAT. Not being able to hold her up with his outstretched arms, he lets her Digitized by Microsoft® LADY’S SEAT. 31 slide through his hands, which is ungraceful and not par- ticularly decent. The question is often asked whether the lady’s cavalier should ride on her right side or on her left? I do not think that a fixed rule on this subject would work well. Under ordinary conditions the man should be on the right, because the lady, in order to turn her head towards him, is obliged to carry the right shoulder back, which is the correct position, as I have already said. Further, the gentleman being on the right, can, in case of necessity, help the lady, to whom he cannot come close enough, on account of the position of her legs, if he is on the left. If it happens that there is danger on the left to the lady, by reason of the presence of horses or carriages, he ought to place himself on that side in order to protect her legs. Digitized by Microsoft® LUNGING A HORSE. I WORK all horses in the same manner. The animal which is to be broken being brought into the school saddled and bridled, I pass the reins of the curb and snaffle through the throat-latch, so that the horse may not catch them with his feet. JI then buckle a lunging rein to the left ring of the snaffle and let the horse walk at ease. For every lesson the horse should have flannel bandages on his forelegs, from the fetlock to the knee, so as to sup- port the flexor tendons and to guard the horse from getting splints which are often caused by a green horse hitting himself. Immediately after the lesson I take the bandages off the forelegs and put them on the hind ones, and leave them on for three or four hours, which period is sufficiently long to Digitized by Microsoft® LUNGING A HORSE. 33 prevent the legs filling and windgalls forming. If flannel bandages are constantly left on, the tissues of the legs are apt to become softened and the tendons to become stretched by the action of the heat. If he tries to get away from me I let him go, my object being to make him keep close to the wall. If he does not try to get away and turns towards me, I show him the driving whip, the sight of which will make him get away as far as the lunging rein will allow him. I hold the whip in my right hand, the rein in my left, and I let the horse free to go at any pace he likes. The object of walking the horse in this way round the school is to make him examine the ground and all the surrounding objects which are new to him. If he is lively he will probably plunge, and then canter or trot, but he will soon steady down. If he is sluggish he will be disinclined to go forward, and it may be necessary to make him go on by showing him the whip. If that does not produce the desired effect, we may touch him lightly on the hind quarters, so as to make him trot or canter for about five minutes. We should most carefully avoid making any abrupt or rough movement which might frighten him. LUNGING A HORSE. I have said that I would give the animal five minutes’ work at a fast pace, but, of course, would not do so unless he was in good condition. If he was not fairly fit, the duration of the work would be shorter during the first few lessons, and would be gradually increased up to the five minutes’ limit. When the horse has circled for five minutes to the left— When the horse has circled for five minutes to the left— that is to say, with the left shoulder towards the inside of the school—I throw the whip down and try to steady the horse by my voice. I then shorten the lunging rein until he is close to me. I speak softly to him and pat him on the neck, which is a form of caress that all horses like. I also stroke his head, if he will let me do so, and then unbuckle the lunging rein and fix it to the off ring of the snaffle. Digitized by Microsoft® 34 ORDINARY ORDINARY RIDING. 34 After a short rest I begin the same exercises to the right for five minutes. When the work is over I again throw down the whip, and call the horse to me, while at the same time gently drawing him towards me by the lunging rein, and then pat and stroke him as before. In my opinion the foregoing exercise is the indispensable first step in breaking, and, as I attach great importance to it, I will give my reasons at some length as follows. A young horse is almost always restless and timid; shadows, walls, and all sorts of trifling objects frighten him. He rushes away from anything that startles him, and goes to the part of the school which is unoccupied, while I remain by myself in the centre. If I show him the whip, while advancing towards his side, he instinctively flies away and goes to the wall, against which he can be easily kept by pointing the whip towards his shoulder. Being placed between the threat which I make and an object which he fears, and from which he has fled, he returns to this fixed object, which appears to him to be less terrifying than the driving whip. Digitized by Microsoft® LUNGING A HORSE. When, thanks to this procedure, he has several times crossed the place of which he is afraid, he will eventually have no fear of it. We may note that it was not necessary to use punishment, which should be our last resort. Further, if the animal is too lively, I would give him a good long turn at the trot, or even at the canter, if he prefers that pace, in order to get rid of his excess of energy. If he is dull, I teach him, by means of a few cuts of the whip, te go forward. We must draw a distinction between a horse that is soft and one that is sluggish. If the former is properly fed and exercised he can become lively; but the latter, although he may be full of muscular vigour, does not like to put forth his strength except when he pleases, a fact which makes him dangerous to an inexperienced rider. We are Digitized by Microsoft® LUNGING A HORSE. 35 never certain except with a free-going horse. Although an impetuous animal may get out of control and run away, I prefer him to a sluggish horse. Pluck is the best quality in a horse. I have always obtained a good result with my equine pupil, who quickly loses fear of the things which surround him, because he has no one on his back to hamper his movements and upset him. I easily obtain my result without a struggle, and without having to put up with the plunges and shies of a horse which has been mounted too soon, and which is liable to roll over on the ground with me, on account of the awk- ward use of his feet. It is of great advantage to teach a horse to know, bear and fear the lunging whip; because if, later on, he refuses to go forward when ridden by an indifferent horseman, the use of the whip will make him obey. The sight of it will often be sufficient to make him go forward. If he refuses to do so, we should touch him lightly and carefully behind, because any roughness or abruptness is apt to make him resist. LUNGING A HORSE. Lunging has the further advantage of enabling us to make Lunging has the further advantage of enabling us to make a horse trot out, by driving his hind quarters forward with the whip. In fact, we can thus make a horse acquire the habit of collecting himself. By being lunged in freedom, he will gain suppleness, confidence, cleverness and sure-footed- ness, which are the best qualities in a horse. A harnessed horse puts his weight on the collar and a mounted animal puts it on the hand of his rider; but a horse which is lunged is obliged to balance himself independently of all support. A horse which is lunged at a fast pace for five minutes to the right, and for another five minutes to the left, gets in good wind, on account of the work his lungs have to do. If he was ridden, he would not get this exercise during his first few lessons; because they would be given at a walk. 3* 3* Digitized by Microsoft® ORDINARY RIDING. 36 During the first two or three lessons, I let the horse go at any pace he likes, provided that it is fast and that he keeps to the wall. I make him trot in the subsequent lessons. It 1s so easy to make a horse trot, that one ought to do so with any horse in the third or fourth lesson without an assistant. The old custom, recommended in almost every book on the subject, of having two men to lunge a horse is faulty, because it is impossible to have perfect harmony in the movements of these men. It often happens that the man with the whip touches up the horse when he ought not to do so, and that the man who 1s holding the lunging rein, stops the animal at the moment when his comrade is making him go on. Of course, such a discord could not happen if the breaker was by himself. Supposing that the horse is at the wall, and going to the left; the breaker, who is at the centre of the school, ought always remain facing the horse and at a line with his shoulder, thus keeping him enclosed in the angle made by the lunging rein and whip, the former held in the left hand, the latter in the right hand. Digitized by Microsoft® LUNGING A HORSE. The breaker should always accompany the horse, but should not fo/Zow him, and should place himself so as always to keep the horse between the lunging rein in front and the whip behind. In order to accompany a horse without following him, while constantly remaining in a line with his shoulder, we need only follow the diagonal, while alternately extending and drawing back the arm. This precaution is essential, because if we describe the circle, when following the horse round the school, we shall get giddy and become unable to carry out the work properly. By moving diagonally we can accompany the horse as long as we like. To make the horse trot, I touch him lightly behind with the whip. It is better to touch the shoulder, but we should not try to do so, especially with a young horse, unless we LUNGING A HORSE. 37 have great experience in using the whip. If, instead of lightly dropping on the shoulder, the lash touches or brushes by the head, the horse will start back, and we shall then cbtain a movement which is the exact opposite of what we wanted. Also, if the horse is a bit lively, we may stimulate him with a click of the tongue, which is a form of stimulus we should not use too much, because it might upset other horses when we are in company. If the use of the driving whip makes the horse plunge or go off into the canter, as it often does, I soothe him by lightly shaking the lunging rein and speaking to him at the same time. The lunging rein should never be kept tight, but should remain in touch with the mouth only by its own weight, and by the vibrations which the breaker gives it. I have already said that the voice is a powerful help in breaking. If, when we shake the lunging rein to make the animal adopt a slower pace, we call out loudly “trot!” no matter how stupid he may be, he will soon connect in his mind the effect produced on his mouth and the sound which falls on his ears. At first he will obey only the combined impression, but he will soon learn to obey the voice by itself. LUNGING A HORSE. When I have obtained a free trot which is kept up for the time required, I bring the horse to the walk by lghtly shaking the rein, in the same manner as I made him change from the canter into the trot. Here also I use the voice rather loudly, though mildly, in saying “whoa!” The next step is to make the horse come up to the breaker. To do this I gently shorten the rein and draw the horse towards me, while walking backwards with very short steps, so that he gradually gets closer to me. When he has arrived within reach of my stretched-out arm, I pat him on the neck and soothe him with the voice. I take great care to avoid making the slightest forward movement with the Digitized by Microsoft® ORDINARY RIDING. 38 body, my wish being to give him confidence. If I were to make a step forward, he would immediately spring back, which result would be the opposite to that which I wished to obtain. If nothing has frightened him while he is coming to me, and if my pats on his neck and my voice have shown him that he need not fear my presence, he will soon gain confidence, and will of himself try to come to me, and all the more readily when he finds that tranquillity is to be obtained only at the centre of the school. He ought to have sufficient confidence to come up to the breaker without fear, but he should do so only at a given signal. Our object is to make him understand this signal without the use of the lunging rein, which will be discontinued later on. I use the driving whip to make the horse come up to me in obedience to my order, and I give him light and repeated flicks on the buttock, ribs, or shoulder, while always seeking to block the side from which he tries to escape. In order to make the animal go forward, I sometimes touch him on the breast, in which case his first movement is to run back; but I continue to hold him tightly with the lunging rein, and while preventing him from running back, I call out “whoa!” If he runs back, he does so because he is afraid of the whip. Digitized by Microsoft® LUNGING A HORSE. An unbroken horse does not fly from a sting, flick, or prod; on the contrary, he goes up to it and lies against it. We shall see further on that the effect obtained on a mounted horse by the pressure of the leg or by a touch of the spur is due solely to education. <A horse in a state of nature will do the very opposite to what he will do when broken. For instance, stung on the right flank by a fly, he will bring himself round to the right, until he meets some object against which he can rub himself or even he upon. The sight of the driving whip makes him run away from it, but its touch makes him go forward. As soon as the tension of the lunging rein has shown him that he cannot get away Digitized by Microsoft® LUNGING A HORSE. 39 from the sight of the whip by running back, his instinct will cause him to go forward. If at that moment the whip is lowered and a pat on the neck given, he will gain confidence, will understand what we want him to do, and will obey our wishes. Although we cannot obtain the result at the first attempt, we shall do so in a few lessons, especially if the breaker does not frignten the horse by some abrupt move- ment when the animal is advancing. We can dispense with the lunging rein as soon as the horse will come freely up to us, when we show him the driving whip. This first lunging work is only preparatory. It has been used in all times, but has been applied and utilised in different ways. It was employed in excess before the days of Baucher, who disliked it. I think it is useful, supposing, of course, that it is not employed to fatigue the horse. In order to make a horse come up to me without the help of the lunging rein, I use the same methods as when lunging. At the beginning of the work I employ the rein a good deal and the whip only a little. LUNGING A HORSE. According as the horse pro- gresses I diminish the use of the rein and increase that of the whip, while always blocking up with the whip the side at which the horse tries to escape from me, so as to make him come up tome. Finally, he gets into the habit of coming up to me on seeing the whip, and without my using the rein in any way. I then teach him to follow me all over the school, while always stopping him with the whip from getting away, and making him promptly go on by light flicks on the hind quarters. Finally I discontinue the use of the lunging rein. If, as always happens, the horse refuses to obey the whip and tries to escape from me, there is a battle, which consists in my flicking the horse on the hind quarters until he comes up to me, which result may at first appear improbable. However, when the horse is pursued by the man round the school for a Digitized by Microsoft® ORDINARY RIDING. 40 sufhciently great number of times, his only idea will be to stop. As the whip follows him all round the track of the school and allows him rest only at the centre, he finishes by coming to it, in the same manner as he learned when being lunged. In order to facilitate this movement on his part, the breaker ought to seize the moment when the horse appears inclined to slacken his speed, to make him come away from the wall, by showing him the whip in front, and at the same time calling out “whoa!” which, from his lunging work, he will have learned to regard as an order to come up to his breaker. sufhciently great number of times, his only idea will be to stop. As the whip follows him all round the track of the school and allows him rest only at the centre, he finishes by coming to it, in the same manner as he learned when being lunged. LUNGING A HORSE. In order to facilitate this movement on his part, the breaker ought to seize the moment when the horse appears inclined to slacken his speed, to make him come away from the wall, by showing him the whip in front, and at the same time calling out “whoa!” which, from his lunging work, he will have learned to regard as an order to come up to his breaker. If the horse refuses to come up and remains at the wall, we should again follow him up, so as to bring him to the centre, and should continue to do so until he obeys. I may add that the breaker, by keeping himself out of reach of kicks and blows with the fore feet, will make his authority felt at a distance, and the horse will learn to obey without being provoked into resistance. I am not a believer in the cavesson, except for really vicious horses. If it is used, it should be light and well stuffed. Having obtained the foregoing important result, I will pass Having obtained the foregoing important result, I will pass « on to “close” work. Digitized by Microsoft® CLOSE WORK; ADVANCING. If my horse refuses to advance, it is evident that, from th position I am in, it will be difficult for him to run back, bu 42 Appropriate punishment and reward are the two great principles of successful breaking. If my horse refuses to advance, it is evident that, from the position I am in, it will be difficult for him to run back, but not impossible, and it is necessary to look out for any move- ment which a green horse may make. To make him go forward, I extend my right arm, while always holding the snaffle reins with the right hand at the chin-groove, over which the curb-chain passes ; and I push, instead of drawing forward, whilst holding the end of the snaffle reins with my left hand behind my back. I lightly touch the horse with the tip of the cutting whip a little behind the girths. If the horse is quiet and not too nervous and excitable, he will go forward without rushing. He will often, however, answer to the cuts of the whip by plunging, rearing, shying violently to one side, or running back, which are the four “defences” which a horse can offer in this case. Let us now examine the best means of thwarting them. If the horse plunges, we have only to raise his head, so as If the horse plunges, we have only to raise his head, so as to put the weight on his hind quarters, taking care to keep close to his shoulder, so as to avoid being struck by his fore feet. A horse cannot plunge with his head high. Rearing is more dangerous, because the horse may hit the breaker a blow on the top of the head with one of his fore feet, to avoid the bad consequences of which accident it is well to wear a chimney pot hat, which has often saved me from a blow on the head. When the horse rears, the right hand should quickly let go the snaffle reins, only the end of which should remain in the left hand, and then, if the breaker turns to the right about, he cannot be hit, as his distance from the horse will be equal to the length of the snaffle reins and that of his left arm. CLOSE WORK; ADVANCING. Having given up lunging, I take hold of the snaffle reins, after having passed them over the animal’s head. I replace the driving whip by a cutting whip, which I use along with the snaffle in the same way as I employed the lunging rein and driving whip. Having the horse with his right side parallel to the wall of Having the horse with his right side parallel to the wall of the school, I place myself close to his left shoulder and take hold of the whip and the end of the snaffle reins with the left hand. It goes without saying, that the reverse aids are used when going round the other way. I conceal the whip from Digitized by Microsoft® CLOSE WORK; ADVANCING. 41 the sight of the horse by placing it along the outside of my left leg. With the right hand I catch the snaffle reins close to the animal's mouth and just below his chin (Fig. 1), and then take a few paces forward. If the horse also advances, I pat him on the neck ; but if he refuses to do so, \| touch him up behind, close to the girths with the whip. Occasionally, a green horse at first refuses to advance and requires a touch of the whip, which is generally sufficient. Some horses refuse Fig. 1.—Leading a horse forward. Fig. 1.—Leading a horse forward. to go forward, especially if we make them bend their necks, in which case I replace the cutting whip by the lunging whip, with which I hit the animal on the hind quarters and always succeed in making him go on. I wish to direct my readers’ special attention to this decisive moment, on which contest depends our future success with the horse. It is important to understand that at this moment the animal pays no heed to the demands of his breaker and is ignorant of the breake1’s means of coercion. He has not yet learned to fear punishment, and knows little about the reassuring nature of pats on the neck. Digitized by Microsoft® Digitized by Microsoft® 42 ORDINARY ORDINARY RIDING. 42 ORDINARY RIDING. Appropriate punishment and reward are the two grea principles of successful breaking. Digitized by Microsoft® CLOSE WORK; ADVANCING. Having allowed him to regain his feet, go up very quietly to him, while carefully concealing the whip Digitized by Microsoft® CLOSE WORK; ADVANCING. 43 If he again rears, bear strongly on the snaffle, but without jerks. When he has tried to rear three or four times without being able to succeed, he will soon give up the attempt, and perhaps may throw himself on his side, which he can do only to the left, as the wall is on his right. A touch of the whip on the left side, and shaking him up with the left snaffle rein will be sufficient to make him get up. When the horse runs back, we should place ourselves in front of him, and should pull strongly on the snaffle reins, while slightly bending the knees and carrying the weight of the body back, so that the horse can drag us back only with difficulty, and consequently will soon become tired. 1 have had so much practice in letting myself be dragged, while keeping on my feet, that I can almost always stop any horse at his second or third step. When he finds that we passively and not actively resist his efforts, he will generally stop and give a deep sigh. Fix him in this position, and try to find out, which we can easily do with a little practice, if he is going to yield or to continue his resistance. The lesson should never be interrupted, and it should on no account be terminated by reason of the resistance of the horse. When the horse has given in, I again gently try, as in the previous manner, to make him go forward and do not stop until I have succeeded. Almost always the animal promptly gives in. In the first part of this breaking, Baucher used to try to make the horse come forward by lightly touching him on the breast with a cutting whip, while he held the snaffle reins at half-length and stood facing the horse. As I have explained in the chapter on lunging, I do not object to this method, although it is open to the serious objection of rendering the man liable to be hit by the horse’s fore feet and of making the animal ticklish. CLOSE WORK; ADVANCING. Besides, touching the horse on the breast is of no further use in breaking; but touching him on the Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 44 sides, as I have described, is the best preparation for the use of the spurs. According to the system of Baucher, one pulls the horse by his forehand, and if the animal is thus made to move, he will drag his hind quarters after him; but by my method the horse gets his hind quarters under him, and by their means he pushes the forehand forward, which is the essential principle of good riding. Digitized by Microsoft® COLLECTING A HORSE AND DIRECT FLEXION. As soon as my horse goes well with me round the school to the left, I make him change and begin similar work to the other hand. Then, when I am satished with him, I com- mence collecting him. While going to the left and keeping myself at his left shoulder, I take the bit reins in my right hand, at five or six inches from his mouth. Keeping the buckle of the snaffle reins in the hollow of my left hand, I seize with its fingers the snaffle reins at about eight or nine inches from the mouth, and I carry the left hand in advance of the animal’s head, in order to draw him forward. It is absolutely indispensable to hold the snaffle reins in this way, if we wish to prevent the horse from stopping, when we feel the curb reins. Merely holding the snaffle reins horizontally will not do, because we want to do more than to simply pull the animal forward. It is also necessary that the pull of the snaffle is in an upward direction ; because it ought to raise his head and neck at the moment when the pressure of the bit prompts him to flex his lower jaw (Fig. 2, jaw contracted ; and Fig. 3, jaw and bit free), which action on his part should be counter-balanced by the pressure of the snaffle, in order that it may not make him lower his head and neck. I feel the snaffle reins in order to raise the head and neck, I feel the snaffle reins in order to raise the head and neck, and I then put equal tension on the curb reins, in order to make him bend his neck and loosen his lower jaw. If the Digitized by Microsoft® COLLECTING AND DIRECT FLEXION. 45 horse does not go freely forward, I take a stronger feeling o1 the snaffle reins, and if he stiffens his neck and lower jaw, I horse does not go freely forward, I take a stronger feeling o1 the snaffle reins, and if he stiffens his neck and lower jaw, I y's hy/ : / its Fig. 2.—Jaw contracted. Fig. 2.—Jaw contracted. Fig. 2.—Jaw contracted. make more use of the curb reins. COLLECTING A HORSE AND DIRECT FLEXION. I particularly recommend that the tension of the reins should not be of a uniform and continuous nature, but should consist of light pressures sufficiently prolonged, so as not to form a jerk, and sufficiently Digitized by Microsoft® ORDINARY RIDING. 40 short, so that the horse may not be inclined to bear his weight on the bridle. If he yields, even in the slightest manner, I give to him and pat his neck. I then try to get him to yield still more, without asking too much from him. I again give to him and pat him ; and so on. We should take particular care to get the horse to give to us, not only with his neck, but also with his jaw, which he will do by opening his mouth. The bending of the jaw is the last stage of flexion. Several horses, in yielding the lower jaw, whether in direct or lateral flexions, bring the jaw from one side to the other side. Although the jaw in this case does not resist the hand, it yields by going to the right or left, instead of yielding in the direction of the axis of the head. This incomplete method of yielding can be corrected only by stimulating the animal to go forward. We can easily understand that if the lower jaw is carried to the right or left, the horse will not be correctly in hand, although his head and neck will be in a good position. His appearance of being in hand 1s not real, because the contrac- tion of the muscles of his lower jaw makes it impossible for his mouth to be light. In these conditions the horse will never go freely up to his bridle, despite the stimulus of the legs. If I require an increase of impulsion, it is because the horse, by escaping from the straight line, brings his head too near his breast, in which case he will have a tendency to get behind his bit. It is therefore necessary to send him up to it. Finally, if the jaw does not yield, the bending of the neck will only cause the weight to be brought back, and con- sequently to make the horse rein back or get behind the bit. The entire principle of direct flexion consists in the alternate opposing actions of the curb and snaffle. Digitized by Microsoft® COLLECTING A HORSE AND DIRECT FLEXION. Whilst the snaffle draws the forehand forward, a light pressure of the curb Digitized by Microsoft® COLLECTING AND DIRECT FLEXION. 47 steadies and bends the head, and causes the jaw to yield with- out stopping the forehand. To obtain this result, combined with lightness, we must con- To obtain this result, combined with lightness, we must con- Fig. 3.—Jaw and bit free. Fig. 3.—Jaw and bit free. great principle of faki Fig. 3.—Jaw and bit free. great principle of faki tinually practise the great principle of faking and giving ; the former to stop resistance, the latter to reward obedience. Having obtained it, we should again take, so as to make the horse yield still more; and so on. tinually practise the great principle of faking and giving ; the former to stop resistance, the latter to reward obedience. Having obtained it, we should again take, so as to make the horse yield still more; and so on. Digitized by Microsoft® 48 ORDINARY R ORDINARY RIDING. 48 A horse should not only champ his bit, but should also relax his lower jaw to it (Fig. 3), which concession proves that the bending of the head and neck is perfect. With this object, when the lower jaw readily yields to the tension of the curb, we should prolong this tension until the horse completely loosens his hold on the curb, and we ought to keep touching him lightly on the side, so as to prevent him stopping (Fig. 4). It is, of course, understood that this work ought to be done with great lightness of hand. We can gauge the sensibility of the mouth by an alternative feeling of the curb and snaffle, and can thus at once find out if the horse has a hard or soft mouth. In this manner we readily get good, that is to say, Zight hands, with which we can manage almost any horse by the continued play of “take and give.” It is a great advantage to have good hands, which will be sufficient for all ordinary work. But we can take and give for all our life without being able to render an account of what we are doing, in which case neither the hand nor the horse makes any progress. Finally, the action of the hand would be limited to giving when the horse pulls, and pulling when he gives. This faculty may be called the possession of a bell in the hand, and is in fact the movement of a bell in all its beauty. The well-trained hand acts in the contrary manner, because its véZe is to break in the horse, that is to say, to advance his education. Digitized by Microsoft® Fig. 3.—Jaw and bit free. great principle of faki It remains fixed in position by strongly closing the fingers when the horse pulls, but the moment the horse yields his lower jaw, the fingers should be relaxed with the rapidity of an electric flash. A good hand gives when the horse takes, and takes when he gives. A weél-trained hand gives when the horse gives, and takes when he takes, and that instantaneously. In all cases we ought to guard against confounding hardness Digitized by Microsoft® COLLECTING AND DIRECT FLEXION. 49 of mouth with the resistance which is due to a faulty position of the head. A horse which carries his head low is always heavy in hand, because he puts all his weight forward, but it does not therefore follow that he has a hard mouth. If we simply change the position of his head, and place it high, it will not bear on the hand, and we will be able to find out what kind of a mouth he has. CNL by Fig. 4.—Going forward when mobilising the lower jaw, so as to prevent the horse getting behind his bit. CNL by Fig. 4.—Going forward when mobilising the lower jaw, so as to prevent the horse getting behind his bit. It is a mistake to think, as many do, that a horse which slavers or foams at the mouth has a good mouth. To produce this soapy foam, a horse must contract his tongue, and will not then be able to have his mouth free, at rest, and ready for the fingering of his rider. A horse produces this foam by con- stantly turning his tongue, or by rubbing it against his hard palate, by passing it over the bit, or by making it into a ball at the back of his mouth. In all these cases, the only 4 4 Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 50 remedy is to put a small movable plate of the shape of a figure of eight, with its centre on the top of the port of the bit, upon which it can revolve. This apparatus will also prevent horses letting their tongues hang out of their mouths. Fig. 3.—Jaw and bit free. great principle of faki Sometimes a horse foams at the mouth by playing with one of the cheeks of the bit, which we can prevent by putting the mouthpiece a little higher up in his mouth and tightening the chin-strap, so that he cannot reach the cheeks of the bit either with his tongue or lips. A good mouth will always continue fresh during work with- out being either dry or wet. If the flexion has been made in the manner I have indicated, and by the methods I have described, the horse will have his neck high and bent at the poll; the axis of the head will be a little beyond the perpendicular, the mouth open, and the curb free (Fig. 3). In order that the flexion may be irreproachable, it 1s necessary that the muzzle should be on a line with the upper part of the shoulder (Fig. 5). The bending of the neck should bring the direction of the head near to, but not behind the perpendicular, which faulty position can be produced only when the neck is bent at a point too near the withers. By this position I have my horse very lightly on the bit. Whereas Baucher, who liked to have his mount Jehind the bit, drew the animal’s head back beyond the perpendicular, and thus put the horse into this position. I will now describe the mechanism of direct flexion, as I understand it, and as I have practised it, with this single difference, that, for the sake of clearness, I take for granted that the horse is halted; although I will explain later on, that at first I make the direct flexion while going forward. The majority of riding masters practise this flexion in an entirely different manner. To give an account of what cught to be the direct flexion, which is the fundamental Digitized by Microsoft® ORJECTS OBTAINED BY DIRECT FLEXION. 51 principle of riding, we must first know what is the desired result to be obtained from making it. principle of riding, we must first know what is the desired result to be obtained from making it. OBJECTS TO BE OBTAINED BY DIRECT FLEXION. 1. To balance the horse by the height of his neck. It is very rare that horses are naturally well balanced. On account of 1. To balance the horse by the height of his neck. It is very rare that horses are naturally well balanced. On account of Fig. 5.—Muzzle on a line with upper part of shoulder. Fig. 5.—Muzzle on a line with upper part of shoulder. their conformation, all horses have a tendency to be heavy in front, and the majority of them have this defect ; the cause being the distance which the head is removed from the base of support. The further the head is away from the centre of gravity the lower it is, and the more weight is on the shoulders. The result of raising the neck, so as to bring the * head near to the centre of gravity, is to more or less equalise the distribution of weight. As good horsemanship depends 4* 4* Digitized by Microsoft® ORDINARY RIDING. 52 on the distribution of weight, as we shall see further on; the first step in breaking ought to be the equal distribution of weight, so that good equilibrium maintained during pro- gression may later on give lightness to every movement. Raising the neck and putting equal weight on the fore- hand and hind quarters will allow them full freedom and energy, and will put the horse in such a position that he will only require to be stimulated. With the neck high, the hocks are easily brought under the centre of the body, and the action of the fore legs becomes lofty. Ina word, raising the neck gives good equilibrium and grace by lightness. In racing, one’s sole object is to gain in length without paying any attention to high action. Consequently, in training, one guards against raising the neck. Here we prove the principle that a horse extends himself as much as possible by “ daisy cutting.” But there are horses which are heavy behind, and one might think that if it is good to raise the neck of a horse which is heavy in front, it would be necessary to lower the neck of the animal which is heavy behind ; but this is not so. * This position is dangerous, because the horse is ready to rear. The danger of being behind the bit is restiveness, which causes rearing, with the probability of the animal falling backwards, Digitized by Microsoft® OBJECTS TO BE OBTAINED BY DIRECT FLEXION. As I have just explained, the conformation of the horse and the relations between his levers are such that the equilibrium of his mass can be obtained only by raising the neck. The horse which is heavy behind is inclined to get behind his bit, and has his hocks either too far removed or too near his centre. In the former case the horse is too much stretched out, and in the latter the croup is unduly lowered, and the points of the buttocks are much further back than the hocks.* Here, instead of a naturally bad distribution of the weight of the mass, as with a horse heavy in front, we have Digitized by Microsoft® OBJECTS OBTAINED BY DIRECT FLEXION. 53 a bad voluntary distribution of the forces in an animal which holds himself back and which does not wish to advance ; and his hocks do not do their work of pushing the mass forward. If we overload the forehand by lowering the neck, we add still more to the weight, and consequently we increase the difficulty of the work required from the hocks.** We must therefore raise the neck in order to lighten the forehand ; but we should raise it from below upwards, and not from front to rear, while taking care not to exaggerate the movement and to keep the hand very light. A high position of the neck is the first condition of good equilibrium, and having obtained it, we should seek to give freedom to the hind quarters, while bringing them into action, and making the horse go freely forward, which we do by the flexions of the well-placed head, by the loosening of the jaw, and especially by the legs. A horse which is heavy in his hind quarters is behind his bit, and if he does not wish to go forward, he is behind the Zegs. With such dh animal we must employ great lightness of hand and great energy of the legs, so as to change his distribution of weight by making him go up to his bit.t We cannot obtain this result by a low position of the neck, which is the chief obstacle to hghtness. 2. To fix the neck in the axis of the body by connecting to the shoulders the head rendered light by flexion. * Without taking into consideration that a depressed position of the neck will give only more spring to the act of rearing. + In such cases the driving whip, which obliges the horse to go forward, is a good preparation for the use of the legs. OBJECTS TO BE OBTAINED BY DIRECT FLEXION. The unbent head is heavy at the end of the neck, which has become too movable. Hence the proverb, “Heavy head, slack neck.” This is a case similar to that of holding a fishing-rod by its thin end. The flexed head, on the contrary, moves with lightness on the high neck, which is kept without stiffness on the axis of the body by the sole action of the good * Without taking into consideration that a depressed position of the neck will give only more spring to the act of rearing. + In such cases the driving whip, which obliges the horse to go forward, is a good preparation for the use of the legs. Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 54 equilibrium of the levers. Head, neck, and shoulders fixed in the axis of the well-balanced body, and making a supple and homogeneous whole. This is the action of flexion. equilibrium of the levers. Head, neck, and shoulders fixed in the axis of the well-balanced body, and making a supple and homogeneous whole. This is the action of flexion. 3. To obtain lightness by the relaxation or flexion of the jaw. Having the body balanced and connected in all its parts, the flexion of the jaw enables us to regulate collectively with extreme lightness all the movements from rear to front, and from front to rear, by receiving on the hand the impul- sion of the mass which the legs throw on the bit, and which the hand, in its turn, partly sent back to the rider’s legs. The hand only retains and sends back to the centre the amount of impulsion which is necessary to maintain equili- brium. The greater portion of the impulsion is naturally employed to propel the body forward. The flexibility from front to rear of the arm of the bent and jointed lever formed by the neck, head, and jaw, progressively increases from rear to front ; that is to say, from the shoulders to the neck, from the neck to the head, and from the head to the jaw. In other words, we hold the fishing-rod by its butt end. * The head ought to oscillate from a position a little beyond the per- pendicular to the perpendicular, but never in rear ofit. The position which I have indicated enables us to get by the shortest way to the end of the lever, and to obtain the greatest effect by the smallest effort. Digitized by Microsoft® OBJECTS TO BE OBTAINED BY DIRECT FLEXION. Thus, all the force developed by the horse is concentrated in the hand, the slightest action of which on the bar bends, first, the jaw, proportionately to the impulsion ; secondly, the head* by the jaw; and thirdly, by the head, the neck; the neck reacting with its greatest effect on the shoulders. The expression employed to define the reciprocal position of the horse and rider is most exact. We have really our horse iz hand. With respect to these remarks it is necessary to point out that the position of the head is singularly favourable to the action of the reins. In fact, the curb, which acts almost like Fig. 6.—Correct preparation for direct flexion. Fig. 6.—Correct preparation for direct flexion. Fig. 7.—Preparation for Baucher’s direct flexion. Digitized by Microsoft® Fig. 7.—Preparation for Baucher’s direct flexion. Digitized by Microsoft® ORDINARY RIDING. 56 a second snaffle, if the head 1s low, presses freely on the bars, and develops all its power when the head is raised, provided always that the head is kept a little beyond the perpen- dicular. The moment the axis of the head comes behind the perpendicular, the action of the curb is false, because it works from below upwards. Then the horse begins to draw his chin into his breast. Such, I consider, 1s direct flexion and its object. We can see that this flexion, as I practise it, is not done by chance or simple routine. On the contrary, I have care- fully given my reasons, and I have touched on all details to justify my practice. Unfortunately Baucher, who was the first to improve the art of flexions, by making it the base of his method, did not give a complete account of its mechanism. This did not matter much to him, because his marvellous equestrian tact remedied every deficiency. Where his theory was false, his hands and legs by themselves rectified, more or less conscientiously, the error of his doctrine. Baucher, however, could not put his tact into his books, in which he left his good and bad doctrines. I consider that by criticising him and by showing where he has failed, I shall render increased homage to the great horseman. Digitized by Microsoft® * Alas! the faults of masters are acquired more easily than their good qualities. OBJECTS TO BE OBTAINED BY DIRECT FLEXION. I maintain that the flexion which Baucher has described, and which is practised every day,* has done much to discredit in the minds of horsemen this most useful exercise, which I consider to be the first condition of good equitation. Baucher’s faulty flexion, which is in very common use to-day, is made at the withers instead of at the poll. It lowers the neck, and causes the horse to place the weight on his shoulders, that is to say, it aggravates the natural fault in equine conformation, and it makes him liable to fall by 57 57 57 Fig. 9.—Lowering the head—a fealty Pp ractice, Digitized by Microsoft® Fig. 9.—Lowering the head—a fealty Pp ractice, Digitized by Microsoft® ORDINARY RIDING. 58 carrying his head low, and to draw his chin into his breast by bringing his head behind the perpendicular. We must note that this fault was originated by Baucher, who, during the greater part of his career, made the flexions at the withers by lowering the neck. Compare Fig. 6, which shows the flexion I have described, with Fig. 7, which illustrates Baucher’s flexion, and which I have taken from his book. It is well to note that Baucher’s horse in Fig. 7 has his legs stuck out in front, and consequently it is im- possible for him to go forward. Nothing could be worse! Compare the correct flexion, shown in Fig. 5, with that in Fig. 8, which is the more frequently practised of the two. Towards the end of his life Baucher recognised this mistake (see his last edition, 1874); but he restricted himself to raising the head of the horse, without making the flexion when it was high. Whatever he did and whatever errors he made, he was an incomparable horseman. The people of to-day who make faulty flexions succeed only in ruining their horses. There is no reason for stopping when one has started on this road. Certain authors have thought it right to systematically lower the neck, than which there is no better means of ruining a horse! As a great curiosity, I have shown in Figs. g and 10 two illustrations which appeared in recently published books, and which show the lesson of lowering the neck. That could be called the art of teaching a horse how to break his knees! Digitized by Microsoft® OBJECTS TO BE OBTAINED BY DIRECT FLEXION. This explains how it is that many people say that they have made their horses perform flexions without any good result, a fact which should not surprise us. As we have just seen, the flexion is such a delicate thing that an incapable horseman who practises it, will often spoil a horse instead of improving him. If, on the contrary, the reader under- stands the principles and practice described in this chapter, Digitized by Microsoft® 59 uae St ES aE Fig. 10.—Lowering the head—a faulty practice. Fig. 11.—Direct flexion whengoing forward. Fig. 11.—Direct flexion whengoing forward. Digitized by Microsoft® ORDINARY RIDING. Go he will always be certain to obtain by flexion the benefits which he has a right to expect. To thoroughly explain the mechanism of flexion, I have been obliged to suppose that the horse was halted. But I must explain, contrary to what is everywhere practised, that I begin the direct flexion while going forward. With this object, I place myself at the shoulder of the horse, which I stimulate by a click of the tongue, while drawing him forward by a stronger feeling of the reins of the snaffle (Fig. 11). Apart from this, the flexion is done exactly as \| have indicated. ~When I have obtained a certain number of flexions of the jaw, I let the horse walk freely beside me for a few moments, and I carefully avoid prolonging the flexions, although I frequently begin them again. When the horse has got into the habit of readily doing the direct flexion at the first indication of the reins by relaxing his jaw, the breaker should modify his method in order to make the animal assume as nearly as possible the conditions he will be under when mounted. This will be a new form of work. The breaker, always in the same position, and being on the near side of the horse, holds the reins of the curb and snaffle in his right hand at about six inches from the jaw. The left hand holds the end of the snaffle reins and the cutting whip, the point of which he keeps on a level with the animal’s side. Under these conditions, at the moment when the right hand demands the direct flexion, the whip performs the office of making the horse go forward, as before described (Fig. 4). Digitized by Microsoft® * We will notice in Fig. 4 the manner in which the right hand of the breaker holds at the same time the reins of the snaffle and those of the curb. The former are held between the thumb and closed index finger, and keep the neck high by their upward pull. The latter are held more or less horizontally, and serve to loosen the jaw, the left curb rein being passed between the middle finger and the ring-finger ; and the right, underneath the little finger. OBJECTS TO BE OBTAINED BY DIRECT FLEXION. We are then placed under the same conditions as when We are then placed under the same conditions as when mounted. No longer, as was recently done, the forehand draws forward the hind quarters, which now get under the body, and propel the forehand on the head held by the Digitized by Microsoft® OBJECTS OBTAINED BY DIRECT FLEXION. 61 reins,* z.¢, by the hand of the rider. In this manner the horse learns to go forward without leaning on the bit, and is then perfectly light, in the same way as we ought to make him later on when he is mounted. The work which I have just described not only prepares the mouth of the horse in an admirable manner, but also makes the hand of the rider skilful. There is no doubt that it is easier to make the jaw supple when standing still than during movement ; but there is the risk of making him keep behind the bit, which inconvenience, or rather danger, is avoided when going forward (Fig. 4). I insist on this point ; because, if the work of loosening the jaw during movement is certainly longer and more difficult, it guards us against the great danger of putting the horse behind the bit, which is always the inevitable result of the first flexions. Therefore, take your time and do well. of the first flexions. Therefore, take your time and do well. Although, as a rule, I maintain that it is of the greatest importance for the whole of this work to be done when going forward, I am obliged to admit that it would be impossible or at least extremely fatiguing to do it with certain horses which throw themselves suddenly on the hand, or which have their heads too low. Personally, I have never found these faults carried to such an extent that I have been unable to obtain direct flexion and relaxation of the jaw while going forward. If the work is stationary, we should take the utmost care to If the work is stationary, we should take the utmost care to prevent, at all hazards, the horse from getting behind his bit. If he reins back ever so little, we should immediately send him forward by touching him on the side with the whip, while holding the end of the snaffle reins in the left hand (Fig. 4). Digitized by Microsoft® * Baucher made the mistake of practising the flexion when mounted at a halt. He naturally proceeded by light touches of the spur. Only his great tact saved him from making his horses restive. ; Digitized by Microsoft® OBJECTS TO BE OBTAINED BY DIRECT FLEXION. If the horse stretches out his hind legs behind, or his fore legs Digitized by Microsoft® ORDINARY RIDING. ORDINARY RIDING. 62 in front, he is also behind his bit, and should be immediately sent forward. A horse may be behind his bit without backing, and this may happen even without the animal moving his feet. He will have this tendency if a perpendicular dropped from the point of his buttocks comes behind his hocks, in which case the weight will be on his hind quarters. We should then act energetically on the snaffle, in order to bring the body for- ward and maintain the balance of the body during flexion, or to avoid a return of the tendency to get behind the bit. After this preparation, direct flexion becomes easy when mounted, especially as the action of the legs in propelling the horse on the hand is more energetic and effective than that of the whip. Here the chief point is to begin, not by the action of the hand, but by that of the legs, which ought to be used progressively. In mounted work, as in flexion on foot, it 1s, of course, necessary that the hand makes the concession at the moment when the horse yields, in order to retake its action immediately afterwards. The entire practice of flexions is comprised in timely faking and giving. It is, of course, understood that the legs should always remain close to the animal's sides, as much for obtaining a definite concession of the jaw as for avoiding the tendency to get behind the bit. It is also taken for granted that the flexion should never be made at a halt, when the horse is mounted, which is a most objectionable practice. As we can obtain relaxation of the jaw only by light touches of the spur, the horse contracts the habit of resting on the spur when standing still. As we have then no means of making him go forward, or of preventing him from reining back, he becomes restive.* This result is all * Baucher made the mistake of practising the flexion when mounted at a halt. He naturally proceeded by light touches of the spur. Only his great tact saved him from making his horses restive. ; Digitized by Microsoft® OBJECTS OBTAINED BY DIRECT FLEXION. Digitized by Microsoft® ORDINARY RIDING. 63 the more certain when the horse no longer dares to go up to his bridle when moving forward, on account of the pre- ponderance which the hand has on the legs, whenever the animal wants to advance. Fig. 12.—Baucher’s flexion when mounted. Fig. 12.—Baucher’s flexion when mounted. Finally, the work of making the horse go forward consti- tutes the great difference between my system of equitation and that of Baucher. My first lesson has been to make the animal go forward. In his Dectionnaire raisonnée @équitation (1833), page 112, Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 64. Baucher writes : “ During the first lessons the entire half-hour should be occupied in stationary work, except the last five minutes, during which the rein-back will be practised.” Twenty-five minutes of stationary work and five minutes of reining back is a deplorable waste of time. For a lesson of Fig. 13.—Correct flexion when mounted. Fig. 13.—Correct flexion when mounted. half an hour's duration I would devote thirty minutes to forward work, without any stationary work or reining back. We shall subsequently see that this difference in method is found in all the work. Naturally, Baucher’s flexion is as incorrect in mounted work as in work on foot. Fig. 12, which is borrowed without any change from his book, enables us to judge his work by comparing it with Fig. 13, which represents correct flexion. Digitized by Microsoft® ORDINARY RIDING. ORDINARY RIDING. 66 OBJECTS OBTAINED BY DIRECT FLEXION. OBJECTS OBTAINED BY DIRECT FLEXION. 65 Fig. 14, which is taken from a recent work, shows in an exaggerated form all the faults of Baucher’s bad flexion. In it the head is low, far from the centre of gravity and behind the perpendicular; the horse has all his weight on his Fig. 14.—Exaggerated example of Baucher’s incorrect flexion. Fig. 14.—Exaggerated example of Baucher’s incorrect flexion. shoulders, and is ready to bring his chin into his breast ; the muscles of the jaw are contracted and the action of the curb is in a downward direction, and is consequently false. This caricature of breaking is simply perfect! The exact opposite of all these conditions is what we should seek to obtain from direct flexion. Digitized by Microsoft® Digitized by Microsoft® MAKING A HORSE QUIET TO MOUNT. To make a horse quiet to mount, we must adopt a method by which we can easily stop him from annoying the rider, or preventing him from getting into the saddle. Let us examine the means we should employ, and let us place ourselves in such a position that we will be able to circumvent any defence which his instinct or bad habits may prompt him to make. The vicious or restive horse rears, lashes out, strikes with his near forefoot, or cow-kicks with his near hind. With such an animal we should make use of the lunging rein and driving whip, and make a feint of putting a foot in the stirrup while standing close to the near shoulder. If he rears, we can hit him thoroughly with the lash of the whip across his buttocks. As he is held by the lunging rein, we can remain sufficiently far away from him to avoid being struck. Every time he rears we should begin again until he yields. If he reins back, we should employ similar methods. If he kicks, raise his head and loudly scold him. If he strikes out in front, cut him with the driving whip on the offending leg. Besides horses which resist in this manner, there are others which are simply timid, nervous, restless, ticklish, or irritable, and which do not remain as quiet as they ought to dao, although they do not really “show fight.” What will they do? They will perform only four movements—namely, go forward, run back, shy off to the right or to the left, against which we should act as follows: I take the near rein of the snaffle in my left hand, and with the same hand I catch hold of the mane at about the middle of the neck in such a manner that there is a slight tension on the rein which I hold. I pass my right hand, in which I have my cutting whip, over the neck, seize with it the off snaffle rein, which I draw up only slightly, and finally take hold of the pommel of the saddle with the right hand (Fig. 15). Digitized by Microsoft® MAKING A HORSE QUIET TO MOUNT. MAKING A HORSE QUIET TO MOUNT. 67 I then face the left shoulder of the horse. If he runs back, a cut on the croup with the whip will bring him forward. This can be repeated twenty or thirty times till he yields. If he goes forward, I bring him back by feeling the reins. If -he shies to the left, I draw his head to that side, and conse- quently bring his hind quarters to the right. Similarly, if he Fig. 15.—Proper way to mount. Fig. 15.—Proper way to mount. shies to the right, I draw his head to the right, so as to make him carry his hind quarters to the left. He makes his defences, when the rider touches the stirrup with his foot, when he puts his foot into the stirrup, or when he raises himself by his straightened left knee, and before he has passed his right leg over the saddle. We should not go to the second or third of these movements until we have made the horse perfectly steady in the preceding movement. We should not place ourselves in the saddle until the animal remains steady during all the time we are standing on the 5 * 5 * Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 68 near stirrup iron, at which morrent it often happens that the horse attempts one of the defences already described. If the movement is only slight, we can correct it by the action of the reins without changing our position. If it is violent, we can place our foot on the ground and correct him with the whip. Finally, we should bear in mind that as soon as we are in the saddle, we should have the snaffle reins in their respective hands, and we should hold them only just short enough to enable us, by a light feeling on them, to steady the horse, in the event of his “ playing up.” I ought to admit that my method of getting into the saddle is contrary to the principles which are generally taught. Digitized by Microsoft® MAKING A HORSE QUIET TO MOUNT. In* fact, it is always laid down that to mount, the rider ought to take both reins of the snaffle and a lock of the mane near the withers in his left hand; place the right hand on the centre of the saddle, raise himself on the stirrap, and, when his left knee is straight, carry his right hand quickly from the cantle to the pommel, while passing his right leg over the horse. He then sits down. By this system the rider is unable to counteract any movement made by the horse. Further, at the moment when the right hand passes from the cantle to the pommel, the equilibrium of the rider is as unstable as possible, and can be upset by the slightest cause. Precisely on account of this want of equilibrium, the rider falls, instead of sits, down on the saddle, and by falling in this manner he can unfor- tunately hurt himself, especially on the pommel, if the horse makes a single step to the rear. With the method which I advocate, the rider is able to avoid any accident, to stop any “playing up,” o1 even any movement on the part of the horse; because he holds a snaffle rein in each hand. to girth him with a When I lunge a horse, I take care to girth him with a moderate degree of tightness before putting him into a trot. Digitized by Microsoft® MAKING A HORSE QUIET TO MOUNT. 69 And as he gets a bit slack from trotting, the girths do not inconvenience him when I mount. I always carry out this procedure when mounting a difficult horse for the first time. And as he gets a bit slack from trotting, the girths do not inconvenience him when I mount. I always carry out this procedure when mounting a difficult horse for the first time. Grooms are in the habit of girthing up their horses very tightly, and it would be contrary to a knowledge of the weak- nesses of human nature to think that they would give up this practice on the very day when I try to succeed at what they have failed to do. On that day they girth up more tightly than ever. MAKING A HORSE QUIET TO MOUNT. They know that the tighter a horse is girthed up the more he will plunge, and therefore they feel certain that they will presently have the innocent pleasure of seeing me chucked over the ears of the horse. Nevertheless I encourage them to girth up more tightly, and after they have squeezed him as tightly as they can I take him by the bridle, walk him about for a few minutes, and the moment before putting my foot in the stirrup I let out the girths one or two holes. J am then in the saddle, and the animal gives a great sigh of relief, which for the moment prevents him from thinking of playing up. I never allow my horses to be held when mounting them. All horses become quiet if not held, and they will gain conf- dence if we mount and dismount several times consecutively, while patting them on the neck. We ought to mount as quietly and lightly as possible. Above all things, we ought to avoid bustling the horse when starting ; because, if he expects we are going to do this, he will never stand quietly to be mounted. I never require anything from the horse which I mount for the first time. I am content if he walks straight on. I keep the reins separated, I feel only those of the snaffle, and I never touch the horse with the spurs for the first few lessons. I ride him a few times round the school to the right and to the left, while leaving him as free as possible, supposing, of Digitized by Microsoft® ORDINARY RIDING. 70 course, that he does not play up, which he will very rarely do if I require almost nothing from him. If he carries his head too low, I try to raise it by almost imperceptible touches on the snaffle reins, which I draw upwards, and not from front to rear. If he holds his head too high, I feel the curb reins very lightly, and in such a way as not to stop his forward movement. If he stops on account of the action of the curb, I let the reins loose and apply my legs behind the girths. In this case the legs have an effect similar to that of the cutting whip during work on foot. Digitized by Microsoft® MAKING A HORSE QUIET TO MOUNT. I keep on at the horse until I have obtained forward movement which is the great end that has to be obtained at any price. Having obtained this forward movement, I prolong it, as I have said, for a few turns round the school, while trying to get the head into a good position; but I work very lightly, and always in such a manner as not to stop the horse. If I obtain only a little play in the mouth, I get off, and make a few flexions on foot until he obeys. I then give him some carrots and send him to the stable. Carrots ought always to be cut lengthwise and never across, in which case they might stick in the animal's throat. I have seen a horse almost choked by swallowing carrots cut across. I do not give sugar to a horse, because if he is bridled and plays with his bit, it will produce a foam that will soil one’s clothes, which is the smallest objection. A more serious one is that sugar given in the stable predisposes a horse to crib- bite. He begins by licking his manger, and, finding the taste agreeable, he finishes by persistently biting it, which is a habit that will often make him crib-bite or wind-suck. In this mounted lesson, I have asked the horse only to go forward, while making him lightly work his jaw. Further, asa general rule,we should not demand from him several things at a time,because he will be apt to confuse them,and we may mistake for disobedience a simple want of comprehension on his part. Digitized by Microsoft® Near curb rein * fnd of curb reins fnd of Of curb rein NéarCorp ein ae AS —_ 7 ] H BB Z Z "ei OO \ je OS" 0) snaft en i: off aff! i } Snaffle Nea Curb” } reins reins Fig. 17 —Action of the curb ; little finger brought towards the body. Curb reins Off snaffle rein pete SETS Curb reins i Near curb rein * fnd of curb reins fnd of Of curb rein NéarCorp ein ae AS —_ 7 ] H BB Z Z "ei OO \ je OS" 0) snaft en i: off aff! i } Snaffle Nea Curb” } reins reins Fig. 17 —Action of the curb ; little finger brought towards the body. Fig. ORDINARY RIDING. é: Digitized by Microsoft® * Is it not strange that in the French army the German method of holding the reins is adopted, namely, the snaffle rein under the curb rein ? MAKING A HORSE QUIET TO MOUNT. 17 —Action of the curb ; little finger brought towards the body. Curb reins Curb reins Off snaffle rein pete SETS Curb reins i ~ Snaffle reins 4 th Fig. 18.—Action of the snaffle ; thumb brought towards the body. Digitized by Microsoft® Off snaffle rein T ORDINARY RIDING. HOW TO HOLD THE REINS. There are three orthodox ways for holding the reins, namely, the Exglish, German, and French. I do not hesitate to say that the French way is the best. As the snaffle is higher in the mouth than the curb, its principal action is to raise the head of the horse, and that of the curb to lower it. In other words, the snaffle is an elevator ; the curb, a depressor. Therefore, the reins should occupy the same respective positions in the hand, as the snaffle and curb do in the mouth, namely, the snaffle reins should be above the curb reins. Contrary to this very simple principle, the English place both reins at the same height in the hand and hold one rein between each finger. The Germans act in a still more con- trary way to the principle in question, by holding the snaffle reins below the curb reins, supposing that the hand is in a vertical position. It appears that the Germans have even less common sense than the English. Reason tells us that the reins should be held in the French manner ;* the hand vertical, the left curb rein under the little finger of the left hand; the right rein between the ring finger and the middle finger, while their ends pass between the thumb and index finger. The two reins of the snaffle are joined in the same hand, and taken between the thumb and index finger (Fig. 16). With the reins held in this manner and without displacing the hand, we are able, by the mere play of the wrist, to obtain the movements which are indis- pensable for acting on the mouth; supposing, of course, that the animal is broken. ist. The hand placed in position exerts an equal tension on all four reins (Fig. 16). * Is it not strange that in the French army the German method of holding the reins is adopted, namely, the snaffle rein under the curb rein ? Digitized by Microsoft® 73 Of snaffle rein SS J a a Wii Tif : inaffle rein HE Y Near y// a z a HY HEH r ; HH Off curb rein sill Fig. 19.--Action of off curb rein ; knuckles lowere1 Curb reins “OF Shaftle rein Near curb rein = es OF curb rein Curb reins Snaffle reins Fig. 20.—Action of near curb rein ; knuckles raised. * Fig. 22 shows the right hand being passed between the off rein of the curb and the off rein of the snaffle, and Fig. 23 shows the right hand being closed on these two reins. Fig. 24 shows the reins separated. HOW TO HOLD THE REINS. ee) Off snaffle re! ——— ae Near snaffle re!? Off curb rein \ Near curb rei: 73 Of snaffle rein SS J a a Wii Tif : inaffle rein HE Y Near y// a z a HY HEH r ; HH Off curb rein sill Fig. 19.--Action of off curb rein ; knuckles lowere1 73 Of snaffle rein SS J a a Wii Tif : inaffle rein HE Y Near y// a z a HY HEH r ; HH Off curb rein sill Fig. 19.--Action of off curb rein ; knuckles lowere1 73 Fig. 19.--Action of off curb rein ; knuckles lowere1 Curb reins “OF Shaftle rein Near curb rein = es OF curb rein Curb reins Snaffle reins Fig. 20.—Action of near curb rein ; knuckles raised. ee) Off snaffle re! ——— ae Near snaffle re!? Off curb rein \ Near curb rei: End of ‘curb reins Fig. 21.--Separating the curb and snaffle reins. Digitized by Microsoft® Fig. 19.--Action of off curb rein ; knuckles lowere1 Fig. 19.--Action of off curb rein ; knuckles lowere1 Curb reins “OF Shaftle rein Near curb rein = es OF curb rein Curb reins Snaffle reins Fig. 20.—Action of near curb rein ; knuckles raised. Fig. 19.--Action of off curb rein ; knuckles lowere1 Curb reins “OF Shaftle rein Near curb rein = es OF curb rein Curb reins Snaffle reins Fig. 20.—Action of near curb rein ; knuckles raised. Curb reins Fig. 20.—Action of near curb rein ; knuckles raised. ee) Off snaffle re! ——— ae Near snaffle re!? Off curb rein \ Near curb rei: End of ‘curb reins Fig. 21.--Separating the curb and snaffle reins. Digitized by Microsoft® ee) Off snaffle re! ——— ae Near snaffle re!? Off curb rein \ Near curb rei: End of ‘curb reins Fig. 21.--Separating the curb and snaffle reins. Digitized by Microsoft® Digitized by Microsoft® 74 ORDINARY ORDINARY RIDING. 74 2nd. Action of the curb; the little finger brought towards the body (Fig. 17). 2nd. Action of the curb; the little finger brought towards the body (Fig. 17). 3rd. Action of the snaffle; the thumb brought towards the body (Fig. 18). 4th. Action of the off curb rein; the knuckles lowered (Fig. 19). 4th. Action of the off curb rein; the knuckles lowered (Fig. 19). sth. Action of the near curb rein; the knuckles raised (Fig. 20). sth. Digitized by Microsoft® HOW TO HOLD THE REINS. Action of the near curb rein; the knuckles raised (Fig. 20). We can obtain all the necessary effects on the mouth of the horse by the rotation of the wrist from rear to front (Fig. 17); from front to rear (Fig. 18); from left to right, z.., pronation (Fig. 19); and from right to left, z.2., supination (Fig. 20). When the reins are held in this manner, they are kept apart as far as possible, supposing that they are in one hand. The effect obtained is almost similar to that which would be produced if the reins of the curb were held in the left hand and the snaffle reins in the right hand, a little above them. We can easily, if required, use both hands; because the nght hand can take up the snaffle reins or put them back into the left hand without disarranging or even touching the curb reins (Fig. 21). Finally, if we wish to have all four reins separated, namely, the near ones of the curb and snaffle in the left hand, and the off ones in the right hand (which is often necessary), we have only to take the off reins in the position in which they are, by placing the right hand between the reins of the curb and those of the snaffle in such a manner that the off rein of the curb will come under the little finger of the right hand, and the off rein of the snaffle between the thumb and index finger of the same hand, in exactly the same position as the reins are in the left hand.* We thus keep in the two hands the Digitized by Microsoft® 75 Snaffle reins Ps 2 “Curb reins — Near snattle POND D) arcee Tue O, 5 by?" snalfle rein WF curb rein WY = ae "ar curs rein “Curb reins Hy J Fig. 22.—Passing the right hand between the off curb rein and off snaffle rei Snaffle reins “Curb reins "a “Curb reins Fig. 22.—Passing the right hand between the off curb rein and off snaffle rein. Near snaffle rein remaining in the left and ; Off snaffle rein held by the “<< right hand a ning in ey” the left nan? Of curb rein taken by the right hand Curb reins Fig 23.—Closing the right hand on the off curb rein and on the off snaffle rein. HOW TO HOLD THE REINS. Curb reins Fig 23.—Closing the right hand on the off curb rein and on the off snaffle rein. Fig 23.—Closing the right hand on the off curb rein and on the off snaffle rein. Near snaffle rein Near curb rein i OFF snaffle rein } OF curb rein l Curb reins Snaffle ee Fig. 24.—Separating the reins. Ry Fig. 24.—Separating the reins. Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 70 same distance between the reins of the curb and those of the snaffle as determined by the little finger and thumb. same distance between the reins of the curb and those of the snaffle as determined by the little finger and thumb. By an inverse movement, we can replace the four reins in the left hand, in the position which they previously occupied. I need hardly add that, until the breaking of the horse is fairly well advanced, we do not draw up all four reins, which would consequently tend to combine and sometimes even to confuse their effects. When we have to deal with a green or insufficiently broken horse, in which case we may require to produce very quickly precise and particularly decisive effects, it is well to separate the reins. t This is what is termed Jateral effects, because the effect on the forehand and that on the hind quarters are produced on the same side. In déagonal eguitation, which is rational equitation and the consequence of good breaking, the effect on the forehand is always on the side opposite to that on the hind quarters. It is the only way to secure the movements in their entirety. Digitized by Microsoft® * The buckle of the bridoon rein ought to remain in the left hand during all the work on foot. TEACHING A HORSE BY THE WHIP TO OBEY LEGS AND SPURS. I invariably begin my lesson by repeating the former exercises, but every day I require something new from the horse. As soon as the horse goes freely in every direction with a slight play of the jaw, I teach him to obey the leg and then the spur. This work should be done on foot and by means of the cutting whip. While facing the horse, I take the snaffle in the left hand* close to the mouth so as to hold the head high. With the whip, which I hold in my right hand, I touch the horse very lightly just behind the girths, at the place where the spur will act, and at the same time I carry his head to the left.t The horse should thereupon carry his haunches to the right. He will show that he obeys by taking one or two steps in that * The buckle of the bridoon rein ought to remain in the left hand during all the work on foot. t This is what is termed Jateral effects, because the effect on the forehand and TEACHING A HORSE BY THE WHIP. 77 direction. I then stop him and pat him on the neck. If, on the contrary, he strikes out in front or kicks, on account of being touched by the whip, I rate him loudly and hold his head very high, which will oblige him to lower his croup. It is of the greatest possible consequence that the patting on the neck should immediately follow the act of obedience on the part of the horse, in the same way as punishment should follow disobedience. This is the fundamental principle of breaking. If the horse throws himself on the whip, that is to say, to the left, which ticklish horses are inclined to do, we must catry his head forcibly to the left, so as to bring his hind quarters to the right; but should not punish the animal, because, as I have already pointed out, his action is instinc- tive. When we have put him several times through this work, which does not fatigue or trouble him, he readily yields, and does it on both sides. I recommend the breaker to be content with two or three steps. He should stop and pat the horse on the neck each time the animal obeys. I recommence this work very often. TEACHING A HORSE BY THE WHIP TO OBEY LEGS AND SPURS. When the horse readily yields to the whip on both sides, we should not turn his head to the side opposite to that towards which he turns his hind quarters. He should be kept straight, so that he may obey only the indication of the whip. When the horse readily yields to the whip on both sides, we should not turn his head to the side opposite to that towards which he turns his hind quarters. He should be kept straight, so that he may obey only the indication of the whip. This work with the whip prepares the horse to bear and understand the action of the leg and spur. At the same time, it places the head in a good position, and it is an excellent and indispensable suppling exercise before coming to the lateral flexions which we will presently consider. Baucher practised the lateral flexions before teaching the This work with the whip prepares the horse to bear and understand the action of the leg and spur. At the same time, it places the head in a good position, and it is an excellent and indispensable suppling exercise before coming to the lateral flexions which we will presently consider. Baucher practised the lateral flexions before teaching the horse to yield his hind quarters to the whip. This method is wrong, because, when we begin the lateral flexions, the instinct of the horse makes him carry his hind quarters to the side opposite to that towards which we bend the neck. If we Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 78 have not taught obedience to the whip, how can we straighten the hind quarters? have not taught obedience to the whip, how can we straighten the hind quarters? Digitized by Microsoft® * Bucking without going forward is the most difficult of all movements to sit. If the horse cannot be made to go on and if he continues to buck without advancing, it will be impossible to sit him. HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. In the preceding chapter I have taken for granted that when I am mounted, the horse will go forward, if I want him to do so. As the animal will not always be obedient, it is well to be forearmed against any probable or even possible resistance which he may make. It is therefore necessary to keep the legs close to his sides, in order to avoid being taken off our guard by an abrupt movement, and, above all, to lean the body well back, so as to put the weight on our seat. We are then ready for anything that may happen. If the horse tries to buck, we should press him forward with If the horse tries to buck, we should press him forward with the legs and hold his head high. It is not hard to sit a for- ward plunge when the head is kept high.* But the rider is easily displaced if the horse bucks without going forward, places his head between his fore legs, and arches his back. In this case, as the rider is not able to make the horse go for- ward, he should turn him to the right or left by means of the snaffle. ach time he tries to stop by lowering his head, he should be turned afresh: but it is not necessary to insist on turning him more to one side than to the other side. All horses have a soft and hard side to their mouths. At first, if the horse resists when we try to turn him to the right, we should content ourselves by trying to turn him to the left. The great thing is to prevent him from bucking without going forward, his bucks being made in order to get rid of his rider. Later on, when the horse has made some progress in his * Bucking without going forward is the most difficult of all movements to sit. If the horse cannot be made to go on and if he continues to buck without advancing, it will be impossible to sit him. HORSE MOUNTED—FIRST DEFENCES. 79 breaking, it would be evidently wrong to let him turn to the right, if we wanted him to go to the left; but at present we should consider only his acts of resistance. HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. It is certainly a great point gained to be able to prove to him that he cannot get us out of the saddle. If he succeeds in doing this, we may be certain he will continue to try on the same form of defence. The danger of at first requiring the horse to turn to which- ever side we wish, consists in the fact that by doing this we run the risk of prompting the animal to add a second act of resistance to the first one. In fact, when we wish to turn the horse to the left, for instance, we are obliged to take a strong pull on the left rein, and may thus make him foznde (half rear) or rear* by throwing too much weight on his hind quarters. The pointe or lancade is a forward spring in which the forehand is kept higher than the croup. Like all other forward movements, it is not dangerous, because its displacing effect is not great.t The movements of the loins and croup are, however, very disconcerting to the rider. When the horse rears, he stands up on his hind legs, which he keeps fixed on the ground. This is a very dangerous form of resistance and may cause the animal to fall backwards. In my youth, when my horse reared, I used to put both arms * We may succeed in preventing the rear or the “‘ pointe’’ by the vigorous use of the spurs. But to do so, we should seize with precision the fleeting moment when the horse holds himself back and is about to throw his weight on his hind quarters. If the spurs are driven in at this moment, they will send him forward in a disordered manner, no doubt ; but that does not matter, because they will have prevented him from fixing his hind legs on the ground. If the rider allows this critical moment to pass, the use of the spurs will be dangerous, because it will still further prompt the animal to rear. + In the half-rear the rider ought to bring his body forward, keep his legs close to the animal’s sides, hold his hands low, and leave the reins slack (Fig. 65). In this illustration, the off reins are lightly felt because I was trying to make the horse canter to the right on three legs. HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. We can see that the near snaffle rein is quite loose. Digitized by Microsoft® ORDINARY RIDING. 80 round his neck and bring my head to the right ; consequently, the horse’s head was against my left shoulder. I subsequently saw the inconvenience of this position, in which one is too far forward on his neck when he brings his fore legs down on the ground. And as we are then obliged to give him his head, he is at liberty to send us over his ears by a strong kick with both hind legs, or to give us a blow in the face or chest with his head, in the event of his throwing it up. From a very long experience I have found the following to be the best means of avoiding accidents which may happen from rearing. Separate the reins—as one should always do, when a horse rears—and take hold of the mane in the left hand at about the middle of the neck. When the horse stands up on end, bring the body well forward by the left arm; and when he comes down again, push the body back into its place by quickly straightening that arm. If the animal makes another attempt at rearing, again bring the body forward, again push it back; and so on, until he ceases to rear. From the moment we stop trying to make the horse go forward, we are able by this means to put up with the horse’s rearing for any length of time without difficulty. If he kicks, our straightened-out arm will act as a prop, and if he springs to the right about or left about, it will furnish a support which will keep us in the saddle, and will enable us to avoid hanging on to the reins, which is the usual cause of accidents. I feel certain that the foregoing position is the best for keeping the rider in the saddle, when a horse rears, or, rather, it is the least bad, because the position of the rider is never good in such a case. I have had good luck with regard to rearing; for although I have ridden a great number of rearers, none of them has upset me. I do not believe that horses voluntarily throw themselves backwards. HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. What is the reason of this silence on their part? Are they afraid to frighten their readers, or do they wish to make them believe that there are no struggles? I cannot say; but in any case it seems to me preferable to speak the whole truth, and to forewarn my readers of the accidents that may happen during breaking, so that they may not be surprised if the animal does not immediately submit to every demand. one always has with horses to a lesser or greater extent. To believe many of them, the breaker is on a bed of roses, and if he will use only their methods, he will be certain to make the horse obey. What is the reason of this silence on their part? Are they afraid to frighten their readers, or do they wish to make them believe that there are no struggles? I cannot say; but in any case it seems to me preferable to speak the whole truth, and to forewarn my readers of the accidents that may happen during breaking, so that they may not be surprised if the animal does not immediately submit to every demand. I have seen a great number of good breakers at work, and they all had battles to fight; Baucher more than any of the others, because he required more. The true talent of the breaker consists in making himself master of the horse, especially in the event of a fight, by suppling him, and by the employment of a rational method of breaking ; because we must not forget that the horse always struggles against his rider, more or less openly, until his education is complete. But we do not arrive at this result without more or less violent struggles. Those who have not had experience of these battles, upon the result of which depends the submission of the horse, begin by provoking the animal to resistance, but they do not dare to continue the attack. In this case the horse quickly understands that he is master. He will renew, as often as he likes, the form of defence which has served his purpose, and thanks to which he is certain to have the last word. This is the way to spoil the breaking. My practice is altogether different. Digitized by Microsoft® HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. As far as I can see, they simply lose their Digitized by Microsoft® HORSE MOUNTED—FIRST DEFENCES. 381 balance. Usually the following happens: The horse by suddenly getting up, causes the body of the rider to go backwards, which is sufficient to upset the animal. When a horse is standing up on his hind legs, he may be compared to the centre rod of a pair of scales which is in equilibrium ; and then the slightest movement of the body of the rider, whether forward or backward, forcibly draws the horse in the former direction or in the latter. I have said that I do not believe that horses voluntarily throw themselves backwards. I mean that a horse will not deliberately throw himself backwards as a means of resist- ance. His instinct of self-preservation will be sufficient to prevent him doing this. But I know that horses which are suffernig from disease of certain nervous centres fall back- wards, and sometimes dash their head against a_ wall. Here we have no concern with animals which are affected by a disease similar to madness in man, and which are unsuitable for any kind of work. I have, however, broken, and seen broken by other breakers, horses which had zwmobilité or megrims. But they were only violent and irritable, and their eyes become injected with blood if upset in the slightest. In fact, they had only the appearance of megrims. To succeed with such animals, we require to have all the good qualities of a breaker and rider, especially pluck. Some horses which are affected with only a certain degree of zmmobilité can be broken. Gaulois, which was a superb Hanoverian horse, was supposed to suffer from this disease. I made him into an excellent school horse, and rode him for several years. It is true that Gaulois, like many other horses which are reputed to have zmzmobilité, showed symptoms of it only when he “ played up.” I am greatly surprised that very few of all the authors who have written on equitation say nothing of the struggles which 6 6 Digitized by Microsoft® ORDINARY RIDING. 82 one always has with horses to a lesser or greater extent. To believe many of them, the breaker is on a bed of roses, and if he will use only their methods, he will be certain to make the horse obey. * Tostop a horse “ playing up,” lean back and lower the hands. Raising the hands, which will also raise the centre of gravity, will be certain to cause a fall. HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. The moment the horse shows fight I shake him up vigorously, but rationally.* We should not tickle the horse with the spur, which would * Tostop a horse “ playing up,” lean back and lower the hands. Raising the hands, which will also raise the centre of gravity, will be certain to cause a fall. HORSE MOUNTED—FIRST DEFENCES. 33 only aggravate the animal’s resistance. On the contrary, our attack on him should be somewhat brutal, so that he may immediately feel that his strength must yield to superior force. Here the real difficulty is to have the pluck to attack vigorously. Very few people make up their minds to do this, in which is the only means of security; because the horse, astonished at the brutality of the attack, will submit, although, if lightly tickled with the spur, he will increase his resistance, and will soon get the best of his rider. It is evident that whereas the attack should be vigorous, it should be made in as rational a way as practicable, that is to say, in a manner which will overcome the horse’s defence by “oppositions.”* But, I repeat, the chief thing above all others is the vigour, the energy of the rider. As regards myself, once in the struggle, I pay little heed to lateral or diagonal equitation. I don’t care if I increase the commo- tion; my great point is to be master, and to make the horse understand that his defence is in vain. To achieve this grand result, when a horse contents himself by merely indicating a resistance, I do not hesitate to provoke him to carry it out fully, so as to bring him to reason. Here is the true secret of breaking, namely, to obtain the absolute submission of the horse. A horse’s breaking is not definitely completed if he has made no show of resistance, because there may be struggles for supremacy in the future. The object of break- ing is the destruction of the free will of the horse. As long as a breaker hesitates to provoke struggles which he feels are close at hand, and which he knows are inevitable, his breaking is not complete. We must also say, in a general way, that the /ac¢ of the * To make an “ opposition ”’ is to do an action contrary to that which the horse wishes to do. Digitized by Microsoft® HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. We succeed in this simply by combining the ““aids,’”’ so as to oppose the forehand to the hind quarters, or the hind quarters to the forehand, namely, to carry one of them to one side, in order to throw the other on the opposite side, 6* Digitized by Microsoft® ORDINARY RIDING. 84 breaker ought to consist, throughout the whole course of breaking, in discovering the defences which the horse is preparing, and in anticipating and counteracting them before they are made. This is more especially necessary in the properly-called defences of the horse, which are not always produced in the form of violent or unruly movements. It constantly happens during breaking, that the horse, in order to refuse to do what is asked of him, obstinately does what he was previously taught. This is the history of every lesson. During all the breaking, the defences of the horse are pro- duced alternately to the right and left. When, by the force of insistence, we have rendered one side supple, we may he greatly astonished to see the horse use, as a defence, what we have just taught him with much trouble, and to refuse to do that which he did without any hesitation the day before. In this case we must recommence the work and carry it on until obedience is equally obtained on both sides. If there is a difficulty in making such a horse canter with, for example, the near fore leading, as soon as we make him do so, we will tind that in every case he canters with that fore-leg leading, and refuses to lead with the off fore. In order to make him canter with the off fore leading, we must begin over again all the work we did in teaching him to canter with the near fore leading, and alternate these two forms of canter, while making’ him do the canter in which there is greater difficulty, more frequently than the other kind of canter. We should con- tinue in this way until obedience is perfect on both sides. When a horse makes a defence always on the same side, we may be sure that he does so on account of pain or of faulty conformation. Digitized by Microsoft® THE WALK, THE WALK, It is most important that a horse should walk with long, regular and free steps, which can be obtained only by allow- ing the animal great liberty of head and neck. If the horse is lazy or dull, he should be made to go on, by closing the legs. If he is impetuous, impatient, or fidgety, he should be patted on the neck and encouraged by the voice. He should be at once stopped if he begins to trot. No mistake should be made between the trot and jog, which are entirely different paces. Jogging, which is often adopted by the horse, is very difficult to correct when it has become a habit. It spoils the long striding walk, and is very fatiguing to the rider. Keeping the horse in hand shortens the steps of the walk, which become higher and shorter, according as the neck is raised and the head is brought into a vertical position. The same thing occurs in the trot and canter. The well-collected liorse is handsome, but he does not cover much ground. HORSE MOUNTED—FIRST DEFENCES—MEANS FOR OVERCOMING THEM. Later on, when the breaking is more advanced, we shall see the horse use, for example, the Spanish trot as a defence against doing the passage or the piaffer. In every case, he will try a less energetic work, in order to escape from a more Digitized by Microsoft® TO HALT AND STAND STILL. 85 fatiguing one. The ruses of a horse are infinite. The breaker has to checkmate them by tact, art and energy. TO HALT AND STAND STILL. It is absolutely necessary to be able to stop the horse when one wishes. As a rule, the halt ought to be made pro- gressively and not abruptly. It can be made at any pace, and often even in cases when a sudden halt is indispensable. The means for stopping the horse is always the same— The means for stopping the horse is always the same— namely, raise the snaffle reins while drawing them back with an equal feeling on both reins, so as to bring the weight on the hind quarters; at the same time, close both legs strongly to bring the hocks under the animal’s body, and feel the curb reins. The horse is then detween the hands and legs. hould be abso- I cannot say that these three movements should be abso- lutely simultaneous. They certainly come one after another, Digitized by Microsoft® ORDINARY RIDING. 80 but so closely together that the intervals are imperceptible. Anyhow, they ought to be executed in the order I have indi- cated. If, for instance, the movement of the legs precedes ever so little that of the snaffle, the effect will be to make the horse go forward, which will be the opposite to what is wanted. This method of stopping the horse is the best and should be the only one used. The halt should be made without con- cussion. It is then painless for the rider, saves the horse’s loins and hocks, and is easy, because the hocks and pasterns bend. To stop himself, the horse acts simultaneously with all the parts of his body, makes no local effort, and preserves all the elasticity of his loins, which bend and become slightly concave. If we stop the horse only by the hand, and without the help of the legs, the forehand will become arched in order to resist the impulsion received, and will push back the hind quarters by a counter stroke in removing it from the centre. In this case the loins become stiff and convex. The shock which results is painful for the rider, and often dangerous on account of its violence, and is very bad for the horse, on whom it inflicts pain in his mouth, shoulders, loins, and fetlocks. A halt, no matter how sudden it may be, should be smooth ; if it is not so, it is badly executed. Digitized by Microsoft® TO HALT AND STAND STILL. The halt ought to be done by the same means at every pace. It is, of course, understood that the faster the speed, the more difficult is the halt, and the more should the rider lean back. The horse ought not only stop himself as quickly as his rider wishes, but should also remain standing still as long as he is required, wherever he may be. It is somewhat difficult to make an impatient, nervous, or excitable horse stand still at this time. We should calm him down, so as to gradually accustom him to his surroundings. We should begin in the school when we are alone, and should pat him on the neck and Digitized by Microsoft® CHANGES OF DIRECTION. 87 speak to him. Each time he wishes to advance or go side- ways, we should put him back in his place, and should not allow him to take a step in any direction. If we allow him to go to one side or the other, no matter how little, the first movement will probably be followed by a second one; and soon. I repeat, that the best way to calm him down and get him to stand steady, is to pat him on the neck and speak kindly to him. If the horse is alone and in an enclosed place, he will readily give in, but to teach him to stand steady in a street, we should bring other horses into the school and make them move about. When he stands steady under these conditions, we should renew the exercise outside, in some quiet place, and should then, little by little, try him in places where there is more traffic. I have already said that habit is everything for a horse. Therefore it is only necessary to accustom him to stand in the middle of the noise and movement of the street, and to make him understand that he will get pats on the neck for so doing. After all, great gentleness, patience and gradual training are the best means for making a horse stand still in any place as long as we like. CHANGES OF DIRECTION. At first, all changes of direction should be taught at the walk. To turn to the right, we should lightly draw the off snaffle rein with the right hand to the right, and not towards our body, so as not to stop the horse. We find that the animal slackens his pace when he turns; We find that the animal slackens his pace when he turns; because the forward reach of his shoulder which begins the movement is decreased, so that the off fore leg of the horse, if the turn is made to the right, covers only about half the distance of an ordinary step. This period of arrest causes Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 88 the croup to swerve, because it cannot go forward, notwith- standing its acquired impulsion. But if, at the moment when the animal yields his head and neck to the pull of the right rein, we slacken the hand, while supporting the legs, we thereby force the right leg to take a pace as long as usual, and shall consequently avoid the period of arrest, by obliging the croup to follow the shoulders. The rider can thus straighten the croup when it swerves in turning, and can do it easily, because he feels to what side the swerve is made, and consequently knows which leg to employ. As we do not know to which side the deviation of the croup may be made, we would run a great risk of making a mistake, if we were to say in advance what leg we ought to employ. The question of using the outward or inward leg in turning has been greatly discussed. The old school recom- mended the inward leg, because they said that it helped the turning movement. Baucher maintained, on the contrary, that we ought to use the outward leg, so as to prevent the hind quarters being swung round to the outside. The practice of Baucher has been generally adopted. The old teachers were wrong. They said that to turn to the right the rider had to bring the animal's shoulders to the right by feeling the right rein, and to push the hind quarters to the left by the right leg. This is lateral equitation in all its beauty. Digitized by Microsoft® CHANGES OF DIRECTION. But they did not take into consideration the fact that the direction, not only of the shoulders, but of the entire horse, had to be changed, and that consequently the animal should remain straight. The following is the simple solution of this problem. We should apply an equal pressure of both legs. Then, if the horse swings his hind-quarters round to one side, we can keep them straight by the pressure of the outward leg. The hind-quarters ought to follow the track of the Digitized by Microsoft® CHANGES OF DIRECTION. 89 forehand without deviating from it in the slightest degree. The horse ought always to keep his hind-quarters in the same direction as the shoulders. It is the rider’s business to decide whether he should apply a more or less strong pressure with one leg or with the other leg, according as the horse swerves to one side or the other. forehand without deviating from it in the slightest degree. The horse ought always to keep his hind-quarters in the same direction as the shoulders. It is the rider’s business to decide whether he should apply a more or less strong pressure with one leg or with the other leg, according as the horse swerves to one side or the other. As a general rule, when we turn to the right, the hind- quarters are inclined to swing round too much to the left ; and vice versdé. Therefore, unless in exceptional cases, we ought to apply the outward lez somewhat more strongly than the inward one when we turn. In turning, the action of the leg, the office of which is to prevent any deviation of the croup, should never precede that of the hand, but should immediately follow it. Another way is at first to turn the croup inwards, by doing which, the movement of the head, and immediately afterwards that of the neck and shoulders, is made difficult Besides, the arched condition of the croup and_ shoulders will prompt the horse to resist the action of the rein. We occasionally meet horses which naturally or by habit go sideways. In this case, they always bring their croup to the same side. Digitized by Microsoft® CHANGES OF DIRECTION. If, for instance, they carry it to the right, that is to say, inwards, when we turn them to the right, we shall be obliged to apply the inward (right) leg more strongly than the outward one. But if, in turning such a horse to the left, he continues to carry his croup to the right, a stronger application will have to be given by the outward (right) leg. Therefore, with the same animal we may some- times have to apply the inward, and at other times, the outward leg. To get the horse to readily change direction, and to give him the required mobility, we should make him perform in the school, different exercises, such as doublers, voltes, deimt- voltes, and changes of hand. Digitized by Microsoft® Digitized by Microsoft® go ORDINARY R ORDINARY RIDING. go The doubler is a straight line which we take, either across or down the centre of the school, after having started from some spot in the wall. Having arrived at the opposite wall, we turn, and continually go on to the same hand. (Fig. 25). Fig. 25.—Doubler. Fig. 25.—Doubler. Fig. 25.—Doubler. The volte is a circle which we describe on some point. But, at the beginning, it is best to do the volte by leaving the end of one of the long sides of the school. As a horse has always a tendency to enlarge the circle, he will keep in bounds by the angle formed by the two walls which face him. (Fig. 26). Digitized by Microsoft® CHANGES OF DIRECTION. QI The demz-volte is made in the middle of one of the small sides of the school. As it finishes on two tracks, we should not ask the horse to do it before teaching him to move on two tracks, which I will explain further on. (Fig. 27). Whatever may be the pace at which the demi-volte is Fig. 26.—Volte. Fig. 26.—Volte. made, the shoulders should be the first to leave, and to arrive at, the wall; that is to say, the horse should always remain on an oblique line. The demi-volte is usually done in a very incorrect manner. Nothing is rarer than a well- executed demi-volte. It often happens that the rider uses his outward leg too Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. Digitized by Microsoft® Digitized by Microsoft® CHANGES OF DIRECTION. 92 much in a change of direction, or that the horse, of his own accord, carries his croup to the inside, or simply goes down the centre. To remedy this, we should start, as for a demi- volte, from the usual place; but should continue to “ double,” while taking care to keep the horse very straight. If this Demi-volte to the left - lig, 27.~-Demi-volte. lig, 27.~-Demi-volte. does not suffice, we should deceive the animal by a demi- volte ; thus doing, as I term it, a counter-demi-volte. (Fig. 27.) We start, for instance, to the right, as for a demi-volte ; but having arrived at the middle of the school, we finish the demi- volte to the left on two tracks. The horse, who was wanting to carry his croup to the right, is obliged to carry it to the left by the vigorous action of the right leg, which at first was Digitized by Microsoft® CHANGES OF DIRECTION. 93 the inward leg, but which in this manceuvre becomes the out- ward one. There is no better exercise than this for keeping the horse always straight and attentive ; because, by alternating the demi-voltes and the counter-demi-voltes, it is impossible for him to become “ routined.” - Fig. 28.—Diagonal change of hand. Fig. 28.—Diagonal change of hand. There are three changes of hand. The most simple is the Wagonal change of hand, in which we go along one of the diagonals of the school, on leaving the wall at the beginning of one of the long sides. (Fig. 28.)* * If we start diagonally at the end of one of the long sides of the school, we will cross the school from one angle to the other, without having changed the hand. Digitized by Microsoft® Digitized by Microsoft® 04 ORDINARY R 04 ORDINARY ORDINARY RIDING. 04 To do the second or reversed change of hand, we start as in the preceding movement, from one of the angles of the school, always beginning at one of the long sides ; but having arrived at the centre, we return by a half circle to the wall of the long side which we left, and which we, of course, take on the left _ th op. Fig. 29.— Reversed change of hand. Fig. 29.— Reversed change of hand. Fig. 29.— Reversed change of hand. Digitized by Microsoft® CHANGES OF DIRECTION. hand, if we had it on the right hand, and wice versd. (Fig. 29.) The counter-change of hand is the most complicated one. The counter-change of hand is the most complicated one. In doing it, we start by entering the long side on two tracks, and, having arrived at the centre of the school, we return to the other end of the long side from which we started, while Digitized by Microsoft® CHANGES OF DIRECTION. 95 keeping on two tracks for the whole time. At the canter this movement requires two changes of leg, the first in the middle of the school, and the second at the wall. If the rider is going on the right, as in Fig. 30, he will begin at the canter with the off fore leading. Having arrived at the centre of the Fig. 30.—Counter-change of hand. Fig. 30.—Counter-change of hand. school, he will make the horse change, and canter with the near fore leading, up to the wall, where he will make him change to the off fore. We can see that in the counter-change of hand there is no change of hand. In a like manner we describe circles and figures of 8 in the middle of the school. This is the best kind of work for sup- Digitized by Microsoft® ORDINARY RIDING. 96 pling the horse, and is also the most certain means for making the rider use his two legs in a timely way. pling the horse, and is also the most certain means for making the rider use his two legs in a timely way. We do not learn to always keep our horse straight by con- tinually “going large”; because in this case the croup can swerve only to one side. Also, I often work my horses, while keeping them at one yard, and in large schools, at two yards, from the wall. We may further note that during the entire period of breaking, the horse tries to keep as little straight as possible. He will understand that if he carries his croup to one side he will escape being collected, which consists in the hind quarters propelling the body in the direction of its axis. CHANGES OF DIRECTION. The best work for suppling the horse is the figure of 8 per- formed a yard from the wall, at the walk, trot and canter; but it should be done with the greatest possible correctness,namely, with a light inward flexion of the neck, an energetic applica- tion of the outward leg, and support from the inward leg. By preparing a horse to readily change the diagonal at the walk, we also prepare him to start into the canter, and to change his leg. On arriving at the centre, we make the horse take two or three steps to the side, and we start him in the required position on a new diagonal. We should take great care to keep him straight, especially at the canter, because at this delicate work, the horse has always a tendency to throw his croup inwards, so as to escape the outward spur, which has helped to straighten him, and against which he tries to guard himself. Hence the necessity, as I have recommended, of opposing the inward leg. Finally, we can obtain impulsion only by closing both legs. Digitized by Microsoft® * We may note that I here recommence the work on foot. I always practise the foot work and the mounted work in the same lesson, 7 LATERAL FLEXIONS. Up to the present I have made the changes of direction only in a rudimentary way. In the same manner, as my first lesson was to drive the horse forward, I only require him at Digitized by Microsoft® LATERAL FLEXIONS. 97 first, for the changes of direction, to turn and follow the line on which [ place him. In every case I go from simple things to complicated ones. When the horse unresistantly obeys everything, from the first indication to all the changes of direction, we have to solve the new problem of displacing the Fig. 31.—Preparation for lateral flexion ; jaw contracted. Fig. 31.—Preparation for lateral flexion ; jaw contracted. Fig. 31.—Preparation for lateral flexion ; jaw contracted. entire body of the horse, while keeping it in equilibrium and lightness. Lateral flexion is the preparation for the movement thus executed. To make the lateral flexion to the right,* for instance, we should stand at the near shoulder of the horse, and take the bridoon reins in the left hand and the bit reins entire body of the horse, while keeping it in equilibrium and lightness. Lateral flexion is the preparation for the movement thus executed. To make the lateral flexion to the right,* for instance, we should stand at the near shoulder of the horse, and take the bridoon reins in the left hand and the bit reins 7 7 Digitized by Microsoft® ORDINARY RIDING. 98 ja the right, in exactly the same manner as for the direct flexion; and, having placed the hand and neck in the same position as we would do for the direct flexion, we make that flexion. When the jaw is relaxed and the flexion complete, we push the head of the horse to the right, by light pressures of the left hand on the bridoon reins, which we hold high, and drawn from rear to front, in order to prevent both the lowering of the neck and any tendency which the horse may have to get behind his bit.* At the same time, the right hand lightly feels the curb reins from behind, while carrying itself to the right in such a manner as to principally tighten the right rein,t until, the lateral flexion of the neck to the poll being complete ; the two reins of the curb acting equally in giving and maintaining the play of the jaw (Figs. Digitized by Microsoft® LATERAL FLEXIONS. 31 and 32), which ought to be the same as in direct flexion. At the beginning, we ought to be content with even the appearance of obedience, and we should not want the horse to do more than to turn his head slightly, while opening his mouth. We must practise him very often at this work, and try to progress by requirmg more exactness each time in these suppling exercises. We will certainly finish in obtain- ing complete flexion, by never employing force and con- tenting ourselves with a small degree of progress at each lesson. In equitation we cbtain much by requiring only a little at * See Fig. 31, preparation for lateral flexion, jaw contracted ; Fig. 32, lateral flexion, jaw relaxed. t The left rein of the curb, however, remains slightly tense. If the right rein only acts, it will pull the muzzle, and the head will no longer be in a vertical position. We may note We may note that I give here the first lesson of obedience to the two similar impulsions in the changes of direction. Up to this I have used the right rein only to turn to the right. I now begin to teach the horse to obey the two simul- taneous impulsions acting in the same direction, namely, the near snaffle rein which pushes, and the off curb rein which pulls, both acting from left to right. Digitized by Microsoft® LATERAL FLEXIONS. 99 atime. Patience then, and no roughness, which will always prevent us from obtaining our end. atime. Patience then, and no roughness, which will always prevent us from obtaining our end. The exact position for the lateral flexion is the same as for The exact position for the lateral flexion is the same as for \ i Fig. 32.—Lateral flexion ; jaw relaxed. Fig. 32.—Lateral flexion ; jaw relaxed. the direct flexion, except that the neck, while remaining flexed at the poll, from front to rear, is also bent to one side (always at the poll), in sucha manner that the direction of the head is perpendicular to what it was in direct flexion, ae ae ae Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 100 and faces the side upon which the flexion was made. LATERAL FLEXIONS. The neck, of course, remains high, as in direct flexion, the muzzle being at the height of the upper part of the shoulder and the head in, or a little beyond, the perpendicular* (Fig. 33). seen by the right flexion, this flexion is Thus, as we have seen by the right flexion, this flexion is generally done in quite a different manner. The only way to find out what the lateral flexion ought to be, is to consider the result we wish to obtain from it. OBJECTS OF LATERAL FLEXIONS. 1. To preserve, by the high position of the neck, the equili- brium of direct flexion in changes of direction. ehand in the changes 2. To strengthen and bind the entire forehand in the changes of direction, by arranging all the parts in such a manner as to make the whole as compact and supple in the turning move- ments, as in the direct movements. In changes of direction, the shoulders naturally cover the ground, while the hind quarters give the propulsion ; but * In doing the lateral flexion we should take care not to alter the distribution of weight. To counterbalance the effect of the flexion, the horse has a natural tendency to make an opposition with the shoulder of the side away from which the head is turned, and to put the weight of the forehand on the left leg if the head is bent to the right. This is inevitable as long as the jaw resists, but the moment it yields, its flexion, involving that of the neck, brings about an equal distribution of weight on both legs. If we allow the horse to contract the habit of opposing the shoulder of the side opposite to the flexion, the equilibrium, and consequently the lightness, will be destroyed. In changes of direction, as in movements, on two tracks, the shoulder of the side opposite to that of the flexion will always be late. The great difficulty in these exercises is to make this shoulder move. Hence we should always try to relieve it by making only a slight bend, whilst the snaffle rein of the side opposite to the change transfers the weight to the inward shoulder (which has less ground to cover), by throwing the weight at each stride to the side towards which the animal is proceeding. This method enables us to obtain great propulsion in work on two tracks. A too complete flexion will stop this propulsion by overloading the outward shoulder. At first one is always astonished to learn that the flexion to the right overloads the near shoulder, which is the natural result of the attempt made by the horse to counterbalance the effort demanded of him. Digitized by Microsoft® Digitized by Microsoft® OBJECTS OF LATERAL FLEXIONS. 101 the rider has no direct action on the shoulders. Digitized by Microsoft® OBJECTS OF LATERAL FLEXIONS. He acts only on the neck through the mouth, and on the shoulders through the neck. Lateral flexion, by strengthening and binding all the parts together, places them in a state of reciprocal dependence, which enables the rider to act on the entire forehand. Without flexion, the fishing-rod, as I have already said, will be held by the thin end; that is to say, Fig. 33. —Correct lateral flexion. Fig. 33. —Correct lateral flexion. there will be a heavy head (because it is far from the centre of gravity) at the end of a slack neck. The action of the reins, instead of directing the mass, is therefore limited to bringing the head towards the forehand, which remains fixed on the ground, in proportion as the horse is on his shoulders. 3. To preserve lightness in the changes of direction by the flexion of the jaw. In all movements, tmpulsion makes a horse a single whole, Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 162 It ts the hind quarters which, by getting under the centre, bind themselves to the forehand.* Tf all this propulsion was accumulated on the bars of the flexed and movable lower jawt—which should be connected to the entire forehand in such a way that the flexibility{ of the arm of the lever would be always increasing from the shoulders to the jaw—the hands, both in the changes of direction and in direct move- ments, would send back with extreme lightness § to the legs, a part of the propulsion which they have received from them. We can thus make the entire body of the horse into an ener- getic and harmonious whole by the good equilibrium of the levers, and by the well-regulated use of the forces in changes of direction. We will thus obtain lightness, and will continue ¢#o have the horse in hand in all the changes of direction. * Many authors speak of connecting the forehand to the hind quarters, which is an absurdity, because the hind quarters throw themselves on the forehand during propulsion. The ‘‘aids’’ keep the body in good position by restraining or regulating the force which comes from the hocks. § In lateral flexion, as in direct flexion, we should work the levers in such a manner as to obtain the maximum effect by a minimum effort, * Many authors speak of connecting the forehand to the hind quarters, which is an absurdity, because the hind quarters throw themselves on the forehand during propulsion. The ‘‘aids’’ keep the body in good position by restraining or regulating the force which comes from the hocks. + When the jaw does not yield, nothing yields, and the horse changes his position all in one piece during the changes of direction. He turns like a boat. ¢ The only lateral movement in /a/era/ flexion is done by the poll; the jaw bends exactly as in direct flexion. § In lateral flexion, as in direct flexion, we should work the levers in such a manner as to obtain the maximum effect by a minimum effort, + When the jaw does not yield, nothing yields, and the horse changes his position all in one piece during the changes of direction. He turns like a boat. OBJECTS OF LATERAL FLEXIONS. It ts the hind quarters which, by getting under the centre, bind themselves to the forehand.* Tf all this propulsion was accumulated on the bars of the flexed and movable lower jawt—which should be connected to the entire forehand in such a way that the flexibility{ of the arm of the lever would be always increasing from the shoulders to the jaw—the hands, both in the changes of direction and in direct move- ments, would send back with extreme lightness § to the legs, a part of the propulsion which they have received from them. We can thus make the entire body of the horse into an ener- getic and harmonious whole by the good equilibrium of the levers, and by the well-regulated use of the forces in changes of direction. We will thus obtain lightness, and will continue ¢#o have the horse in hand in all the changes of direction. In the foregoing remarks I have described what I consider to be the nature and object of lateral flexion, and have tried, as in the description of direct flexion, to give reasons for my practice. Unfortunately the greater number of those who teach horses flexions, do the work in a haphazard way, with- out accurately understanding the nature of the result which they ought to try to obtain. I must say that Baucher did not give a better account of the mechanism of lateral flexion than he did of direct flexion. Or, rather, he committed the same fault in lateral + When the jaw does not yield, nothing yields, and the horse changes his position all in one piece during the changes of direction. He turns like a boat. ¢ The only lateral movement in /a/era/ flexion is done by the poll; the jaw bends exactly as in direct flexion. § In lateral flexion, as in direct flexion, we should work the levers in such a manner as to obtain the maximum effect by a minimum effort, Digitized by Microsoft® OBJECTS OF LATERAL FLEXIONS. 103 flexion as in direct flexion, because the latter is a preparation for the former. This faulty flexion, which is adopted by those who have followed him, is made at the withers instead of at the poll. Here, again, as in direct flexion, we find the exciting cause of the lowering of the neck, which changes a useful exercise into an injurious one. OBJECTS OF LATERAL FLEXIONS. In it the head is low, the bend of the neck is at the withers, instead of at the poll, and the head of the horse is in profile instead of facing one. Fig. 34.— Incorrect lateral flexion. Fig. 34.— Incorrect lateral flexion. A comparison of Fig. 34 (wrong flexion) with Fig. 33 (correct flexion) shows us at a glance the advantages of the lateral flexion which I have described, and the faults of the flexion which is commonly practised. To complete this criticism, I need only repeat all what I have said about the wrong method of direct flexion and its faults. We need not be surprised that flexions have been blamed for softening the neck ; that is to say, for rendering it movable, independently of the remainder of the body, which Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 104 is precisely the result of lateral flexion at the withers; whilst lateral flexion at the poll, on the contrary, stiffens the neck, and binds all the forehand in such a manner as to dis- place it from the whole, which is an indispensable result, as I have explained, because the rider has no direct action on the shoulders. Head low and isolated from the body by a movable neck, which yields of itself without drawing the shoulders, and allows the horse to oppose every movement of the neck by a movement of the shoulders in an opposite direction, with consequent impossibility of directing the fore- hand, and the results of a lateral flexion at the withers and of a lowering of the neck which follows it. Contrary to direct flexion (which I never practise, either on foot or mounted, except when going forward), lateral flexion at first is done on foot, when the horse is standing still; on account of the difficulty of putting the hind quarters in movement. In mounted work, I practise lateral flexion only when going forward. I make it an absolute rule, once I am mounted, never to ask my horse anything except when he is advancing, and I have consequently avoided making my school horses inclined to get behind their bit, which is the usual danger in high school breaking.* In mounted work, lateral flexion is made by the same mechanism as on foot. * T have already said that a high position of the neck can be obtained only during forward progression, and that the reason I keep the necks of my horses very high is because I continue to drive them forward during all the work I give them. In fact, the greater the forward impulsion, the more do the hind quarters get under the centre, and the more is the forehand lightened. Digitized by Microsoft® Digitized by Microsoft® OBJECTS OF LATERAL FLEXIONS. In order to bend the neck to the right, the near snaffle rein, tightened and drawn to the right, keeps the head high, and, being pressed against the upper part of the neck, it pushes the head from left to right, while the off curb rein, slzghtly tighiencd, aids this movement, and loosens the jaw (Fig. 35). OBJECTS OF LATERAL FLEXIONS. 105 As the first care of the rider should be to keep his animal straight, he should use both legs to obtain impulsion, the leg of the side opposite to the bend of the neck being applied more strongly than the other leg. When the legs act simultaneously, they are agents of pro- When the legs act simultaneously, they are agents of pro- pulsion; but when one acts stronger than the other, it is an Fig 35.—Correct lateral flexion when mounted. Fig 35.—Correct lateral flexion when mounted. agent of direction. One leg should never act by itself: the simultaneous action of both legs is propulsion; the pre- dominance of one over the other is direction. Finally, as the hands are much more effective for guiding than the legs, they should be used in a very light manner. The fundamental error of Baucher’s lateral flexion is as agent of direction. One leg should never act by itself: the simultaneous action of both legs is propulsion; the pre- dominance of one over the other is direction. Finally, as the hands are much more effective for guiding than the legs, they should be used in a very light manner. The fundamental error of Baucher’s lateral flexion is as well marked in mounted work as in foot work. Compare Fig. 36, which I have taken from Baucher’s book, with Fig. 35, which represents correct flexion. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® 106 ORDINARY RIDING. ORDINARY RIDING. 106 Fig. 37, taken from a recent book, shows clearly that many persons of the present day who claim to practise lateral flexion have no idea of its mechanism or object. lateral flexion have no idea of its mechanism or object. Fig. 36.—Baucher’s lateral flexion when mounted. The lateral flexion I have described is a suppling Fig. 36.—Baucher’s lateral flexion when mounted. The lateral flexion I have described is a suppling exercise, whether it is done on foot or when mounted, and Digitized by Microsoft® Digitized by Microsoft® OBJECTS OF LATERAL FLEXIONS. * As long as the jaws work we have lightness, and the slightest indication is sufficient for changes of direction. OBJECTS OF LATERAL FLEXIONS. 107 is of great importance. Equilibrium, lightness and mobility depend on the flexions and on getting the horse in hand, and until he has learned all this, it would be folly to ask for more. It is therefore of the highest importance to make the lateral flexion complete, so as to obtain extreme obedience from the horse. Fig. 37-—Incorrect lateral flexion. Fig. 37-—Incorrect lateral flexion. But when we practise lateral flexion in later work ‘changes of direction, work on two tracks, etc.), we ought to content ourselves with a very slight lateral flexion of the neck,* which like the head, should always be well placed. We can understand that a too strongly accentuated flexion checks the effect of propulsion by throwing all the weight on the outward shoulder. * As long as the jaws work we have lightness, and the slightest indication is sufficient for changes of direction. Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 108 The suppling exercise, which we term lateral flexion, is not the less necessary, because it demands much in order to obtain little. But we must recognise the fact that in later work it is enough to obtain a slight bend of the neck, provided that the head and neck are always well placed, and above all things that the jaw is loose. Fig. 38.—Lateral effects in rotation of croup and shoulders. Fig. 38.—Lateral effects in rotation of croup and shoulders. * I have already explained that /ateral egudfation is only a preparation for diagonal eqguitation, which is the only rational method, and by which alone we can obtain combined effects. Everyone understands that the action of the rider should be effected diagonally, for the simple reason that the movements of the horse are produced diagonally. + For the better explanation of the action of the reins, I have omitted the snaffle in Fig. 4o. I may remind my readers that the proper way to hold the ends of the snaffle reins is to have the buckle in the hollow of the hand (Figs. 38 and 39). Digitized by Microsoft® * I have already explained that /ateral egudfation is only a preparation for diagonal eqguitation, which is the only rational method, and by which alone we can obtain combined effects. Everyone understands that the action of the rider should be effected diagonally, for the simple reason that the movements of the horse are produced diagonally. ROTATION OF THE CROUP AND SHOULDERS. When I have successively made the horse readily yield to the whip, and to perform the lateral flexions with equal facility, I have then to combine both these movements into a single movement. I have at first taught him to yield to the whip from left to right, while helping the movement with the near snaffle rein, which draws the head to the left, so as to carry the croup to the right. This work is called “lateral effects” (Fig. 38) ; because the two effects are produced on the same side (left rein and whip to the left). Having done this, I succeed, Digitized by Microsoft® ROTATION OF CROUP AND SHOULDERS. 109 little by little, in making the horse give way, by holding the head straight (Fig. 39), which is called a “direct effect.” Now it is necessary for him to yield, by adopting “ diagonal effects” in the same way, namely, with the whip on the left and the flexion on the right (Fig. 40).* For that purpose, without using the snaffle rein, the end of which is in my left Fig. 39.—Direct effects in rotation of croup and shoulders. Fig. 39.—Direct effects in rotation of croup and shoulders. hand,t I catch hold of the near curb rein, quite close to the mouth, with the left hand, while the right hand, at the height Digitized by Microsoft® ORDINARY RIDING. TIO of the horse’s breast, holds both the whip and the off rein, for which the withers act as a pulley (Fig. 40). The horse will then make the direct flexion. I then try to obtain the beginning of the right lateral flexion by raising the head by means of slight upward jerks on the curb, and by pressing the head to the right, while at the same time I make the croup give way by working the whip from left to right. Fig. 40.—Diagonal effects in rotation of croup and shoulders. Finally, I increase these effects, until the yielding of the neck, jaw and croup is complete. Finally, I increase these effects, until the yielding of the neck, jaw and croup is complete. I thus succeed I thus succeed in making the horse pzvot, while I do the complete lateral flexion. I I purposely use the word “ pivot,” which expresses my idea in an incomplete manner, but which accurately describes Baucher’s method of doing this rotation. ROTATION OF THE CROUP AND SHOULDERS. In fact, during the rotation of the croup, according to his plan, the forehand Digitized by Microsoft® ROTATION OF CROUP AND SHOULDERS. 111 remains stationary and serves as a pivot, which I have found to be a mistake. During breaking, none of the parts of the horse should be stationary, because immobility often degener- ates into a means of defence. Therefore, instead of making the horse pivot, I try to make him describe, a very small circle with the forehand round the centre, and a large concentric circle round it with the hind quarters. Of course, I do not pass abruptly from lateral effects to direct effects, or from direct effects to diagonal effects. On the contrary, I go imperceptibly from one to the other, so that the horse may well understand what I want him to do, and that he may not evince any surprise or confusion. The employment of lateral effects has been a preparatory step towards the execution of the movement by direct effects. In the same way, direct effects are only a preparatory measure for diagonal effects. This work would be of no use if its only object was to make the horse give way to the whip. Its great end is, with- out frightening the horse, to prepare him to obey the legs at first, and the spurs subsequently and by degrees. We now come to mounted work, which I do by again We now come to mounted work, which I do by again passing from lateral effects to direct effects, and from direct effects to diagonal effects, as during work on foot. If, when in the middle of the school, I wish to obtain the rotation of the croup (reversed pirouette) from the left to the right, I bring my left heel close to the animal’s side. As a green horse does not know what I wish him to do, his first movement will be to lean against my leg, at which movement the good effect of the preceding work will come to my aid. I touch him lightly with my whip on the left side, as near as possible to my heel, and I avoid above all things touching him too far back, which would be almost certain to make him kick. At the same-time I use the left snaffle rein to make him carry his croup to the right. * The great art in breaking is to reward and punish in an appropriate manner, in order to do which we must seize the exact moment of obedience or resistance. Here we must bear in mind the fundamental principle of breaking, namely, that reward should follow obedience as quickly as punishment follows disobedience. Digitized by Microsoft® ROTATION OF THE CROUP AND SHOULDERS. I can assure my readers Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 112 that no horse will resist these indications, if they are gently employed. I use the near snaffle rein according to the amount of resistance which the horse often makes at first. I draw the head a little more to the left, while continuing to touch the animal’s side with the heel and whip if the resistance is great. The horse is forced to yield to these three forces acting on the same side. As soon as the horse takes a step to the right, I stop him and pat him on the neck. I then let him go quietly round the school, in order that he may think over what he has just done. It is most important always to let a horse be free and quiet after he has obeyed. A horse will accept this as a reward, which we should not be chary in giving him. Stop- page of work and pats on the neck are the only means to make him understand that he has done well. We are so often obliged to have recourse to punishment during break- ing, that we ought to eagerly seize the opportunity of patting him on the neck, when he shows the slightest sign of obedi- ence. The more we pat him on the neck, the less will we be forced to punish him.* As I have just said, by letting the horse walk at ease for a few moments, we give him time to understand the movement which he has just done and the indications which cause him to do it. Apparently the horse yields only physically, but in reality it is his intelligence, or, to speak more correctly, his memory to which we appeal. We must therefore work on his memory, and for that reason I allow him the necessary time to permanently remember this fact. Having let the horse go quietly round the school, I do the ROTATION OF CROUP AND SHOULDERS. 113 same exercise over again, twenty or thirty times, without changing the side, until he obeys the moment I close my heel to his side. I then give him similar work with the right leg. When the horse accurately obeys both legs, I put on dummy spurs (stump spurs), in order to accustom him to obey something more severe than the heel. * T have already said that I discard the whip when mounted, and I take it up again only to make the horse obey the leg and to obtain the first extension of the legs in the Spanish trot. In both cases I continue the use of the whip for only two or three lessons. \ 8 Digitized by Microsoft® ROTATION OF THE CROUP AND SHOULDERS. Then, each day I increase the effect of the leg, while diminishing that of the whip, which I gradually discard.* Later on I come to the spur. I use the whip. only to aid the memory of the horse, and to induce him to obey the spur without frightening him. We should not forget that the effect of a touch of the spur op a green horse at the beginning of the breaking is exactly similar to that of the sting of a fly, which he at first tries to drive away with his tail. If he does not succeed in this, he will cow-kick on the side he is pricked. If the insect does not fly away, he will look out for some object, such as a wall or tree, upon which to lean or lie, in order to crush the author of his sufferings, the fy. Therefore, when we give him the first touch of the spur, how can we expect that his first move- ment will not be to cow-kick or to lean against a wall? We may see that it is a grave mistake to use the spur with- out having prepared the horse for it, by making him succes- sively obey the whip, leg, heel and dummy spur. If we spur a horse which is neither prepared for nor accustomed to it, he will not understand its meaning, and will not obey. If we try to force him to do so, he, not knowing what we want him to do, and feeling the pain which we inflict on him, will instinctively defend himself. The more severe the attack, the more stubborn will be the resistance at first, * T have already said that I discard the whip when mounted, and I take it up again only to make the horse obey the leg and to obtain the first extension of the legs in the Spanish trot. In both cases I continue the use of the whip for only two or three lessons. \ 8 8 8 Digitized by Microsoft® ORDINARY RIDING. 114 and the more energetic the subsequent defence. If the horse is soft, he will lean against the spur; but if he is impetuous, he will immediately make a most violent defence, and the breaking will have failed in every way. * For this reason, I have already said that in the same lesson we must care- fully avoid requiring the horse to do two or more things which might confuse him. As his comprehension is very slow, we ought to guard against perplexing him. + It often happens that the horse throws himself on the spur, sometimes to one side and sometimes to the other. In this case we ought to effectively correct him with the spur, for which object I place him in the middle of the school, and drive my heel and spur into his rebellious side, so as to make him bring it round. When he has thus made two or three pirouettes, I stop and begin again the work T left off. If he again resists, I recommence the work, until he has thoroughly given in. Digitized by Microsoft® * For this reason, I have already said that in the same lesson we must care- fully avoid requiring the horse to do two or more things which might confuse him. As his comprehension is very slow, we ought to guard against perplexing him. + It often happens that the horse throws himself on the spur, sometimes to one side and sometimes to the other. In this case we ought to effectively correct him with the spur, for which object I place him in the middle of the school, and drive my heel and spur into his rebellious side, so as to make him bring it round. * For this reason, I have already said that in the same lesson we must care- fully avoid requiring the horse to do two or more things which might confuse him. As his comprehension is very slow, we ought to guard against perplexing him. + It often happens that the horse throws himself on the spur, sometimes to one side and sometimes to the other. In this case we ought to effectively correct him with the spur, for which object I place him in the middle of the school, and drive my heel and spur into his rebellious side, so as to make him bring it round. When he has thus made two or three pirouettes, I stop and begin again the work T left off. If he again resists, I recommence the work, until he has thoroughly given in. ROTATION OF THE CROUP AND SHOULDERS. Some animals will become restive, others will become maddened at the mere approach of the leg, and the breaker, instead of having taught anything, will have rendered education impossible. It is always thus in breaking, the great difficulty being to make the horse understand what we want him to do. As we can appeal only to his memory,* the means which we employ with him should be simple, and should be invariably the same. In riding, the horse ought to be taught to understand that the spur is simply an “aid,” and that it becomes a punish- ment only when he plays up.t Many persons who have only a superficial knowledge of equitation imagine that, instead of touching the horse behind the girths with the spur, it is more rational to keep the knee tightly pressed against the flap of the saddle, and by drawing back the heel to spur the horse on the side. Nothing can be more faulty than this method, by which the spur slides along a large extent of the side. By it we succeed only in tickling the animal and provoking him to defend himself, and we are unable to spur him with sufficient power to drive him forward and to paralyse his defence. The further back the + It often happens that the horse throws himself on the spur, sometimes to one side and sometimes to the other. In this case we ought to effectively correct him with the spur, for which object I place him in the middle of the school, and drive my heel and spur into his rebellious side, so as to make him bring it round. When he has thus made two or three pirouettes, I stop and begin again the work T left off. If he again resists, I recommence the work, until he has thoroughly given in. Digitized by Microsoft® ROTATION OF CROUP AND SHOULDERS. 115 spur is drawn away from the girths, the nearer it approaches the most ticklish spot. In fact, horses are not ticklish near the girths, but all of them are ticklish further back. Besides, in this kind of attack, the lower part of the leg cannot remain close to the animal’s side, because the foot swings backwards and forwards; the result being that the attack is sudden, instead of the horse being prepared for it by a gradual pressure of the leg. * The fact that all beginners and bad riders instinctively assume this position, in order to get the greatest possible firmness of seat, prevents them from being able to use the spurs. Besides, every rider instinctively adopts this position when his horse plays up. 8* Digitized by Microsoft® ROTATION OF THE CROUP AND SHOULDERS. It should, on the contrary, be capable of being regulated with precision, so that it can be light, well-marked, or extremely severe, according to the resistance offered by the horse to the indications given by the rider’s legs. When spurring a horse just behind the girths, the knee should be turned a little outwards, so that the spur, like a sword, may be brought at right angles to the side, pro- gressively and with certainty. The scratching and fraying form of attack is made without precision and by jerks. Those who believe that the fact of the knee being slightly turned outwards diminishes the firmness of the rider’s seat, should be taught that the rider gets his strongest grip by means of the upper and back part of the calf of the leg. Therefore the position which the leg occupies when it spurs the horse just behind the girths, is the one which is most favourable to the rider’s firmness of seat.* Gripping too tightly with the knees pushes the thighs upwards, and causes the rider to be more or less raised out of the saddle. By gripping with the hollow of the leg just behind the knee, we have, on the contrary, perfect adherence from the buttock to the heel. Besides, we can do fine work with the horse only 8* 8* Digitized by Microsoft® ORDINARY RIDING. 116 by always keeping the heels close to his sides. With the knees and toes turned inwards, the heels are too far from the sides, and can work only by jerks. Good horsemanship cannot be practised without progressive and delicately effected transitions. When the horse readily yields to the legs, I change the feeling on the reins by degrees. It is always necessary to finally place the head of the horse on the side to which it is directed. Nevertheless, the change should be made so gradually that the animal will not notice it. At first he will yield to my leg only when I use it, while feeling the rein of the same side—a lateral effect. I gradually diminish the tension of the rein, until he will obey the leg by itself, and I then employ both snaffle reins to keep his head straight—direct effect. Fuinally, I succeed little by little in using the opposite rein—diagonal effect. Digitized by Microsoft® * When the horse readily yields to the action of the legs, it is well to use them alternately ; but only as an indication, to make the hind quarters slightly yield, in which case a step or two will be sufficient. The rider will thus succeed in sending the hind quarters from one leg to the other leg, and to measure this action. This is the beginning of ‘‘ equestrian tact.” * When the horse readily yields to the action of the legs, it is well to use them alternately ; but only as an indication, to make the hind quarters slightly yield, in which case a step or two will be sufficient. The rider will thus succeed in sending the hind quarters from one leg to the other leg, and to measure this action. This is the beginning of ‘‘ equestrian tact.” t This work can be properly done only when mounted, on account of the necessity of supporting the hind quarters and of pressing the horse forward. t This work can be properly done only when mounted, on account of the necessity of supporting the hind quarters and of pressing the horse forward. ROTATION OF THE CROUP AND SHOULDERS. Here progress will be similar to that which was obtained when doing the same movement on foot; solely by the aid of the whip, that is to say, the horse will finish by rotating his croup to the right, while keeping his muzzle to the right ; and vice versd. At this work I never require complete lateral flexion when mounted. A very slight bend towards the side to which we are going is sufficient.* Having obtained this result, I teach the horse to do the simple pirouette, in which he turns his shoulders round his hind quarters.t We should not only supple the hind quarters, but should t This work can be properly done only when mounted, on account of the necessity of supporting the hind quarters and of pressing the horse forward. ROTATION OF CROUP AND SHOULDERS. 117 also give to the shoulders the greatest possible mobility, which is an indispensable quality for every kind of work. We can exert no direct action on the shoulders, as we can on the mouth and hind quarters. The impulsion to put them in movement is given by the hind quarters, which are put in action by our legs; and the direction is given by the mouth, which receives the indication of the reins. I shall now describe how I make the shoulders rotate, namely, how I do the pirouette from left to right. Having halted the horse in the middle of the school, I carry both hands to the nght and close the legs, in order to prevent a retrograde movement; the left leg being pressed more strongly to the side than the right leg, so as to prevent the horse from bringing his hind quarters round to the left. I work the snaffle reins simultaneously with the legs, the off rein being lightly drawn to the right, but not backwards, while the near rein pushes the shoulders to the right. The near rein performs another office, as follows: If the horse, having carried his shoulders to the right, wishes to throw his hind quarters to the left, a slight pull of the near rein gives immediate help to the left leg to keep the croup in its place, by carrying the head more or less to the left, according to the extent the hind quarters have swung round to the left. ROTATION OF THE CROUP AND SHOULDERS. At the beginning we should make a large circle rather than a small one, which would be a true pirouette. In this way we keep the horse in hand, and we easily prevent him getting behind the bit, which we should always take the utmost care to do. The absolute necessity of avoiding any tendency in the horse to get behind his bit, makes it dangerous during the first period of breaking to teach simple or reversed pirouettes, which are excellent exercises for both man and horse, when they are sufficiently advanced in their education. The reversed pirouette has the great fault of leaving the Digitized by Microsoft® ORDINARY RIDING. 118 shoulders inert; and the ordinary pirouette, that of fixing the hind quarters on the ground. shoulders inert; and the ordinary pirouette, that of fixing the hind quarters on the ground. We cannot get a horse to do a pirouette, if we drive him forward. At the beginning of breaking we should not employ any movement which will leave one part of the horse stationary, for the great point is to make the horse move with his whole body. Instead of pirouettes, it is better to do ordinary and reversed vol/es, in which we can always press the horse up to his bridle. The volte is, in fact, only a pirouette described on a large circle. In the reversed volte, the croup describes the great circum- ference. A horse which enlarges the circle has a tendency to get behind his bit. In the ordinary volte the shoulders describe the great circumference. Then, if the horse diminishes the size of the circle, he has a tendency to get behind his bit, to avoid which fault he should be kept be- tween the legs. We should ask him to do only movements which bring his whole body into play, and we should keep pressing him forward. * By this I mean the movements of the horse as a whole which bring him into hand, namely, to send him by the legs on the hand and to senda part of the impulsion from the hand to our legs. Digitized by Microsoft® SCHOOL WALK. When the horse is well in hand at the walk, we can make him do the “school walk,” which is a shorter, lighter, and more cadenced pace than the ordinary walk. In it, the feet are put down in the same succession as in the trot, from which it differs only by the fact that its steps are shorter. To obtain the school walk, we should use the legs ener- To obtain the school walk, we should use the legs ener- getically and the hands moderately ; should make numerous movements with the horse’s whole body by means of these aids ;* and should cover as little ground as possible, by THE REIN-BACK. 119 shortening the steps. Too long steps show that the horse has not begun to collect himself, without doing which his steps cannot be in cadence. The school walk is an excellent form of gymnastics, in which the combined “aids” of the rider bring all the powers of the horse into play. It makes him supple, graceful, light and well posed; in other words, it puts him into perfect equilibrium, and it prepares him finally for the artificial paces, and especially for the rassembler, which of itself brings him to the gassage and piaffer. I teach the horse to do all the changes of direction at this school walk; but only when he can do them easily at an ordinary walk. * Reining back is the only work during which I put extra weight on the shoulders, Digitized by Microsoft® THE REIN-BACK. People generally, when trying on foot to make a horse rein back, commit the great fault of holding his head as high as possible while pushing him back. The exact opposite of this should be done; because, by raising the head while pushing the horse back, we overload the hind quarters, which require to be light in order to perform this move- ment. In fact, the hind quarters are the first to move, which they cannot do freely if they are overloaded. Even if we try to push back the horse only a little, we will make him get behind his bit and rear, which is a most serious vice. To make a horse rein back properly, we should, on the contrary, put the preponderance of weight on his shoulders. In order to do this, I slightly lower the head of the animal* by light, downward pressures on the snaffle reins. ize with each hand, I stand directly facing the horse, and seize with each hand, * Reining back is the only work during which I put extra weight on the shoulders, ORDINARY RIDING. 120 close to the mouth, a snaffle rein, with which I push him back. I need hardly say that in this position the rein-back is very easy, the horse being on his forehand and his loins and hocks relieved. The hind feet, like those in front, will be easily raised instead of being dragged, and when the animal is pushed back by the snaffle he will not be able to arch himself. We ought to be contented with two first steps to the rear, and should then pat him on the neck and bring him forward. We should frequently recommence this work, which 1s better than prolonging the rein-back, which at first will fatigue the horse, who, not having his loins and hocks suppled, will have his joints more or less stiff at this new work. The more we prolong the work, the less will we impress the memory of the horse; because we do not stop to pat him on the neck and to make him understand that he has done well. Finally, we must bear in mind that all work which fatigues the horse discourages him, if he is not brought up to it by degrees and prepared by successive suppling lessons. Digitized by Microsoft® THE REIN-BACK. I never require from a horse more than ten or twelve steps when reining back, and I then bring him forward the same distance, while keeping him continually in hand. I never make him do this movement more than three or four times consecutively. We rarely fail to obtain the rein-back by the means which I have described, and which should be invariably combined with great gentleness. It sometimes happens that the horse refuses to rein back, either from stubbornness or from pain. I have seen horses resist all known means, even the most violent ones, which, unfortunately, are nearly always employed with these animals, In such cases I stand directly facing the horse. I hold in each hand a snaffle rein close to the mouth, and I simply step Digitized by Microsoft® THE REIN-BACK. 121 on his feet while pressing him backwards. I have never met a horse which would not yield to this method. on his feet while pressing him backwards. I have never met a horse which would not yield to this method. When the horse reins back without stiffness, and with the head lowered, I try to make him do the same movement with the head more and more raised, for which object I place myself at his shoulder, and I do the direct flexion in reining back. I thus endeavour to obtain a very free rein-back with the horse well in hand. If the horse reins back too quickly, in order to escape from the bit, I draw the snaffle reins forward so as to make his retrograde movement more slow. This is the normal rein- back, for which the preceding work is only the preparation. It is evident that if the horse is ready to rein back at the very first sign of the direct flexion, there will be no necessity to lower his head, and still less to tread on his feet. The lowering of the head is useful only to prevent him getting behind his bit. I therefore do not practise it unless the animal shows signs of wanting to rein back. When the horse readily reins back by means of direct flexion when held by the hand, I begin to put him through the same work while on his back. In mounted work I do not force the forehand. THE REIN-BACK. In fact, it is dangerous to pull on the mouth, which action throws too much weight on the hind quarters, and may consequently lead to his getting behind his bit and rearing. Also I never attempt the mounted rein-back unless I am certain that my horse will go freely forward by a pressure of the legs, which, in my opinion, is the best means of mobilising the hind quarters, and with which I always begin the rein-back. I feel the bit reins only lightly, in order to make the horse lower his head after I have halted him. I then close my left heel, and the horse, being already obedient to the legs, raises his near hind as if he was going to take a step to one side, because he was taught to give way to the spur. At this Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 122 instant I gently feel the off snaffle rein in a backward direction, but not to one side, which would have the effect of turning his head. The near hind foot, which is in the air, places itself firmly down on the ground behind the off hind, at the moment when the old snaffle rein makes the off shoulder go back. I then close my right heel. At the instant the horse obeys the indication I give him—namely, when he raises his off hind, as if to take a step to one side—I utilise this moment to give a backward pull on the near snaffle rein, the off hind being of necessity placed in rear of the near hind at the instant the near snaffle rein brings the left shoulder back. Having obtained two steps of the rein back, I am content, and I hasten to pat the horse on the neck to show him that he has done well. When the horse has often done two steps, and then four, he will be able to rein back easily. I do not use spurs at the beginning of the rein-back, so as to avoid exciting the animal unless he is very sluggish and does not answer readily to the legs. I have just shown the way to proceed at the beginning, when teaching a horse to rein back. But we must not conclude that we should always continue the same effects of the legs separately. Digitized by Microsoft® THE REIN-BACK. That would, in fact, make the horse sway his body from right to left, which would be wrong, because, when he reins back, he should always remain as straight as he does when he goes forward. When the horse understands the movement, and when he can easily take his first step backwards, we should use both reins and both legs. The rein-baclz will then be correct; and if the haunches have a tendency to deviate out of the straight line, we can easily straighten them by pressing a little more strongly with the leg of the side towards which the croup deviates, than with the other leg. Digitized by Microsoft® Digitized by Microsoft® EQUESTRIAN TACT. 123 When I say that I use a certain rein or a certain leg \| mean the leg or rein which ought to have the stronger action. During the entire work both reins should be lightly felt, and both legs ought to be kept close to the side. Continual co-operation ought always to exist between the hands and legs. When I say that I use a certain rein or a certain leg \| mean the leg or rein which ought to have the stronger action. During the entire work both reins should be lightly felt, and both legs ought to be kept close to the side. Continual co-operation ought always to exist between the hands and legs. It is certain that a horse can rein back without the help of the diagonal aids, and that a rider who knows nothing about the principles which I have just enunciated, can make him do so. But he will never succeed in having his horse in hand, with the head high, as if he were going forward, the hind legs being raised as high as the fore ones, and, above all things, with the points of the buttocks not further to the rear than the hocks.* We should thoroughly understand that these conditions are essential in retrograde movements. EQUESTRIAN TACT. Before going further, we may recapitulate as follows the results we have obtained: The horse carries himself very freely forward on the legs being brought close to his sides ; he correctly does the direct and lateral flexions of the jaw; is well in hand ; yields immediately to the action of each leg ; easily executes the respective rotations of the haunches and shoulders; and performs all the changes of direction with facility. It must be well understood that during all the time my horse does these suppling exercises while I am on foot, I had also given him the same work mounted, and that I only seek by general effects to confirm and improve the results obtained on foot. Respecting flexions and collection when mounted, I ought to remark that it is more easy to profit by acquired pro- pulsion than to create it. By this I mean that, if I am at * Tf this condition is not fulfilled the horse will be behind the bit, Digitized by Microsoft® ORDINARY RIDING. 124 the walk, \| have two things to do, namely, first to create unpulsion by the legs; and, second, to make the horse do direct or lateral flexion. At the end of a turn at the trot and canter I am, on the contrary, full of impulsion, and to get the horse in hand \| need only a fingering of the reins Gn direct or lateral flexion), while keeping the legs close to the sides. Under these conditions there is no risk of the horse getting behind his bit, and then he comes most easily to hand.* We now come to the ramener, collection, and rassembler. If the ramener and collection belong to ordinary riding, the rassembler pertains only to scientific equitation. I therefore crave the forbearance of my readers for discussing at present the vassembler, which 1s the last stage of the general effects, of which the ramener at first, and subsequently the collection, are only the beginning. The word ramener, which is borrowed from Baucher, means nothing else than direct flexion. * When the horse will not obey the diagonal effects, it is an excellent lesson for him to finish the trot or canter by a well-marked lateral flexion, with the opposite leg very close and with great impulsion. The direct flexion is made naturally, if the time of halting is correct ; because we ought to stop the horse between the legs and hands (with both legs close) when he is full of forward movement. Digitized by Microsoft® EQUESTRIAN TACT. The vamener is only the first part of getting the horse in hand, and signifies that the horse’s head is high, his head perpendicular, and that he champs and plays with his bit, when the rider feels it by means of the reins; but owing to deficient impulsion he is not light in hand. The effect obtained is localised in the jaw and in the upper part of the neck. It is therefore only partial and leaves the general equilibrium incomplete. It is the first step towards perfect distribution of weight ; collecting the horse is the second, as I have just said; and the vassembler is the last. JI mention the vamener only out of respect for the authority of Baucher, who, working on the horse while standing still, brought him Digitized by Microsoft® EQUESTRIAN TACT. 125 back (vamener) ; but I try to obtain direct flexion only during forward movement, which at once enables me to get the horse in hand. I therefore exclude from my vocabulary the word ramener, which indicates a backward action, and is therefore entirely opposed to my system of riding.* back (vamener) ; but I try to obtain direct flexion only during forward movement, which at once enables me to get the horse in hand. I therefore exclude from my vocabulary the word ramener, which indicates a backward action, and is therefore entirely opposed to my system of riding.* Direct flexion should always be preceded, sustained, and completed by the action of the legs pressing the hind quarters on the forehand. The legs ought to zake and give like the hands, and with the hands, that is to say, simultaneously and in the same pro- portion. This constitutes general movement. If the hands give and the legs continue their action, the horse will be ows of hand, because the propulsion developed by the legs will no longer be received by the hands. If the hands act without the legs sending them any impulsion, the horse will bring his chin into his breast or will get behind his bit; because his hocks have been left too far behind him. The expression “take and give,” as I have explained it when speaking of direct flexion, therefore applies as well to the action of the legs as to that of the hands. Legs and hands should always act in harmony, according to the desired result. * A sluggish horse which does not go up to his bridle, and which answers badly to the action of the legs, would be ramené’d if his neck was bent at the withers according to the system of Baucher. The ramener never conduces to good equilibrium, but on the contrary destroys it, and does not help to get the horse in hand, Digitized by Microsoft® * When the hind legs are well under the body, the croup is low, and con- sequently the forehand is high. + Naturally, the greater part of the force of propulsion is employed to send the horse forward. { People often make the mistake of saying that a horse which ‘‘ cracks nuts ”’ is well in hand. A horse that “cracks nuts,’’ continually snaps with his teeth, whatever may be the position of the neck, but more often when it is high. It is true that this horse is light, but he is not in hand. For a horse to be in hand he should obey the indications of the hands on the reins, which he cannot do unless he lets go the bit. A horse which ‘‘cracks nuts’’ gives himself up to this trick, but he never releases the bit. Nevertheless, the mobility of the jaw caused by this habit is a proof that the horse does not stiffen his jaw, and he is therefore always light. From which we can conclude that the horse which ‘‘cracks nuts”’ is generally well balanced. For all ordinary riding he has a sufficiently delicate mouth, but if we want to do high-school work with him, it is indispensable that he should champ and yield to the bit each time the rider requires him to do so, that is to say, he should be in hand. For that object it is absolutely necessary that he should be made to give up his nut-cracking trick, by progressive and rigidly correct flexions. We will thus succeed little by little in making him release the bit and get into hand. EQUESTRIAN TACT. We get the horse in hand by this combination of the alternate actions of the legs and hands acting on the whole. Getting the horse in hand, which is an excellent term of the old school, is the result of equilibrium during propulsion, obtained and preserved by direct flexion, resulting from the action of the legs impelling the hind quarters on to the fore- hand. Here we are in the best conditions of good horseman- ship. The hind legs, being well under the body, drive it forward and maintain equilibrium by the high position of the ORDINARY RIDING. 126 neck.* The momentum of the mass ends at the bit, namely, at the end of the arm of the lever (of which the flexibility from front to rear increases from rear to front), whence the hand of the rider sends back, in its turn, the amount necessary to maintain equilibrium,} towards the hind quarters, which by a fresh spring again impels all the mass forward ; and so on. This horse is thus truly in hand.£ In my opinion, he ought at the same time to be on the hand. The horse is on the hand when, being in direct flexion, he closes his jaw on the bit from time to time, so as to remain in constant communication with the hand of the rider. \|\| A horse that “cracks nuts,’’ continually snaps with his teeth, whatever may be the position of the neck, but more often when it is high. It is true that this horse is light, but he is not in hand. For a horse to be in hand he should obey the indications of the hands on the reins, which he cannot do unless he lets go the bit. A horse which ‘‘cracks nuts’’ gives himself up to this trick, but he never releases the bit. Nevertheless, the mobility of the jaw caused by this habit is a proof that the horse does not stiffen his jaw, and he is therefore always light. From which we can conclude that the horse which ‘‘cracks nuts”’ is generally well balanced. Digitized by Microsoft® EQUESTRIAN TACT. For all ordinary riding he has a sufficiently delicate mouth, but if we want to do high-school work with him, it is indispensable that he should champ and yield to the bit each time the rider requires him to do so, that is to say, he should be in hand. For that object it is absolutely necessary that he should be made to give up his nut-cracking trick, by progressive and rigidly correct flexions. We will thus succeed little by little in making him release the bit and get into hand. I wish to draw my readers’ particular attention to the following important dis- tinction: When the horse ‘‘ cracks nuts ’’ he retainscommand of his lower jaw ; but when he is in hand, the rider has control over it. The horse which pulls at the hand is not ox the hand; he is beyond it. When a horse which is ov the hand seeks to force it and go beyond it, we should, according to Baucher’s teaching, pull him up, put him into flexion, and set off again. My advice is to press him up to the hand by an energetic use of the legs, at the risk of upsetting him, and I thus succeed in getting him into hand by impulsion, Digitized by Microsoft® EQUESTRIAN TACT, 127 Finally, the horse comes up again on the hand when the impulsion communicated by the legs brings the hocks strongly under the animal’s body and sends him freely on the bit, which is possible only when the horse is in hand to a maximum extent, which form of control is the rassembler. It is necessary that the tension of the reins is light enough to allow the propulsion to pass, but great enough to establish contact between the bit and the hand, and to give us the feeling that as the impulsion comes freely on the hand, we can dispose of it as we like. As the neck in this case is necessarily high, and as the action of the horse is lofty, the meaning of the expression, “the horse comes up to the hand,” is perfectly clear. We can now understand what is meant by the horse being between the hands and legs, both of which send back impul- sion to each other, so as to preserve equilibrium while going forward. * Straight or horizontal equilibrium is the distribution of weight for a hack, and is between that of the race-horse, which is too much on his forehand, and that of the school horse, which is tog much on his haunches, Digitized by Microsoft® EQUESTRIAN TACT. The school horse should be completely enclosed between the hands and legs; and the hack should be in front of the legs and on the hands, so that he can lean a little with the bars of his mouth at fast paces. The horse which does not answer to the legs is behind the legs. He has too much weight on his haunches; in other words, he is behind the bit. Every horse is not capable of being perfectly rassemblé'd, Every horse is not capable of being perfectly rassemblé'd, which is the extreme limit of ezug in hand ; but every horse ought to be trained to get into hand with good equilibrium, no matter what may be the work for which he is required. The hack, hunter, charger, and even the carriage horse, acquires a good position only by being got into hand, in which case the equilibrium is straight or horizontal.* It is generally thought that the object of getting a horse ORDINARY RIDING. 128 in hand is to give him a fine carriage, which no doubt is valuable, but is its smallest advantage. Equilibrium, which is the result of getting the horse in hand, gives mobility, namely, the facility of instantly doing, without effort or fatigue, every required movement at all paces. Also, this equilibrium keeps the horse sound during severe work; because it requires from each part of the horse only those efforts which come naturally to him. We thus avoid wearing the animal out prematurely ; because no special strain is put on any particular organ. If army horses were sufficiently suppled by a first course of breaking, and if the soldiers who ride them understood equestrian equilibrium, and knew how to take advantage of opportunities, our cavalry would gain in appearance, firmness of seat, and staying power. The men would have more confidence in themselves and their horses, and they would be more active, more skilful and more energetic. The horses would stand more work, and they and the government budget would be relieved from needless expense. We must not think that the horse should be always kept in hand. I certainly do not advise that this should be done the whole time one is hacking, hunting, foraging or chargitig. So far from that, I am the resolute enemy of keeping the horse always collected. Digitized by Microsoft® EQUESTRIAN TACT. We should know how to be able to get the horse in hand whenever we wish to do so, and at all paces; but only from time to time, and when occasion demands. It is of the utmost necessity at certain moments of difficulty, as, for instance, when we think the horse is going to play up, and when the animal, from fatigue, softness of constitution, or some other cause, goes in a lumbering, unconnected manner. Getting him into hand restores his balance and is of great use in every respect. As I have already said, all horses can be got into hand, but only some are sufficiently well shaped to be rvassenzdlé'd. EQUESTRIAN TACT. 129 The rassembler, which is the act of getting a horse into hand to a maximum extent, is the complete equilibrium of the animal in all his movements. It is the perfect form of collecting the well-suppled horse. In it the loins, hind- quarters and hocks are flexible; the hocks stoutly press the mass forward ; the shoulders are free and movable; the neck is high and the jaw readily obeys the feeling of the rider’s hands on the reins, and all the parts of the horse being in action and equally enterprising, combine to form an energetic, harmonious and light whole. The equilibrium is so perfect and so unstable, that the rider feels that he can make his horse do whatever he desires by the slightest indication of his wishes. Both of them, so to speak, are in the air. They are ready to fly! How can we succeed in bringing to perfection and in refining the art of getting a horse into hand, so as to obtain this ideal of equilibrium? If we have well understood the action of collecting a horse, and the coming and going of the forces of the legs to the hands, and of the hands to the legs, we will remember that the hands allow the amount of impulsion necessary to the forward progress of the mass to pass, and throw back towards the hind quarters only the amount of impulsion required to preserve balance. This is obtaimed by the deli- cate and constant play of the fingers, which may be compared to the fingering of a piano as regards delicacy and speed. EQUESTRIAN TACT. The question is: what proportion of the force ought the hand allow to pass through it, and what proportion ought it retain? We should measure this proportion with absolute precision at each stride, by the correct combination of the “aids,” so as to send to the hind quarters only the amount of force necessary to maintain equilibrium with a maximum of propulsion. Eguestrian tact consists in doing this. If the fingers do not work with enough decision, the centre of 9 9 9 Digitized by Microsoft® ORDINARY RIDING. 130 gravity will be carried to the front a little too much, and the horse will be ready to go beyond the hand. If they act too strongly, too much weight will be put on the hind quarters, and the hocks will be brought too far back. In both cases there will be no rassembler. The fingering of the reins should regulate with absolute precision the distribution of the propulsion.* We have to solve this problem at each stride, which is not identical to the preceding one or to the following one. Here is the end we have sought. We can succeed by work and perseverance to get the horse in hand in a manner which approaches the vassembler,and even to occasionally obtain the rassembler, but very few riders can keep up the vasseméler by a scientific fingering of the reins.t The propulsion and the effects which the whole sends from the rider to the horse, and from the horse to the rider, are like an elastic ball. The spur, so to speak, goes to seek for this ball in the hind legs of the horse, and makes it come up close to the heels of the rider, whence, passing by the seat, it ascends to the withers, follows the upper part of the neck to the poll, falls into the mouth, where the hands receive it, and, following the lower part of the neck, it returns to its starting point, where it is picked up and sent on again by the legs. Therefore, this ball continually goes round a circuit when the horse is rassemblé'd. To make this comparison perfectly exact, we should say that it is a football which leaves the legs and arrives at the mouth, and a billiard ball which comes back. * Tn order that the rider may properly feel his horse—that is to say, when the tassembler is perfect—the harmony and union between him and his animal should be such that the force of propulsion and the effects of the whole should be transmitted without intzrmission or interruption. The propulsion and the effects which the whole sends from the rider to the horse, and from the horse to the rider, are like an elastic ball. The spur, so to speak, goes to seek for this ball in the hind legs of the horse, and makes it come up close to the heels of the rider, whence, passing by the seat, it ascends to the withers, follows the upper part of the neck to the poll, falls into the mouth, where the hands receive it, and, following the lower part of the neck, it returns to its starting point, where it is picked up and sent on again by the legs. Therefore, this ball continually goes round a circuit when the horse is rassemblé'd. To make this comparison perfectly exact, we should say that it is a football which leaves the legs and arrives at the mouth, and a billiard ball which comes back. * Tn order that the rider may properly feel his horse—that is to say, when the tassembler is perfect—the harmony and union between him and his animal should be such that the force of propulsion and the effects of the whole should be transmitted without intzrmission or interruption. Digitized by Microsoft® EQUESTRIAN TACT. + It is impossible to obtain and preserve a good rassemdler unless the horse has been kept perfectly straight during his course of breaking. If we do not succeed in holding the animal in this straight line, which begins at the poll and finishes at the tail, the horse will escape being vassemélée’d. If any part deviates — haunches, shoulders, or jaw yielding laterally, instead of yielding in a straight line—the result will be spoiled propulsion, and without complete propulsion there can be no vasseméler. Being able to feel that the horse is straight is the first manifestation of eques- trian tact. The moment the slightest deviation is perceived, the legs send the hind-quarters to each other, whilst the effects of the hand, which combine with the effects of the legs, straighten the forehand. At this moment the rider succeeds in getting the egeestrian feeling (equestrian tact) by the more or less fine perception of the successive positions of the forehand and hind quarters, until the animal is perfectly straight. Digitized by Microsoft® Digitized by Microsoft® EQUESTRIAN TACT. 131 Although I have ridden horses for fifty years, I did not obtain perfect vassembler until the last ten years. It is true that I worked for a long time according to the some- what inexact data of Baucher. But the fact is that for many years \| continually felt the vassembler escape me, by the dis- placement of the centre of gravity, whether to the front or to the rear. I had to greatly refine my tact and consequently my “aids” to obtain the complete rassembler, and to pre- serve it with a maximum amount of propulsion. But this is not all. There are not only direct movements, but also those to the side and when turning. In these move- ments one leg always predominates, in which case the impulsion which comes on the bit is not equally distributed between the two hands. The right leg throws more impulsion on the left hand, and vice versd. ? * The difficulty is so great that Baucher acknowledges that the ‘‘ lightness ’ (read rassembler) escaped him in changes of direction. The fault was less his, than that of the bad position of the neck which I have pointed out. * Digitized by Microsoft® 9 Digitized by Microsoft® * High school work naturally demands very complicated efforts which the rider ought to feel on account of the precision which it requires. The most difficult effort to perceive is that which is called “ the magpie jump,” which the horse makes by simultaneously placing his two hind feet on the ground, so as to relieve himself. When the movement is softly made and the pasterns bend, it is difficult to catch. However, if we allow a horse to contract this habit, he will lose all regularity of action, Digitized by Microsoft® EQUESTRIAN TACT. It is therefore necessary, in order to preserve equilibrium in turning to the left, that the left hand, while remaining bound to the right hand, sends back to the centre a larger amount of force, which is all the more difficult to measure ; because this hand, while keeping up the rassembler, has to regulate the change of direction.* If we now reflect that in all work of equitation, the hose, whether going forward or keeping himself back, constantly tries to escape to the right or to the left, by his haunches or shoulders, we will see that in order to maintain perfect equilibrium we have to simultaneously perceive all the actions of the horse and all those which are being prepared, so as to combine them by opposing them, by the simultaneous action of the “aids,” and to produce from them the desired ideal of equilibrium. I said a short time ago that the vassembler in direct move- * 9 Digitized by Microsoft® ORDINARY RIDING. 132 ment is the end of equitation. The continual rassembler, not only in side movements and turning, but in all movements, whatever may be their combinations, is the supreme refine- ment of riding—the full possession of the ideal. Thus the two organisms are so combined that ‘ke man is one with his horse, the former perceives the efforts of the latter so directly and rapidly that each action of the man responds so surely and rapidly to a corresponding action of the horse, that the animal expects it, and lends himself to it instantaneously. Then the horse has only reflex actions. The only brain he has is that of his rider. I am right in saying that it is the ideal of which we dream. ment is the end of equitation. The continual rassembler, not only in side movements and turning, but in all movements, whatever may be their combinations, is the supreme refine- ment of riding—the full possession of the ideal. Thus the two organisms are so combined that ‘ke man is one with his horse, the former perceives the efforts of the latter so directly and rapidly that each action of the man responds so surely and rapidly to a corresponding action of the horse, that the animal expects it, and lends himself to it instantaneously. Then the horse has only reflex actions. EQUESTRIAN TACT. The only brain he has is that of his rider. I am right in saying that it is the ideal of which we dream. How can we obtain this tact, this keenness of perception, this refined and rapid feeling of all the efforts of the horse in every degree, preparing the efforts which are going to follow? This cannot be taught in a book. For these faculties we require practice, work, and, above all things, natural aptitude and love for horses. By his seat and legs the rider ought to feel with absolute certainty everything that goes on under him, as for instance, if the hocks are brought more or less under the centre, or if they remain behind ; what legs are raised, and to what height, and if the croup is going to deviate from the straight line. By his hands and legs, helped by his eyes, the rider ought By his hands and legs, helped by his eyes, the rider ought to be able to feel the actions and above all things the ten- dencies of the jaw, head, neck, and shoulders. As the fore- hand begins the movements desired by the animal, we can say that “the hand ought to feel the ideas of the horse.”* SIDE STEPS AND TWO TRACKS. 133 Thus the rider will have the feeling of the complete equili- brium of the horse, and the free disposal of his forces at any moment. I cannot say more on this subject, except to advise my readers to devote themselves to practice.* * Pictures of a rassembl/’d horse can be seen in photographs in this book. I attach great importance to these photographs, because they are free from all trickery. If we study them with attention, we will see even in the most energetic school work, that my horse maintains straight or horizontal equilibrium. In ordinary school equilibrium, the horse is often too much on his haunches. The great impulsion which I try to obtain always keeps my horse in horizontal equilibrium, however high the action of his fore legs may be. + The great utility of this movement is that it enables us to place our horses where we like, and at all paces, when riding in the open. Digitized by Microsoft® SIDE STEPS AND TWO TRACKS. I was obliged to discuss the rassembler, which belongs to scientific riding, when referring to collecting a horse, which subject leads up to the rassembler. I have even to speak at the same time of the side step and the “two tracks,” because these two movements are closely connected; although the former pertains to ordinary riding,t the latter belongs ex- clusively to high school riding. The first remark to make, is that in side steps—and still more in the “two tracks,” because the speed is faster—the rider ought to freely carry his weight to the side towards which the horse is proceeding. The necessity of this is emphasised by the fact that the side movement of the horse naturally displaces the rider towards the side away from which the animal is going. Consequently, when the side movement is rapid, the rider can be very easily thrown off to the side away from which the movement is made. Therefore the rider ought to lean to the left, on the saddle and stirrup, during side steps from right to left. This posi- tion, which unites the rider to the horse, by giving them the same impulse, has the further advantage of relieving the right + The great utility of this movement is that it enables us to place our horses where we like, and at all paces, when riding in the open. ORDINARY RIDING. 134 shoulder, which has more ground to cover than the left one. We require a certain amount of practice to attain this result ; because, as I have already said, the side movement of the horse naturally puts the rider into the opposite position. I never begin to teach the side steps when the horse is going along the wall, which would uselessly increase the difficulty, by the want of impulse resulting from the change in direc- tion. By removing the horse from the wall, I forcibly stop his forward movement. I make the horse do the first side steps at the end of a change of hand, from right to left for instance. I am on the track, going to the left, with the wall to the right. On arriving near to the wall, which, at the end of the change of hand, will be to my left, I carry both hands to the left, while closing the legs and acting vigorously with the right leg. Digitized by Microsoft® * In other words, I have recourse, as I always do, to lateral equitation, in case of resistance proceeding from the incomplete education of the horse. But at the stage of breaking to which we have arrived, his educa- tion ought to be sufficiently advanced for diagonal equitation to give us all its results. SIDE STEPS AND TWO TRACKS. The near snaffle rein draws to the left, and the off rein applied against the neck presses the shoulders equally to the left. We can see that these are absolutely the same “aids” as those for the rotation of the shoulders, although the move- ment is made while gaining more ground to the front. If the horse resists the right leg, I have recourse to the off rein, to make him carry his haunches to the left.* Even if the animal makes only two or three side steps I am content with his progress, I pat him on the neck, and slacken the reins. After that, I make the horse do a change of hand from left After that, I make the horse do a change of hand from left to right when leaving the wall, which is on my left. While thus going obliquely, I hold him as straight as possible, and when I arrive near to the wall, which will be on my right, I carry my hand to the nght and I apply the left leg, while pressing him up to the hand by means of both legs. I may add that the hand ought to profit by the supplemental! * In other words, I have recourse, as I always do, to lateral equitation, in case of resistance proceeding from the incomplete education of the horse. But at the stage of breaking to which we have arrived, his educa- tion ought to be sufficiently advanced for diagonal equitation to give us all its results. SIDE STEPS AND TWO TRACKS. 135 impulse, to transform the forward movement into a movement from left to right, with the least possible resistance.* I continue this work for a sufficiently long time, and accentuate it according to the progress obtained, by which I wish to say, that at first I am content to make the horse take two or three side paces. Later on, when he gets more expert, I require more from him, always at the moment when we arrive close to the wall, so as to obtain five or six side steps. Further on, I begin the side steps at the middle of the school, so that I can make him do twelve or fifteen steps: I finally make him do the shoulder-in.+ + I do not put the horse’s shoulder to the wall until he has been well trained. * Energetic impulse is the first condition of correctly executed work on two tracks. It results from the firm support of the inward leg, providing that the outward leg has a predominant action. If, during work on two tracks, the inward hind leg of the horse is carried away from the body instead of being brought under its centre—as would be the case if there was sufficient impulsion—the horse will be inclined to get behind his bit ; the cause of this fault being the insufficient energy of the inward leg of the rider, the action of which ought to be supplemented by a corresponding action of the outward leg. Digitized by Microsoft® SIDE STEPS AND TWO TRACKS. By the expression “ to give cadence to the horse,” I mean the school walk. At this pace a horse has great mobility, and the side movement is made more easy, because the fore legs and hind legs can move more readily, without touching each other, which is impossible at the ordinary walk.* Up to this, I have purposely used the expression “side step,” and not “two tracks,” because we always begin by going to one side, which is far from the two tracks. We are never completely master of a horse which we have allowed to contract the habit of always keeping close to the wall, which is a bad preparation for riding in the open, and also for high school work, the first condition of which is that the horse should be always betwee the legs of the rider, or, to speak more precisely, should be solely guarded by the ‘‘aids.”’ It is therefore well to work the horse at a distance of one or two yards from the wall. When the horse has got into the habit of pressing his shoulder into the wall, how are we to put him straight ? And if we wish to take him away from the wall. to go down the centre, to do a demi-volte, or to set off on ‘‘two tracks,”’ what ‘‘aids ’’ should we use ? Let us suppose that the rider is on the right hand. He will then instinctively feel the off snaffle rein to bring the left shoulder away from the wall, which is a mistake, because in pulling the off rein he will bring the head and neck to the right, but the more he draws them to the right, the more will the lateral flexion of the neck push the left shoulder to the left. The proper way to bring the left shoulder away from the wall is to draw the near snaffle rein rather high and forward on the neck, then carry it to the right, while lightly feeling the off rein, and we will then bring the whole neck along with the shoulders to the right. * To make side steps from left to right, the horse should pass his near fore and the near hind, one after the other, over their respective off legs, in order to gain ground to the right. SIDE STEPS AND TWO TRACKS. eA horse has only too great a tendency to allow himself to be guided by the wall, instead of giving himself up exclusively to the ‘‘aids.”’ I greatly dislike con- tinual work close to the wall. If the horse is straight and limits himself to following the wall, whatever the pace may be, he will let himself be guided by this permanent obstacle, much more than by the rider’s ‘‘aids.’”? He will even take a kind of moral support from it, and his natural tendency will always be to slightly carry away his croup, and to get the shoulder close to the wall ; hence the difficulty of holding him straight, and of keeping him well between the legs. In movements on two tracks, the wall leads the horse so well and helps him so much in keeping his shoulders in the proper direction, that he quickly begins to shave the wall too closely, which often becomes a ‘‘ defence ’’ on his part. If we remove the horse from the wall, we will be obliged to direct the shoulders with the hand, which ought to be the only guide. If the horse, when doing the shoulder-in, gets behind the bit, and brings his hind quarters too close to the wall, we should stop the movement on two tracks without changing the position of the horse, should push him forward with the legs, and make him go down the centre of the school. No exercise is better than this to give impulse in the movement on two tracks, and to make the horse independent of the wall. In movements on two tracks, the wall leads the horse so well and helps him so much in keeping his shoulders in the proper direction, that he quickly begins to shave the wall too closely, which often becomes a ‘‘ defence ’’ on his part. Digitized by Microsoft® Digitized by Microsoft® 136 ORDINARY RIDING. The number of side steps which we succeed in obtaining is of little consequence. The essential thing is to see that the horse is well placed—the shoulders being always more advanced than the croup—which is the only position that facilitates the side movement. Position is everything. At the beginning of the work I try to give cadence to the horse, but only at the moment I am going to get him to do side steps. Digitized by Microsoft® * A horse which his rider persists in working only by means of lateral effects can never become a good school horse: he is ungraceful, and his work is disunited. In fact, in lateral equitation, he carries his head and neck to the opposite side to which he is moving ; and further it is impossible for him to do the rassembley, because when we work with the two “ aids’”’ of cne side we have nothing to support the other side, which consequently escapes us. We should therefore continually work both reins and both legs ; but it is principally the double action of the opposite rein and leg which should SIDE STEPS AND TWO TRACKS. When the horse is at the ordinary walk, the pace is too slow and too low for the legs to pass over their fellows without touching them. In the side step, at the school walk, each of the near legs passes successively its corresponding off leg, and (this is the important point and results solely from the cadence) is put on the ground only at the moment when the off leg is just raised. They therefore cannot hit each other. Digitized by Microsoft® SIDE STEPS AND TWO ERACKS. 137 When a horse does the work I have just described, it is said that he goes stdeways, no matter how bad may be the position of his head and neck. But for this work to merit being called “work on two tracks,” the position should be correct. Yet the position is correct, and a horse moves truly on zwo tracks when he goes obliquely forward on two parallel lines, the one made by the forehand, the other by the hind quarters. He thus advances from the side, w7th the head and forehand always leading the hind quarters. The head and neck ought to be kept high and slightly bent to the side towards which the horse is going. Above all things, he should be well in hand, light, and in a cadenced pace. In my opinion, this is the longest and most difficult work. If we seek to obtain it complete and correct from the beginning, we will either get no good result, or we will cause the horse to at once resist, because he had not passed through the “mill” of lateral, direct and diagonal efforts which I have described.* My reason for laying considerable stress on the subject of “two tracks,” 1s that this work has a great influence on all the subsequent breaking, where we always meet during pro- pulsion the same combined action of the diagonal “ aids.” When the horse knows how to go on two tracks, he will seek every means to escape from the rassembler. At frst, he refuses to yield to the direct leg; he then yields too much and forces the opposite leg. He escapes by going too quickly zo one side. * Every horse which knows how to go on ‘‘two tracks’? makes an abuse of it. When horses are accustomed to do demi-voltes and changes of hand on ‘‘two tracks,”’ they often put themselves sideways when their rider wishes them to do these movements while holding them straight, and by this defence they escape being rassembld. By holding the horse straight, we make him bring his hocks under his body, which is the very thing he tries to avoid doing by placing himself sideways. The remedy is in the legs of the rider. Later on, when the breaking nas been finished—the horse being kept at the vassezb/er in all the school move- ments—he will not be able to obtain help from this defence. Digitized by Microsoft® * The delicacy of the diagonal effect combines admirably with the energy of the legs, because it results from the preponderance of one ‘‘ aid” over the other. Besides, the true action of the ‘‘aids’’ is: legs energetic, heels delicate, and hands light. SIDE STEPS AND TWO TRACKS. We should therefore continually work both reins and both legs ; but it is principally the double action of the opposite rein and leg which should play the chief part in all movements. Digitized by Microsoft® 138 ORDINARY RIDING. I will suppose that the rider is placed with his shoulder to the wall and is going to the right. The horse, which tries to checkmate all the efforts the rider makes to keep him in hand and in good position, begins for instance, to lean against the rider’s left leg (outward leg or direct leg). The spur pricks him and forces him to give way. He then tries to get behind his bit. In order to drive him forward, it is necessary to employ the right leg (outward leg or opposite Zeg). As we hardly ever use the spur of the opposite leg, from fear of straightening him, the horse will finish by press- ing against this leg and by saving himself by running to one side, which will prevent us getting him in hand and regulating his work on “two tracks.” The remedy is simple. It is sufficient, when the horse throws himself on the opposite leg, to attack him vigorously with the spur of that side, in order to make him straighten himself. We must adopt this plan each time the horse throws himself to one side, which he will soon give up doing. As we may see, the best system is to throw into disorder, in order to establish order. This procedure, which is criticised by all those who have not the audacity to practise it, is the only one which incontestably establishes the power of the breaker over the animal. By it, the horse learns that there are insurmountable obstacles to the carrying out of his wishes. That point being settled, if we keep him balancing between reward and punishment, he is ours.* Digitized by Microsoft® SIDE STEPS AND TWO TRACKS. SIDE STEPS AND TWO TRACKS. 139 According as we quicken the pace at the work on “two tracks,” in order to pass from the school walk to the ordinary trot, or even to the fast trot, it becomes more and more difficult to employ the “aids” in an appropriate manner. We should, in fact, hold the horse very straight in the hand, because every effort to straighten the hind quarters, or to support the forehand, diminishes the propulsion, which ought to be extremely energetic. To obtain a maximum of impulse at the quickest pace, we must keep the forehand and hind quarters absolutely on their own respective tracks, and must combine the energy of our legs, which give the impulse, with the delicacy of the constant action of the diagonal effects,* which enable us to keep the horse in position, without diminishing the impulse in any way. This movement is perhaps the most inconvenient one, on account of the extreme difficulty of correctly combining the effects of the “aids” with great propulsion. Not the least difficulty is to hold the horse always straight. The direction of the impulse, even in work on two tracks, will always pass between the two ears, if the head is well placed. The correct position during work on two tracks at a fast trot is very difficult to keep. In fact, the energy of the pace increases the natural disposition of the rider to carry his body to the side opposite to that to which he is going. Hence the necessity of continual watchfulness on his part. The great difficulty as regards the horse is to make him quicken the trot without breaking into the canter. This is the touchstone of impulse, and the proof that the animal answers freely to the “aids.” Work on two tracks, as I do it, in no way resembles the Digitized by Microsoft® ORDINARY RIDING. 140 sleepy work on two tracks which we generally see in nding schools. I pay particular attention to demanding energetic work, and I keep my horse full of enterprise, which is the exact opposite of what is usually done. If my mount is enterprising, he becomes so on account of my legs having been enterprising in the first instance. SIDE STEPS AND TWO TRACKS. In sleepy work the horse obeys conditionally ; in energetic work he obeys without conditions ; he keeps nothing in re- serve and he gives himself up to his rider, which is the first condition of equitation. Figs. 41 and 42 show Germinal (a thorough-bred by Flavio out of Pascale) at work on “ two tracks” at the school walk. In Fig. 41, the horse begins by putting himself into posi- tion, at which he is perfect in Fig. 42. We can see how far the correct position is from the crossed position, which many riding masters adopt in work on “two tracks”; the result being that they stop all impulse. The impulse appears greater in Fig. 42 than in Fig. 41, because the horse, which is going on two tracks from left to right, was photographed at the moment when he rested his right hind foot on the ground, the right being the side to which he was going. In Fig. 41 (two tracks from right to left), the near fore foot, which forms the support, belongs to the side (the left) to which the horse is going. A comparison of these two illustrations enables us to thoroughly understand the movements of the horse's legs during work on two tracks. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® THE TROT. To make a horse trot, we must begin by slackening the reins, and increasing the pressure of the legs a little. We ought to avoid striking him with our heels, which might upset him. But if he is sluggish, we may at first make him feel the heels, and afterwards the spurs, but only after a pressure Digitized by Microsoft® Fig. 41.—On ‘‘ two tracks ”’ at the school walk, from right to left. Fig. 41.—On ‘‘ two tracks ”’ at the school walk, from right to left. Fig. 41.—On ‘‘ two tracks ”’ at the school walk, from right to left. Fi 41.—On ‘‘ two tracks ”’ at the school walk, from right to left. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Fig. 42.—On “two tracks ”’ at the school walk, from left to right. Fig. 42.—On “two tracks ”’ at the school walk, from left to right. Fig 42.—On “two tracks ”’ at the school walk, from left to right. walk, from left to right. d by Microsoft® ize ti igi Digitized by Microsoft® Digitized by Microsoft® THE TROT. 145 of the legs. At first we must limit ourselves to the short trot, and, above all things, we should make the animal do it in a correct and cadenced manner, that is to say, the foot-falls should be equal as regards time. As the horse is supple, and has been trained to obey the “ aids,” he will soon do what we require. At first it is well to allow the horse to be as free as possible, in order that we may see if he trots level naturally. If at first we keep him in hand when trotting, he will not go freely, and if there is an inequality in his gait, it will be difficult to judge whether it is due to a fault of conformation, unsound- ness, or bad horsemanship, namely, erroneous effects produced by the “aids.” The reins, especially those of the curb, ought to be only very slightly felt. If the horse carries his head too high, we should use the curb reins. The fact of the head being carried too high and in a backward direction, crushes the hind quarters. If, on the contrary, the head is too low, we should use the snaffle reins; but should not draw them to the rear, because that would stop the propulsion. * This naturally increases the height of his action. THE TROT. We should give light quick pulls to these reins alternately, with- out jerking them, while taking care always to keep the hands high. We had best trot the horse only twice round the school, stop him, bring him into hand, and do the same things over again several times. When we have got the horse into a good position at the trot, with his neck high,* and the line of his face nearly per- pendicular, but a little farther advanced than the vertical line, we can keep up this pace for a longer time. We should gradually work up to this, because the more tired a horse becomes, the lower will he carry his head. If we prolong the work, he will become heavy in hand, in which case we should stop him by strongly closing the legs, and, having got him completely in hand, should set off again. * This naturally increases the height of his action. Digitized by Microsoft® 46 ORDINARY ORDINARY RIDING. 146 When we have succeeded in trotting for five minutes to each hand without fatigue, and, above all things, without the horse “breaking,” we can make him go faster. We shculd, however, avoid abruptly passing from the short cadenced trot to the fast long trot, which would upset his balance, and would unexpectedly throw a considerable weight on his shoulders. At first we should increase the speed of the pace only towards the end of a turn at the trot. During the last couple of turns round the school, for example, we may act vigorously with the legs, while taking only a slight support on the snaffle reins, the action of which should be limited simply to keeping the head in place. By practising these exercises we shall succeed in making the horse do all he can at the trot, as regards high action and speed. Above all things, we ought to avoid trying to make the horse trot faster than he can do; for such an attempt will probably teach him the false and ugly gait of trotting in front and cantering behind. There are two ways of riding at the trot, namely, the French (bumping in the saddle) and English (rising in the saddle). Digitized by Microsoft® THE TROT. The former is of no practical use, although it 1s an absolutely indispensable school exercise,* for giving a good seat to beginners when they trot without stirrups; but I disapprove of it for all other purposes. It is fatiguing to the rider, and still more to the horse. I cannot understand why it has been used for such a long time in the army. When we rise at the trot there are neither jerks nor re- When we rise at the trot there are neither jerks nor re- actions. The rider should have his loins slightly bent, and consequently the upper part of his body should be inclined a little forward. He should not try by rising to follow or to anticipate the movements of the horse, but should let himself be raised. His ankle joints and knees acting together will * It is the foundation ofall good riding. Without it there would be no seat. Digitized by Microsoft® THE TROT. 147 sustain his movement, and will make him descend softly into the saddle, and into the cadence marked by the pace of the horse. He should always rise from under himself, that is to say, he should let the horse raise him, while helping the movement with the knees and ankle joints; but the upper part of the body should do nothing. Otherwise, the muscles of the loins and shoulders will be contracted, the rider will become stiff, and will not be firmly united to his horse. The body ought to rise and fall as a whole. sustain his movement, and will make him descend softly into the saddle, and into the cadence marked by the pace of the horse. He should always rise from under himself, that is to say, he should let the horse raise him, while helping the movement with the knees and ankle joints; but the upper part of the body should do nothing. Otherwise, the muscles of the loins and shoulders will be contracted, the rider will become stiff, and will not be firmly united to his horse. The body ought to rise and fall as a whole. The rider who hollows out his back, in place of using only his legs, necessarily carries his abdomen forward when he rises, and backward when he descends into the saddle, than which nothing can be more ungraceful. * In equestrian language, the diagonal always takes its name from front to rear. Thus, the right rein and left leg is the right diagonal, and the left rein and right leg is the left diagonal. It is the same with the legs of the horse, the right fore and left hind forming the right diagonal, and the left fore and right hind the left diagonal. Io* THE TROT. Only one-third (the ball) of the foot should be placed in the stirrup. If the foot is “home,” the ankle will lose all its elasticity, and consequently the trot will become stiff and painful. The natural trot of a horse which is not upset or suffering, is an alternate and absolutely identical movement of the two diagonals. At the rising trot, the rider can trot either on the left or right diagonal biped.* The rider is said to trot on the left diagonal biped, when he rises at the same moment that the horse raises his left fore foot, and comes down on the saddle, when the horse puts that foot on the ground. In the well-executed English trot, the rider rises and comes down only once during the succession of the two bipeds. He rises and descends along with the left biped, for instance, without the right biped having any influence on his move- Io* Digitized by Microsoft® ORDINARY RIDING. 148 ments. But if he is not in rhythm, he will come down too soon on the saddle, and will receive a shock from the right biped, as the result of the straightening of the left hock. He will come twice in the saddle and will ride incorrectly. The rider ought to be able to ride at the trot equally well on one biped, as on the other biped, and should be able to change from one to the other, so as to relieve himself, and especially the horse, during a long journey, but this requires a certain amount of practice to do. A rider ought to learn how to know on what biped he is, which is difficult at first. It is best to begin a preliminary study at the walk, while raising ourselves in the stirrups at each step the horse takes, as if he were trotting. We have thus time to see what movement of the horse we are following. After a little practice at this exercise, we can start again into the trot. It is worthy of note that each rider naturally adopts one particular biped, and almost always without knowing that he does so, and he becomes so habituated to it that he feels ill at ease when he changes to the other biped. Digitized by Microsoft® THE TROT. If we wish to have a fine trotter, we should complete his education in the open, after having made him do in the school the exercises I have just described. On a road a horse lends himself more readily to the work, and goes freer than in a school. As we have space in front of us in the open, we are able to keep up the speed of the pace for a longer time ; but in a school the corners oblige us to slacken a little at frequent intervals. All horses do not trot in an equally free manner. Certain animals readily maintain this pace, if the speed is moderate ; but if the speed is increased, they will break into a short canter. It is correctly said that such horses keep themselves back. It is very important not to allow a horse to change his pace without giving him the indications to do so, but it is not less Digitized by Microsoft® THE TROT. 149 important to make the horse, whenever we like, exert himself to the utmost at his trot. important to make the horse, whenever we like, exert himself to the utmost at his trot. If a horse starts into the canter when we want him to do the fast trot, we can at first try gentleness in order to correct this habit, which is only idleness. We can stop him and pat him on the neck, to reassure and calm him, and then put him again into the trot. This plan generally succeeds with impetuous horses, but it has no good effect on lazy ones, with which we should do just the contrary. When an animal breaks into the canter to avoid the fast trot, we should vigorously push him forward into the gallop, and should keep him at it for a certain distance, say for 500 or 600 yards, which is to be the punishment of his resistance and laziness. After a few experiences of this kind he will perceive that, so far from obtaining relief by breaking into the canter from the trot, he lets himself in for a severe and trying pace. This plan is within the reach of everyone. There is another which I can recommend, but which requires a greater knowledge of riding. * Tt will be noted that I am doing lateral equitation here ; my reason being that I take for granted that the horse is either imperfectly broken or unbroken. Digitized by Microsoft® THE TROT. When the horse of his own accord breaks into the canter, in order to escape from the fast trot which his rider wants him to do, he naturally leads with the easier leg of the two. I have already said that every horse has one side more easy than the other. We can therefore thwart him in the pace he has taken by pressing him forward at the canter, while making the other leg lead ; for instance, with the right leg if he has of his own accord struck off with the left leg. We will then employ the near snaffle rein, in order to keep back the left shoulder, which takes the lead, and the left leg to press the haunches to the right.* We should apply the opposite “aids” if the horse leads with the right fore leg. It is self-evident that this plan, like the Digitized by Microsoft® ORDINARY RIDING. 150 preceding one, is applicable only to horses which are im- perfectly broken. When a horse is well broken, he never breaks into a pace which we do not want him to adopt. If a horse does not readily take to the trot, we should not be too ready to think he is lazy or bad-tempered. The fault is often due to the fact that the rider’s hands are bad for a sensitive mouth, because they are either too heavy or they move about too much. It may happen that the bit is too severe,* or that the horse’s mouth is sore, from having been bruised. Also, the horse may be suffering in his loins or other parts, and his reason for changing the pace may be only to relieve himself. In all these frequently occurring cases, the best remedy is to remove the cause. In place of first putting the blame on the horse, which is only natural, the rider ought perhaps begin by trying to find out if he himself is not the culprit. The following is an excellent means of finding out if soreness of the mouth is the cause of the horse not trotting true. Instead of allowing him to bear on the snaffle, leave its reins perfectly loose and catch hold of a good-sized handful of the mane near the middle of the neck, and draw it towards you. Horse dealers use this plan every day. * Thin mouth-piece, long cheeks, high port, each one of which conditions will of itself make the bit severe; and combined, they will convert it into an instrument of torture. Digitized by Microsoft® THE TROT. The trainers of trotters, whose only object is a maximum The trainers of trotters, whose only object is a maximum of speed, generally let their horses take a very strong bear- ing on the hand. The regularity of the pace and lightness of mouth are of little matter to them. Their horses, being im- petuous, always pull very hard, and the rider, on his part, pulls no less vigorously on the mouth ; because he thinks that the more he pulls, the faster will the horse go. This is a great error; because, by pulling too strongly on the mouth, we throw the weight of the body on the hind quarters, and we Digitized by Microsoft® THE TROT. 151 thus fatigue the loins and hocks. To make a horse trot well, we should simply try to get him to lean on the hand. No doubt, in a flat race, as at the trot, the rider to some extent carries the head and neck of his horse; but he should take care to carry it wpwards and not backwards, by doing which he would infallibly check the power of the loins and hocks. thus fatigue the loins and hocks. To make a horse trot well, we should simply try to get him to lean on the hand. No doubt, in a flat race, as at the trot, the rider to some extent carries the head and neck of his horse; but he should take care to carry it wpwards and not backwards, by doing which he would infallibly check the power of the loins and hocks. To study the exact conditions of trotting races, I once went and lived at Dozulé, which is a small village in Normandy. I had previously ridden on the flat and across country, and as 1 had also broken three or four school horses, I thought I was going to astonish the Norman lads by my science. I admit, in all humility, that the most surprised one was your humble servant. At Dozulé there was a brave boy called Pascal, who was very well up in everything about horses, and especially about trotters. I had known him for a long time, and we had previously arranged together the plan of my sojourn at Dozulé. Pascal had charge of the training of two remarkable trotters, which belonged, if I remember rightly, to the Marquis of Croix. THE TROT. This was about the end of 1864. On the morning after my arrival, we were in the saddle, and at first we walked on the trotting track, which was a mile and a quarter in length. Then we began to trot, and Pascal beat me easily. I took my defeat with a smile, because I thought that his horse was faster than mine. On the follow- ing day we changed horses, and Pascal again beat me. I admit that I was all the more annoyed, because the same result was reproduced the following fifteen days, on each one of which Pascal rode the horse on which I was beaten by him the previous day. He pulled with all his might at the reins and jerked them, which he called “ ringing” his horse. I told him that his way of riding was not rational ; but he replied that it was the only Digitized by Microsoft® ORDINARY RIDING. i152 way to get all the speed out of a horse, and apparently he was right. I then asked him to let me ride the same horse for a fortnight, during which time we ran no races together. During this period, I succeeded in making the horse take only a light bearing on the hand, and to hold his head steady, and finally I beat Pascal four times running. I afterwards obtained, in an equally short time, the same result with the other horse. The two horses having been placed with confidence on the hand, and having the free play of their hind quarters, trotted as a whole without breaking and almost without fatigue. According to the system of Pascal, on the contrary, they broke, trotted almost always disunited, and also were soon knocked up. I was also closer to ‘he horse than he was; he made violent movements, while I made none; and finally, having succeeded more rapidly than he could in getting my horse at full speed, I was able to maintain it longer. I do not claim that I gave more speed to my horses, but with my handling, they did their work with confidence and ease, whilst he hurt their mouths and fatigued them, which fact was to be seen at the winning post. Not having to make the same efforts when I rode them, they were neither blow- ing nor sweating after the trot. Digitized by Microsoft® THE TROT. Trotters which are trained according to Pascal’s method, are generally unpleasant and sometimes dangerous to ride. In fact it is difficult, when they have once started, to stop them quickly. Nevertheless, if taken out of training and given suppling exercises, they will very often do excellent Service. Pascal had the great advantage over me of knowing his horses, and he knew—at least, I like to think he did—when they were at their top speed. This knowledge is much more important than is generally thought. If a jockey who has not a knowledge of pace, is riding a trotter which is going at Digitized by Microsoft® THE CANTER. 153 full speed, he will probably try to make him go faster, and by pressing him on, will make him break into a gallop. full speed, he will probably try to make him go faster, and by pressing him on, will make him break into a gallop. The feeling which makes us press on a horse under ail conditions is very natural. Nothing unnerves us more than to ride a race alongside a horse which keeps a neck in front of us. Nevertheless, if the trotter we are riding is at the top of his speed, we should take care not to force him beyond it, which would cause him to gallop. In order to put him back into the trot, we would have to slacken speed, and would consequently lose several lengths. Finally, when we ride a trotting race, we should know the moment the horse has attained his highest speed, which we should keep up as long as possible. The result of the race will then become a question of staying power; because the animal which can keep up his highest speed for the longest time, will have the best chance. We should note that in a trotting race a rider should keep his mount at the top of its speed from the start. We will see further on that this is not the case in dat-race riding. THE CANTER. Of all paces, the canter is the most difficult and complicated. Tew riding masters succeed in making a horse do the move- ments they require at the canter. I never begin the canter before getting the horse thoroughly under control, by which I mean that he should be physically, and, if I may say, morally obedient to me in all his paces; that he should be supple, well in hand, and should readily yield to the legs. I expect, above all things, the loins, haunches and hocks to be perfectly supple, and the horse to obey the effects of the whole without hesitation, in order that I may be able to utilize the resulting forces as I wish. Digitized by Microsoft® Digitized by Microsoft® 54, ORDINARY R 54, ORDINARY ORDINARY RIDING. 154, Having obtained these conditions, I am certain to presently succeed in placing my horse in a proper position for the canter, and to immediately get the canter which I wish the horse to do, and not the one he desires to perform. If the horse obeys the legs, I shall be able to prevent him from going szdeways, which is an extremely bad habit, and which is better to prevent than to punish. It is much more difficult to straighten a horse which is accustomed to throw his hind quarters to the right or left, than to teach him to canter straight from the beginning.* At the canter, the rider ought to keep his body straight. If he leans too much forward, each stride of the canter will throw him on to the animal’s neck. If he is too far back, the loins will become hollow, and the body will consequently become stiff. The horse canters either on the near fore or off fore. He is said to lead with the off leg, when the right legs are in * In the school, when a pupil wishes to make his horse strike off into the canter with the outside leg, he generally puts him too much sideways. _I take for granted that he is going to the left, and wishes his horse to strike off with the off fore. He will then carry his hands to the left, which will be all right, if he does it only just enough to put the weight on the left shoulder. Digitized by Microsoft® THE CANTER. But if he does not succeed, he will probably carry his hands more and more to the left and he will turn his horse into the position of shoulder-in. The horse at this period of breaking does not know how to do the shoulder-in at a canter ; because his teacher should have begun by making him canter straight on the inward and outward legs before doing that. The pupil thus succeeds only in destroying all impulse, and in making the canter impossible, by causing the horse to get behind his bit. He should, therefore, carry the hands to the left only just enough to put the weight on the left shoulder, but not sufficient to place the animal sideways. If the horse puts himself in an oblique position, he should not insist on making him canter, but should straighten him, and should begin starting him off again, while holding him straight. Starting well is not everything. We should keep the horse straight. When the horse canters with the outward leg leading, the rider, at startin When the horse canters with the outward leg leading, the rider, at starting, and also to keep up the pace, has always a great tendency to place the horse sideways by bringing the shoulders in, and by placing the horse on two tracks. If we persist in this fault, all progress will be impossible ; because the horse can never canter correctly on two tracks, if he has not learned at first to canter straight on the outward leg. Digitized by Microsoft® Digitized by Microsoft® THE CANTER. 156 advance at the moment they come on the ground. In the canter to the left, the left legs do this. advance at the moment they come on the ground. In the canter to the left, the left legs do this. To teach a horse to canter on the off fore, we ought to close both legs, carry both hands to the left, and feel the off reins a little stronger than the near reins.* In this way we put the weight on the near shoulder, without bringing to the left the head, which should always remain straight, with the end of the muzzle slightly inclined to the right. As soon as a horse has learned to keep in a good position, he should be put into movement. t By means of the left leg, the rider throws, so to speak, the weight of the horse’s body on his right leg, which sends it to the hands. * I always begin by using the snaffle, whatever may be the pace I am teaching. Digitized by Microsoft® THE CANTER. The hind quarters, which act the part of a propeller, ought to push and carry the fore hand forward. To bring the hocks under the body, I increase the effect of the legs, while pressing equally with them. Finally, I use a_ stronger pressure with my left leg, and I carry it a little further back than my right one.t+ If the horse has some “go” in him, the effect of the legs will be sufficient to send him up to the hands (bridle). At this moment, I raise my hands while feeling the reins, and I profit by the impulse given by the legs, to raise the horse, while preventing him extending himself and breaking into a trot. If the horse is sluggish, we should prolong and increase the action of the legs, and if need be, we should use the spurs. Under these conditions, it is difficult for the horse not to lead off with the off fore. It may however happen that he leads off with the near fore, or trots. In either case, I stop him as quickly as possible, and I replace him in the position I have previously described. I then begin again, and continue Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 156 to do so, until he leads with the off fore.* As soon as he has done three or four strides on this leg, I stop him, pat him on the neck and let him walk round the school in perfect freedom. After that I do the same exercise three or four times.t to do so, until he leads with the off fore.* As soon as he has done three or four strides on this leg, I stop him, pat him on the neck and let him walk round the school in perfect freedom. After that I do the same exercise three or four times.t Baucher, by a singular error, recommended lateral equitation for starting at the canter. In his edition of 1846, page 219, he advised for starting with the off fore leading, to carry the hands to the left—which would put tension on the right rein —and to apply the right leg. I attribute this error to the bad flexion of the neck which he practised. * If the horse starts disunited, that is to say, if the forehand canters to the right, and the hind quarters to the left, we should increase the action of the “aids ’’ to obtain the canter which we require, and if the hind quarters in this case are disunited, we should use the left leg energetically. I advise beginners who find some difficulty in understanding the canter, to exercise prudence, for there is nothing worse than to try to remedy a fault which does not exist. At first, lean forward a little, so as to see with which leg the horse canters. If the right shoulder is in advance when the fore feet come down, we may be certain that he is leading with his off fore. We should now feel our seat, and the regularity and suppleness (ease) of the displacement of the body will tell us if the canter is well united, in which case the horse will be leading with the off hind.as well as with the off fore. But if the displacement of the seat is done in a jolting manner and with a double shock, the canter will be dis- united, and the near hind will be leading. We can then, but only if we are absolutely certain, use the left spur to drive Digitized by Microsoft® * If the horse starts disunited, that is to say, if the forehand canters to the right, and the hind quarters to the left, we should increase the action of the “aids ’’ to obtain the canter which we require, and if the hind quarters in this case are disunited, we should use the left leg energetically. THE CANTER. The neck being low and bent at the withers to the right, causes the left shoulder to be rounded, and the effect of the left leg would bend the two ends of the horse towards the inside, that is to say, to bring his head and croup towards each other. The effect of the right leg, on the contrary, is to partly remedy a bad equi- librium resulting from the faulty flexion of the neck. How could Baucher reconcile this practice with his great principle— in turning—of “ applying the leg of the side opposite to which the turn is made” (Edition of 1846, page 189)? We can then, but only if we are absolutely certain, use the left spur to drive the hind quarters to the right, and keep the left leg close to the animal’s side, in order to force him to remain united. t I have already said that we should always finish with the new work, so as to impress it on the memory of the horse. Digitized by Microsoft® THE CANTER. 157 According to this system, it is necessary when turning to the right at the canter to use a stronger pressure of the right leg, in order for the horse to lead with his off fore; and the left leg, in order to turn to the right. The inconsistency is so evident, that it is sufficient to merely point it out. Everyone knows that if a horse has learned to canter with the near fore leading, by means of the support of the left leg, the effect will be to make him change his leg, and not to turn to the right. I think it is useless to describe the means by which we make a horse lead with his near fore, because they are naturally the opposite ones to those which we have just described. We ought always to begin the canter with the off fore * leading, and for this object we ought to make the horse circle to the right in the riding school. I never ask the horse to lead with the near fore until I can readily make him canter freely with the off fore leading. This is a matter of a few days, the number of which we cannot exactly fix. * The rule in riding is to begin all movements to the right. On « straight line in the open we generally canter with the off fore leading, which is the style adopted by ladies. For all reasons it is best to begin the canter in this way. Digitized by Microsoft® * All Arab horses are left-footed, with respect to the fact that they always turn to the left and never to the right, which peculiarity is simply the result of education. If we place ourselves on the right of an Arab horseman, he cannot touch us. He must turn to the left-about in order to reach us. t Almost all authors advise that when a horse pulls at the canter, he should be stopped and made to rein back, than which nothing is less rational. A horse pulls because his hocks are far from the centre of the body. In making him rein back we push the hocks still further to the rear, and we directly counteract the object we desire to attain. On the contrary, without stopping the horse, we should press him well up to the bridle with the legs, should keep him in hand while he is moving forward, should make him do movements as a whole, and take and give with the hands and legs in order to collect him, which is the only means for Digitized by Microsoft® * All Arab horses are left-footed, with respect to the fact that they always turn to the left and never to the right, which peculiarity is simply the result of education. If we place ourselves on the right of an Arab horseman, he cannot touch us. He must turn to the left-about in order to reach us. THE CANTER. Some horses easily lead with the off fore, but others find this difficult to do, although they may be very clever at starting with the near fore leading. Horses are like men—some are naturally and by habit right-handed, and others are left-handed. It seems probable that some horses are naturally right-footed; because, if we did not take account of contracted habits, all horses would be left-footed. In fact, when we lead a horse by the bridle on foot, we always keep on his left side, which is also the side on which we feed, saddle, and bridle him. As the horse likes to take notice of what goes on around him, the left is the side towards which he constantly turns his head, and consequently he ought to be more supple to the left than to the right; but he ORDINARY RIDING. 158 is not so. I have met as many right-footed horses as left- footed ones. We can find out which he is by working him ; for there is nothing in his conformation that would enable us to decide that question.* As a right-footed horse will remain all his life much more supple and easy to the right; the left side will naturally be the one to which we should work him in flexions, side steps, voltes, canter, etc.; and wzce versa. Before requiring the horse to do more, I wait till he can canter as readily with one leg as with the other, while keeping him straight all the time. I then try to get him completely in hand, which I certainly will not be able to do at the canter, if I have not first made his mouth supple and light at all paces and during all the preceding exercises. The best definition of the action of the “aids” in the present case, is that the hands make the walk and the legs the canter—namely, that whilst the legs press the horse with energy, the hands lightly restrain him. He then finds him- self controlled by two opposing actions; propulsion being the dominating one. They oblige him to raise himself and canter. THE CANTER. The riding master will find that he has attained his object when the horse canters slowly without effort, and, above all things, without trying to escape from the hand.t t Almost all authors advise that when a horse pulls at the canter, he should be stopped and made to rein back, than which nothing is less rational. A horse pulls because his hocks are far from the centre of the body. In making him rein back we push the hocks still further to the rear, and we directly counteract the object we desire to attain. On the contrary, without stopping the horse, we should press him well up to the bridle with the legs, should keep him in hand while he is moving forward, should make him do movements as a whole, and take and give with the hands and legs in order to collect him, which is the only means for THE CANTER. 159 There are three ways of seeing that a horse canters with the off fore leading: First, it is sufficient to look at his shoulders or feet. If he canters with the off fore leading, the off shoulder, and First, it is sufficient to look at his shoulders or feet. If he canters with the off fore leading, the off shoulder, and especially the off fore foot, will respectively be in advance of the near shoulder and near fore foot. Second, the horse has always his croup a little turned to the side of the leading fore leg. Third, when the off fore leads, the right leg of the rider is more shaken than the left leg,* and consequently it is more difficult to keep that knee close to the flap of the saddle than the other knee. After a series of attentive observations, we ought to be abie to tell with what leg a horse leads, even with our eyes shut. Before going further in the teaching of breaking, it is important to explain the movements of the canter.t The canter of the school horse and hack ought to be in well-marked three time.t Three times (beats) are, therefore, necessary to form a stride of the canter. Let us take, for example, the canter with the off fore leading. bringing his hocks under his body. Thus the forehand will be lightened in proportion as the hind quarters drive the body forward. Digitized by Microsoft® THE CANTER. At this time, half-turns on the haunches will be very useful, provided that the rider has enough ¢acé to keep the haunches well engaged and the horse well up to his bit. * The right hock, placed under the body, gives more impulse in the canter to the right than the left hock, and it is the one which displaces the right knee of the rider. The action of the left hock, which is much more to the rear, and is consequently a less powerful propelling agent, has necessarily a much less displacing effect on the left knee. t This study is necessary for the changes of leg. t The long-striding gallop, like that of the racecourse, is in four time. The very short canter of a rassemblé’d horse, of which pace I shall speak later on, is also in four time. Digitized by Microsoft® ORDINARY RIDING. 160 FIRST TIME.—Near hind in support (Fig. 43). ort, the ne FIRST TIME.—Near hind in support (Fig. 43). the n SECOND TIME.—Left diagonal support, the near hind and off fore having just quitted the ground (Fig. 44). SECOND TIME.—Left diagonal support, the near hind and off fore having just quitted the ground (Fig. 44). THIRD TIME.—Off fore in support (Fig. 45). e the action of the “ aids ” wh THIRD TIME.—Off fore in support (Fig. 45). the action of the “ aids ” wh I will now indicate the action of the “ aids ” which the rider should employ during this stride. The horse FIRST TIME.—Strong support from the left leg. The horse finds himself resting for a moment only on his near hind, but the period is so short that it cannot be seen without great practice. Fig. 43.—First time of the canter ; near hind in support. Fig. 43.—First time of the canter ; near hind in support. SECOND TIME.—Left diagonal. The two legs of the rider ought to exert their pressure to try to get the horse in hand. THIRD TIME.—The horse’s off fore is in support. The rider ought to receive the horse lightly on his hands ; at first to support him, and immediately afterwards to complete the task of getting him in hand, which work was initiated by the legs during the second time. ” I will now explain the effects of the “aids” during the three times which make a stride. Digitized by Microsoft® THE CANTER. First TiMeE.—tThe rider's left leg ought to be strongly pressed against the horse’s side, because the horse is standing, Digitized by Microsoft® THE CANTER. 161 so to speak, on his near hind, while his body is inclined forward and his other limbs are not much raised from the ground, The entire weight of the horse and rider are, therefore, carried on this leg, and if the rider does not support the left side of the horse, it is certain that this leg will give way under the excess of weight, and that the croup will swerve to the left. SECOND TIME.—The two legs of the rider exert their SECOND TIME.—The two legs of the rider exert their pressure in order to press the horse up to the hand. This is Fig. 44.—Second time of the canter ; left diagonal in support. Fig. 44.—Second time of the canter ; left diagonal in support. the only moment of the canter when the horse has two supports, and we ought to profit by the impulse he has just received, to get him into hand at the following time. THIRD TIME.—The rider receives the horse on the hand. This is the easiest moment at which to get the horse completely in hand; because, at the second period, the right hock being under the body, gives the greater part of the propulsion, which our hands should utilise to rassembler the horse. The hands ought also to support the horse a little during the third [period ; because the animal’s off fore is then his II II Digitized by Microsoft® ORDINARY RIDING. 162 only support. At this moment horses usually make mistakes, and are apt to fall if they are not supported. only support. At this moment horses usually make mistakes, and are apt to fall if they are not supported. Of course I point out only the chief “aids.” We under- stand that the hands and legs should always co-operate with each other. It might appear, after what I have just said, that the near hind is the leg which becomes most fatigued at a canter to the right. In fact, this is the usual opinion of many of those who have written on this subject. Digitized by Microsoft® THE CANTER. Nothing could be more erroneous ; because the off hock, being under the body and having to raise all the weight, does the most work during each stride, which is a fact beyond dispute. I am aware that the near hock, being placed at the end of the arm of the lever, is under a much less favourable condition for action. But precisely because the position which the off hock occupies under the body, is more favourable to the complete utilisation of its force, it makes a greater effort than the near hock, and consequently it becomes more fatigued. If we attentively watch a horse which is cantering with his off fore leading, we will quickly see that the off hock and fetlock become much Digitized by Microsoft® Digitized by Microsoft® VOLTES AND DEMI-VOLTES. 103 more bent than the near ones, and consequently their upward action is better marked and their spring more energetic.” more bent than the near ones, and consequently their upward action is better marked and their spring more energetic.” action is better marked and their spring more energetic.” If we canter to the right, a horse which has an unsound off hock, caused, for instance, by a spavin or curb, he will immediately become disunited, on account of the movement causing him pain. But if we make him canter with the near fore leading, he will not change his hind legs. The off hock therefore exerts more force in the canter to the right, and the near hock in the canter to the left. On this account, the rider ought to keep his legs firmly closed, especially during the second period of the canter. By doing this, the hock, which is under the body, is quickened in its action, the horse is pressed forward, and is prevented, at the second period of the canter, from remaining longer on the ground than at the first or third period. If we do not act thus, the second period will be slower and heavier than the other two. 1 * Among ladies’ horses which always canter with the off fore leading, the off hock always becomes prematurely worn out. Ladies do not like to canter to the left, because it displaces their seat too much. As they are seated on the near side of the horse, the propulsion given by the off hock is not made exactly under their seat, hence the displacement is less than in the canter to the left. TL Digitized by Microsoft® Digitized by Microsoft® VOLTES AND DEMI-VOLTES AT THE CANTER. When I have taught a horse to canter in a light and easy way, I make him do great voltes. It is best to describe large circles at first, because it is more difficult to keep a horse straight in small ones. Horses generally carry their haunches either to the outside or to the inside ; the former fault being more easy to remedy than the latter. Let us suppose that the horse is cantering with the off fore leading and is turning to the right. After having closed the legs, I carry the hands forward and to the right,—forward to prevent him stopping, and to the right to bring his shoulders * Among ladies’ horses which always canter with the off fore leading, the off hock always becomes prematurely worn out. Ladies do not like to canter to the left, because it displaces their seat too much. As they are seated on the near side of the horse, the propulsion given by the off hock is not made exactly under their seat, hence the displacement is less than in the canter to the left. TL TL Digitized by Microsoft® ORDINARY RIDING. 104 away from the wall. The off snaffle* rein should be pulled to the right, while the near rein presses the neck and conse- quently the shoulders to the right, and at the same time it helps the left leg to support the haunches, and thus assists to hold the horse straight. If the horse tries to throw his croup to the left, my two legs ought to increase their action to push him forward, the left leg acting a little more vigorously than the right, to keep his right leg leading. The right leg pushes the body forward and forces the haunches to follow the same line as the shoulders. away from the wall. The off snaffle* rein should be pulled to the right, while the near rein presses the neck and conse- quently the shoulders to the right, and at the same time it helps the left leg to support the haunches, and thus assists to hold the horse straight. If the horse tries to throw his croup to the left, my two legs ought to increase their action to push him forward, the left leg acting a little more vigorously than the right, to keep his right leg leading. VOLTES AND DEMI-VOLTES AT THE CANTER. The right leg pushes the body forward and forces the haunches to follow the same line as the shoulders. It is very easy to understand why I prefer a horse which carries his quarters to the outside, to one which carries them to the inside. In the former case, the animal presses on my left leg, and I then use the spur, which has two objects. First, to prevent the croup deviating to the left; and, second, to force the animal to keep cantering with the off fore leading. But if he carries his quarters to the inside of the circle, I am obliged to use my right leg, and sometimes the spur, with more or less force, and I thus run the risk of making the animal change behind. Also, the horse which carries his haunches towards the inside of the circle, is behind his bit. These faults seldom appear, if we begin on large circles, which we should only gradually make smaller, and in propor- tion to the ease with which the horse does the work. I may add that even in small circles we should keep the horse very straight. Immediately after, I begin demi-voltes, which present no difficulty if executed without precision, but which are very hard to do in an absolutely correct manner. I have rarely seen them thus done. * T have already said that I always work with the snaffle when beginning any new exercise. It is the means for direction ; and the curb, for getting the horse in hand. In proportion as the snaffle is brought into play, the curb aids more and more in getting the animal into hand. This continual action of the curb is found in all exercises to be associated with the action of the snaffle in giving direction. Digitized by Microsoft® VOLTES AND DEMI-VOLTES. 105 \ The horse being placed straight and in hand, as in the voltes, ought to leave the wall. When he has made three strides, he ought to cover the rest of the ground on two tracks, while coming up to the wall. To go on two tracks, while the horse is cantering with the off fore leading, the off snaffle rein pulls the head and neck a little to the right, whilst the near rein, being applied against the neck, presses the shoulders to the right. * The shoulders should arrive first at the wall, so that the movement may be correct. Digitized by Microsoft® * A horse which crosses his legs may escape falling, but only if he is lucky. If a horse which is cantering with the near fore, falls on Leing turned to the right, he does so because he is abruptly put into the position for the canter to the right. In high school riding we succeed very well in turning to the right, while our horse canters with the near fore. To do this we should make a large circle when turning, should always keep the horse in the position of the canter to the left, and should even exaggerate this position, by using the right leg to push the croup a little more to the left. It is easier to change the leg. Digitized by Microsoft® VOLTES AND DEMI-VOLTES AT THE CANTER. The left leg causes the haunches to move to the right, and the right leg helps to press the horse forward and to keep him in hand, which is important, because he has to advance while going to the side. On arriving at the wall, which is on the right of the rider, we should straighten the horse,* stop him, get him into hand at the walk, and start him into the canter with the near fore leading. We can do the same work on the other foot. When the horse can do demi-voltes well, I prolong them for some strides at the canter, without changing the leg. Thus, I begin the demi-volte at the wall, which is on my left. While cantering the horse with the off fore leading, I turn to the right, and continue to keep up the canter on the off fore, even after the demi-volte, but only during two or three strides at the first attempt. 1 increase the number of these strides only little by little, according to the lightness and cleverness of the horse. This is the most simple way to teach a horse to canter with the right leg when turning to the left; and vice versdé. This exercise is indispensable if we wish to readily obtain changes of leg when cantering round to the right or left. When the horse goes round the school correctly on the leg which is next to the wall, we should start several times with the off fore leading, and also with the near fore leading, both Digitized by Microsoft® ORDINARY RIDING. 166 tc the right and to the left, while always keeping him along the wall. CHANGES OF LEG. When a horse at the canter leads equally well with both legs, it is necessary that the rider should make him strike off with whichever leg he likes. He should also know how to make the horse change his leg, namely, to pass from one canter into the other canter without stopping. This is not high school work, but belongs to ordinary riding, whether practised in the school or outside. If, for example, we are at the canter with the horse’s near fore leading, and we wish to turn to the right, it is impossible to make this change of direction without danger. In fact, the near leg, which is in advance, will cross the off leg, and the horse will probably fall, in which case the rider will be almost certain to blame the horse; but the fault is his own.* If, when cantering with the near fore leading, we wish to turn to the right, while maintaining the same pace, we should first of all make the horse change his leg, in which case the turning movement will be made easily and without danger. In fact, the horse will have his head and body bent towards the side to which he is turning. Further, it is the forward leg which covers the ground of the side to which the change of direction is being made.t t With respect to changes of direction, I give the following advice: always enlarge the circle as much as possible ; slacken the speed a little when turning ; and if not quite certain of the change of leg—which is difficult in propor- tion to the speed—it is best before turning to make the horse change into the trot and not to put him into the canter again until he has completed the turn. CHANGES OF LEG. 167 We should exercise great care in teaching the horse to change his leg. It is very difficult to lay down in a book the exact time when a horse is ready to learn changes of leg. I can only say, ina general way, that the proper time is when he has become free, supple, light, and well balanced in all his paces, obedient, and above all things, attentive to the “aids,” by means of the exercises we have described. * T again repeat that I always begin a new work at the end of a lesson. t The difficulty is not in starting, but in keeping the horse at a uniform distance from the wall while holding him straight. Digitized by Microsoft® t The difficulty is not in starting, but in keeping the horse at a uniform distance from the wall while holding him straight. * T again repeat that I always begin a new work at the end of a lesson. t The difficulty is not in starting, but in keeping the horse at a uniform CHANGES OF LEG. We have now arrived at the point when the horse will start freely into the canter with the off fore leading when going to the right, and with the near fore when going to the left, and that he will do so with equal freedom on both legs. To obtain a change of leg, I proceed in the following manner :* I start the horse into the canter on the off fore while going to the right. When he has gone some strides on this leg, I stop him and make him start off on the near fore while circling to the right, and taking care to hold him as straight as possible. I repeat this work several times, and continue it until I feel that the horse is perfectly light in hand, and that he will start off into the canter at the slightest pressure of the legs, without hurrying himself and without trying to carry his haunches out of the straight line. To make sure that the horse does not carry himself sideways, we should start him at about a yard away from the wall, although it is difficult to do so.f The result of this is to make the rider keep the horse very straight, without the help of the wall, and, at the same time, to make him more precise in his work. We should not try to get the horse to change his leg away from the wall, before making him do so, while going along it. The horse should also very readily start into the canter on the Digitized by Microsoft® ORDINARY RIDING. 168 leg we wish, when we have placed him in the position to do so, and have given him the proper indication with the legs.* I then start him into the canter on the off fore, and keep him at it until he becomes quiet. I then walk him for a minute or two, and put him into the canter on the near fore, and keep him at it, the same as before, until he is light and quiet, after which J again walk him for some moments. Finally, I start him into the canter on the off fore, and so on. I there- fore make successive starts at the canter, each one being on a-different leg. } In this case he is cantering ‘' false,’’ which is the term used by English cavalrymen. * Position by legs and hands and stimulation by the legs are two most important principles in riding. Digitized by Microsoft® CHANGES OF LEG. Little by little I shorten the periods during which I let the horse walk between the starts at the canter, in such a way that he does not take, for instance, more than five, four, three, two, and finally, one step between the respective starts. At last he learns to start into the canter alternately from the stationary foot to the other foot, the starts being interrupted only by the halt. Thus, the horse having been started into the canter on the off fore, is stopped, and is immediately started again into the canter on the near fore, this change of leg being helped by the halt. As the true change of leg is done without halting, it is called a change of leg in the air. At this period of his training the horse is ready for the change of leg, which I ought to be able to make him do without upsetting him in the least. I start him into the canter on the near fore, while going to the right. He therefore canters on the outward leg,t and I support him at that time with the near rein and right leg, which gives a stronger pressure than the left leg. When I come to a corner of the school I completely change my “aids,” and use the off rein and left leg. This * Position by legs and hands and stimulation by the legs are two most important principles in riding. } In this case he is cantering ‘' false,’’ which is the term used by English cavalrymen. } In this case he is cantering ‘' false,’’ which is the term used by English cavalrymen. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® CHANGES OF LEG, 169 change of “aids” ought to be done with great decision and perfect combination. In order for the movement to be well done, it should be executed with extreme quickness, and with- out the slightest jerk. This quickness and smoothness are possible only if the rider has constantly taken the precaution of lightly feeling the off rein, and of keeping the left leg very close to the animal’s side, so that he need only feel this rein a little stronger and press the leg a little more. CHANGES OF LEG. As the horse has already lightly felt the off rein, and as the left leg has been kept very close to his side, there will be neither jerk nor irritation to the horse when this hand and this leg predomi- nate in their turn. Finally, as we have practised our horse to start on the off leg, by the action of the off rein and left leg, and as we ask him to do this first change of leg when turning to the right, which will be easier for him to do than when turning to the left, he will very rarely refuse to do so, even at the first time. If, however, he does not obey, we must not persist in roughly forcing him, because that would cause him to throw his haunches to the right, and make him afraid of the change of leg, which he would not understand. He should be stopped, brought into hand at the walk, made to start on the near fore, and then asked to change his leg. But before doing this we should wait until he has got quiet. The fact of the horse failing to do a change of leg several times, is a proof that his preparation is insufficient ; that being surprised by the “aids” he tried to escape or throw himself to one side; or that he did not understand what we wanted. In ali these cases we should begin starting him afresh. Any failure in this respect will be extremely rare if the horse has ‘ been properly prepared. Whenever we meet with a difficulty we should go back to the start, with the near fore leading, or with the off fore lead- ing. By these repeated starts with a halt, we shall succeed in Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 170 making the change of leg so easy, that the horse will often do it on his own account, when prompted merely by the preparations to which he has been submitted. In fact, when we stop him on one leg, the “aids” ought to slightly prepare him to start off on the other leg. Whenever I have had time to prepare a horse, I have never failed to make him change his leg at the first attempt. * T have said that in all things horses acquire habits with great facility. Therefore, during breaking, we should most carefully avoid giving him bench-marks (if \| may use the term), whether by putting him to the same work at the same place, or by repeating different exercises in the same order. This advice is, I think, all the more important, because the majority of riding masters persistently give bench-marks to their horses, which makes the breaking apparently more easy. Although the horse by routine does his work at a given moment, at certain spots, and according to a prearranged programme, he is not properly trained, because, so far from being submissive to the will of his rider, the rider has to accommodate himself to the habits of the animal. Consequently the horse is habituated, or, as we may say, ‘‘ routined,”’ but he is not broken in. Digitized by Microsoft® CHANGES OF LEG. What- ever kind of horse he may be, he will always do one change correctly, after having failed to do several. We should then get off, pat him on the neck, and send him back to his stable. At the following lesson we ought to repeat and prolong the same lesson, until the animal changes easily from the near fore to the off fore. Having then turned round, so as to go to the left, we should, in the same way, make him change from the off fore to the near fore. We should always avoid making him change at the same place, as that would always make him want to change when he passes it. It would therefore become impossible to make him change as we wish, because our will would be subordinated to his.* When I have got the horse to readily change from the out- ward to the inward leg in the corners, I put him to do the same work on a straight line. The change of leg should be required only at a certain period of the stride, when it is easiest for the horse to do. As * T have said that in all things horses acquire habits with great facility. Therefore, during breaking, we should most carefully avoid giving him bench-marks (if \| may use the term), whether by putting him to the same work at the same place, or by repeating different exercises in the same order. This advice is, I think, all the more important, because the majority of riding masters persistently give bench-marks to their horses, which makes the breaking apparently more easy. Although the horse by routine does his work at a given moment, at certain spots, and according to a prearranged programme, he is not properly trained, because, so far from being submissive to the will of his rider, the rider has to accommodate himself to the habits of the animal. Consequently the horse is habituated, or, as we may say, ‘‘ routined,”’ but he is not broken in. Digitized by Microsoft® Digitized by Microsoft® CHANGES OF LEG. 171 I have already said, each stride is composed of three distinct periods, which are marked by the feet of the horse coming down on the ground. In reality there is a fourth, namely, the period of suspension, which is not marked. “Paris, 27th August, 1890. “To THE Epitor Gil Blas. “Paris, 27th August, 1890. “To THE Epitor Gil Blas. “Paris, 27th August, 1890. Gil Blas. CHANGES OF LEG. The canter to the right may be divided as follows: 1st period, near hind; 2nd period, off hind and near fore; and 3rd period, off fore. The fourth period begins at the moment the off fore leaves the ground, and ends when the near hind is placed on the ground. At this moment the horse is in the aur, between the third period of one stride and the first period of the next stride. The best time to cbtain the change of leg is at the fourth period, because the horse is then in the air.* * Baucher gives no explanation of this subject. The majority of riding masters make this change of leg during support, and not when the horse is in the air, as I have advised. Their plan gives rise to an inevitable halt, which destroys the rhythm of the canter, and consequently alters the canter. My method, on the contrary, maintains the canter with all its impulse, and allows the rider to lengthen or shorten the stride, as he may wish, in all the changes of leg. A correctly executed change of leg at each stride thus forms a true pace, at which I obtain so much impulse that I am ready, without false modesty, to challenge anyone to have a race at it. On this subject I sent the following letter to the editor of Gil Blas, who declined to publish it. I fortunately got it inserted in the Echo de Paris, but it received no answer :— “ As Baron de Vaux knows many riding masters whom he favourably criticises, I would like to get one of them to accept my challenge. “Tam ready to accept my adversary’s conditions as regards ground and length of course. He can also fix the stake, which should go toa public charity. “This will be a good business for the poor, if not for riding. Ten thousand francs, for instance, will give them a great deal of pleasure. “T am, Sir, “Your most obedient servant, Digitized by Microsoft® “To THE Epitor Gil Blas. ““ Baron de Vaux has severely criticised my book and my riding ina long article which recently appeared in Gil Blas. It has in no way annoyed me, and I, being a horseman, have not the rashness to find fault with the criticism of a penman. ‘There is, however, one point which I think is worthy of notice, ‘There is, however, one point which I think is worthy of notice, because it can be submitted to practical proof. “The writer of the article tries to make out that I do not hold my horses straight in the changes of leg. If this be true, I must necessarily lose ground in forward progression. That being the case, I propose a race of changes of leg at each stride, whichI believe is an original idea that has never been tried. The winner will, of course, be he whose horse is the straighter of the two. Digitized by Microsoft® Digitized by Microsoft® 72 ORDINARY ORDINARY RIDING. 172 To obtain the change of leg during the fourth period, we should give the signal to the horse during the second period, when the diagonal is in support. When cantering to the right, the effect of the spur, at the moment when the left diagonal is in support, will be a vigorous straightening of the olf hock, which will forcibly send the near shoulder in advance of the off shoulder when the horse is in the air. The near hind leg will go in advance of the off hind leg all the more easily, because it is off the ground when the off hock gives its push. Under these conditions, after the spur has touched the horse during the second period, the third period (support by the off fore) loses the greater part of its impulse in the stride, and will be accentuated only as much as the equilibrium may require, because it is the off hock which has given the increased propulsion necessary for a change of leg. Everyone can now understand the meaning of change of leg im the air. The reversed indications are used for changing from the near fore to the off fore. in this chapter \| have spoken of changes of leg only from an ordinary point of view. Later on I will discuss this subject with respect to high school work. * We should not only watch a skittish horse, but should also engage his atten- tion as soon as he shows any signs of nervousness. In such cases many riders will not use their legs for fear of exciting the animal, and they continually hang on to the reins, especially those of the snaffle. By allowing him to be a prey to his fear, the rider does the very thing to increase it. By giving the animal a support on the bit, he is placed in the most favourable condition to play up. We should act in an entirely different manner, and, by the energetic use of the legs, should drive the horse forward ; because, at the first show of uneasiness, his tendency will necessarily be to get behind his bit, which is a preparation for shying, spinning round, and rearing. We should at the same time profit by the impulse given, in order to place the horse vigorously but lightly in hand, by means of the alternate effects of the curb and snaffle. Having got him in hand, we should give him a lateral flexion to the side opposite to which his object of terror is on. Finally, while keeping him well in hand, we should press him boldly forward, which is, in every case, the least dangerous thing to THE HACK. I will not try to describe the proper type of hack, because every riding man or woman has his or her own ideal, which “ JaMEs FILuis.”’ Digitized by Microsoft® THE HACK. 173 the riders choose according to their habits, temperament, and style of riding. Horses have certain innate qualities which cannot be given or replaced by breeding, rearing, or breaking. the riders choose according to their habits, temperament, and style of riding. Horses have certain innate qualities which cannot be given or replaced by breeding, rearing, or breaking. The first thing to require is surefootedness, which is a sine gud non. It is absolutely necessary that the animal will make no mistake at any of his paces. Hence we like our horses to raise their feet freely and to place them in a well- balanced manner on the ground. A horse which drags his iegs, or “daisy cuts,” is apt to make a false step by striking a stone or other inequality on the ground, and may conse- quently fall down. A horse which brings his toe first on the ground will be very apt to stumble, which is a fault we find among animals which “daisy cut.” A horse which raises his feet properly, cannot dig his toes into the ground, and must necessarily place them flat. ‘\ hack should have pluck. That is to say, he should not be restless or nervous. No amount of training can cure a horse of stumbling, or give pluck to an animal which 1s always ready to shy or spin round. As long as a good rider holds his horse between his hands and knees, and is on the alert,* he will save him from break- * We should not only watch a skittish horse, but should also engage his atten- tion as soon as he shows any signs of nervousness. In such cases many riders will not use their legs for fear of exciting the animal, and they continually hang on to the reins, especially those of the snaffle. By allowing him to be a prey to his fear, the rider does the very thing to increase it. By giving the animal a support on the bit, he is placed in the most favourable condition to play up. THE HACK. We should act in an entirely different manner, and, by the energetic use of the legs, should drive the horse forward ; because, at the first show of uneasiness, his tendency will necessarily be to get behind his bit, which is a preparation for shying, spinning round, and rearing. We should at the same time profit by the impulse given, in order to place the horse vigorously but lightly in hand, by means of the alternate effects of the curb and snaffle. Having got him in hand, we should give him a lateral flexion to the side opposite to which his object of terror is on. Finally, while keeping him well in hand, we should press him boldly forward, which is, in every case, the least dangerous thing to Digitized by Microsoft® Digitized by Microsoft® ORDINARY RIDING. 174. ing his knees or from meeting with any other accident. But, if he relaxes his attention, he will run a good chance of bringing back a blemished horse to his stable. Under these conditions, hacking ceases to be a pleasure and becomes a labour, a continual preoccupation, and an exnuz which soon makes one hate riding. We see that the two first good qualities of a hack are surefootedness and pluck. In my opinion there is a third quality which excels the first two, because it includes them and everything else which we desire from a horse. This supreme quality in every horse is impetuosity, namely, energy, fire, courage, and constant readiness to go forward. A horse which is not surefooted knows his defect, will always be hesitating, and will not dare to rush forward. A horse which is not plucky is always ready to get behind his bit. A horse which is a free goer is confident in being able to keep his feet, and, though glad to go forward, he does not get disturbed by the things which he passes. No matter what may be the breed or shape, a horse, like a man, takes his value from his mental qualities. Energy and heart make the horse. What use is the most beautiful railway engine, if it has no steam to drive it along the rails? do. These manceuvres will turn the attention of the startled animal from his cause of fear, and will place him in the most favourable position to avoid violence or disorder. It is evident that we should use these means in proportion to the skittishness shown by the animal. As a general rule, when a horse becomes impatient, we should send him up to his bit by the vigorous application of the legs, and he will instantly become quiet. Digitized by Microsoft® THE HACK. Above all theories, there is the fact that the only good horse tor every kind of work, whether for high school equitation or outside, is the horse which will go forward: that is to say, an impetuous horse. By this, I of course do not mean an animal which has irregular paces, is restless, vicious, apt to “play up” on no provocation, and is ready to run away. do. These manceuvres will turn the attention of the startled animal from his cause of fear, and will place him in the most favourable position to avoid violence or disorder. It is evident that we should use these means in proportion to the skittishness shown by the animal. As a general rule, when a horse becomes impatient, we should send him up to his bit by the vigorous application of the legs, and he will instantly become quiet. THE HACK. 175 The impetuous horse is one which wishes only to go forward, and in consequence of his being well balanced and having plenty of natural energy, he goes freely up to his bridle. True balance and natural energy are innate qualities in him, because they are conditions of his being: this in- cludes everything. Equitation cannot exist without energy. In the horse I speak of, the energy is already made, and he gives it to his rider. It is easier, as I have said, to profit by impulse than to create it. The fault of being behind the bit is the stumbling block in every kind of riding, and the natural disposition of an impetuous horse is directly opposed to this tendency. In every case, no matter what happens, a forward move- ment is the least displacing one for the rider. The fact of Arab horses being hot, generally makes them easy and agreeable to ride. These animals canter high, and always bring the hocks well under the body. For the above reasons I am right in saying that every horse which is not hot is fit only to be put between the shafts. As a rule, people are afraid of an impetuous horse, and they are wrong; because he is the only one which will be faithful, and will not do them a bad turn. He is the only animal which, by reason of his constant desire to go forward, is plucky even in his defences. * The tendency of every horse, no matter how impetuous he may be, is to return to the stable quicker than he left it. In order that the rider may not be obliged to come back quicker than he went out, I advise him to begin his ride ata fast pace, so as to quieten the animal down, which is most important later on for preventing him jibbing, Digitized by Microsoft® * The tendency of every horse, no matter how impetuous he may be, is to return to the stable quicker than he left it. In order that the rider may not be obliged to come back quicker than he shying, kicking, etc. If we take the trouble to make the horse return at a slow pace, during a certain time, we shall succeed in being able to regulate his paces as we wish for the entire period of the ride, and we shall thus prevent him pulling or starting off at a canter the moment his head is turned home. THE HACK. which is generally preferred, can The sluggish horse, which is generally preferred, can neither be reliable nor plucky. In every case, so far from giving himself up to his rider, he continually restrains him- self, and is always ready to keep behind the bit, which, in my eyes, is the worst of all faults. He thinks only of returning to his stable,* and tries to take advantage of every chance to In order that the rider may not be obliged to come back quicker than he went out, I advise him to begin his ride ata fast pace, so as to quieten the animal down, which is most important later on for preventing him jibbing, Digitized by Microsoft® ORDINARY RIDING. 176 gratify this caprice. If we wish to go fast, and quiet means are not sufficient, we should use the spurs or whip. When the sluggish horse defends himself, he does so with all the more spite, because he has kept back all his energy to oppose the will of the rider. There is a great difference between the two, because the impetuous animal gives all his strength for the advantage of the rider; and the sluggish horse, for the detriment of the man in the saddle.* A cold horse does not take us into his confidence, because when riding him we are obliged to vigorously use the “ aids,” which are a punishment to him. A hot horse which gives himself up wholly to his rider, gets only pats on the neck, and appreciates all the shght differ- ences in the indications of the “aids.” There is neither fear nor distrust between the two. Many riders find fault with hot horses, because they are impatient, and start off at a quick pace immediately the reins are touched. The fault is on their side. In fact, when- ever we wish to go rather fast, we begin by shortening the I also advise that the horse should not be turned sharply round to come back to his stable by the road he came out. Such a habit would prompt him to pull immediately he was turned. * In 1857, at Chalon-sur-Sa6ne, two horses were brought into the riding school to be broken. One was so skittish and lively that I found it ex- tremely difficult to put my foot into the stirrup. shying, kicking, etc. If we take the trouble to make the horse return at a slow pace, during a certain time, we shall succeed in being able to regulate his paces as we wish for the entire period of the ride, and we shall thus prevent him pulling or starting off at a canter the moment his head is turned home. I also advise that the horse should not be turned sharply round to come back to his stable by the road he came out. Such a habit would prompt him to pull immediately he was turned. * In 1857, at Chalon-sur-Sa6ne, two horses were brought into the riding school to be broken. One was so skittish and lively that I found it ex- tremely difficult to put my foot into the stirrup. After having made several unsuccessful attempts, they took me by the seat of the trousers and threw me into the saddle. When I got there, the horse did nothing wrong, and went forward wellin hand as soon as I stopped holding him back. As the other horse held his head down, did not move, and looked like a sheep, the master of the riding school thought he was quiet, and mounted him without any trouble, but he did not remain in the saddle long, because this supposed co/d-tempered horse bucked him off at the first attempt, but the hot horse carried me gaily round the school. The conclusion which I draw from this, is that we should distrust a horse which looks too quiet, and that we should not be afraid of an impetuous animal. * I wrote this book at the time when I made my début at the Hippodrome with Germinal and Markir. Remarks were made on the apparent con- tradiction between my statement that I broke only thorough-breds and the fact that I had carried the breaking of the half-bred Markir as far as with any other horse, no matter what his breed might be. The truth is that I bought Markir, not only on account of his good points, but also for his strength and activity, which I greatly admired. While breaking him, I often asked myself if he would do all I expected, and I frequently feared that his abilities would not come up to my require- ments, which he amply fulfilled to my great surprise, and to such an extent that I determined to find out if the pedigree he had been given was correct. I therefore wrote to my friend Lenoble du Theil at the Haras du Pin, and told him that Markir had been sold to me as being out of the thorough- bred mare Thérésine by the Norfolk half-bred Weighton Merrylegs, and I begged him to see if this was right. Imagine my surprise and joy on receiving the following answer: ‘‘ The thorough-bred mare Thérésine was put to Merrylegs, but did not hold, and was covered two months later by Cyrus, which is a thorough-bred Anglo-Arab, and the sire of Markir.” * I wrote this book at the time when I made my début at the Hippodrome with Germinal and Markir. Remarks were made on the apparent con- tradiction between my statement that I broke only thorough-breds and the fact that I had carried the breaking of the half-bred Markir as far as with any other horse, no matter what his breed might be. THE HACK. After having made several unsuccessful attempts, they took me by the seat of the trousers and threw me into the saddle. When I got there, the horse did nothing wrong, and went forward wellin hand as soon as I stopped holding him back. As the other horse held his head down, did not move, and looked like a sheep, the master of the riding school thought he was quiet, and mounted him without any trouble, but he did not remain in the saddle long, because this supposed co/d-tempered horse bucked him off at the first attempt, but the hot horse carried me gaily round the school. The conclusion which I draw from this, is that we should distrust a horse which looks too quiet, and that we should not be afraid of an impetuous animal. Digitized by Microsoft® THE HACK. 177 reins, and the horse quickly establishes a connection between these two acts. But if, in place of starting off the moment we adjust the reins, we keep the horse at the walk, he will not contract this bad habit, or he will give it up if he has acquired it. The signal for a fast pace should be given only with the heels. With respect to breed, we find in the first line in this book, the cry of my heart: “I break only thorough-breds.”* I unhesitatingly put thorough-breds above all others, whether for hacking or for high school riding. They are pre-eminently the best for all kinds of work. Besides, a man who has got into the habit of riding thorough-breds, will not care to ride any other horses. For me the ruling qualities of the thorough-bred are the lightness, the elasticity of the fine steel spring which puts them into action, and the suppleness which will be developed by breaking. If we listen from afar off, for the sound of his foot-falls, we shall hardly hear them on account of the light- Markir is therefore thorough-bred. The curious thing about this matter is that it was proved by practical experiment before any investigations had been made. I2 Digitized by Microsoft® ORDINARY RIDING. 178 ness with which he puts his feet on the ground. He skims the ground, which he treads with a delicacy full of energy. The feet of other horses, compared to his, clatter and hammer the ground, and their paces are much heavier. * Nothing is more deceptive, even for speed, than pedigrees. Paternal and maternal atavism jumble up everything. Nevertheless, certain sires transmit characteristic points to the majority of their progeny. * Nothing is more deceptive, even for speed, than pedigrees. Paternal and maternal atavism jumble up everything. Nevertheless, certain sires transmit characteristic points to the majority of their progeny. I have ridden four horses sired by Vermouth and broken two of them. All four were sulky and required punishment to make them pass their stable. As long as they were ridden vigorously they went on a little, but no one could make them move freely except when they were playing up. Ikept my eye on them for a long time, and found that they all turned jibbers. The progeny of Zut are nervous, ticklish and restive. The stock of Parmesan and Gantelet are excellent horses, supple, energetic, and good-tempered. The stock of Clocher are good, but not handsome; they can stay and carry weight. The same can be said of the sons and daughters of Bra- connier, which are not so good-tempered as those of Clocher. Horses got by Castillon are slugs. Flavio sires good, supple and very wiry, though hot-tempered horses. It is clear that observations of this kind, which could be multiplied to infinity, are all the less exact, because the influence of the dam has not been taken into consideration. Nevertheless, they are partly true, and are consequently worthy of mention. I have ridden four horses sired by Vermouth and broken two of them. All four were sulky and required punishment to make them pass their stable. As long as they were ridden vigorously they went on a little, but no one could make them move freely except when they were playing up. Ikept my eye on them for a long time, and found that they all turned jibbers. The progeny of Zut are nervous, ticklish and restive. Digitized by Microsoft® The progeny of Zut are nervous, ticklish and restive. * Nothing is more deceptive, even for speed, than pedigrees. Paternal and maternal atavism jumble up everything. Nevertheless, certain sires transmit characteristic points to the majority of their progeny. I have ridden four horses sired by Vermouth and broken two of them. All four were sulky and required punishment to make them pass their stable. As long as they were ridden vigorously they went on a little, but no one could make them move freely except when they were playing up. Ikept my eye on them for a long time, and found that they all turned jibbers. The progeny of Zut are nervous, ticklish and restive. THE HACK. On the other hand, there are many excellent half-breds. We sometimes meet with horses which show no signs of breeding, but which develop marvellous qualities in the same way that certain thorough-breds of illustrious pedigrees are mere garrons.* This prompts me to again say that a horse should above all things be fot. I may add that the thorough-bred is incomparable in courage and energy. As the thorough-bred has the reputation of being coéd, I shall no doubt be asked how I can reconcile my love for him with the superiority which I claim for the ot horse? This contradiction is only apparent, and I find that I have already explained it. There are cold horses and fod horses among thorough-breds, as there are among other breeds. I will even say that the innate energy of a thorough-bred will more easily make him generous, if he is ridden as he I have ridden four horses sired by Vermouth and broken two of them. All four were sulky and required punishment to make them pass their stable. As long as they were ridden vigorously they went on a little, but no one could make them move freely except when they were playing up. Ikept my eye on them for a long time, and found that they all turned jibbers. The progeny of Zut are nervous, ticklish and restive. The stock of Parmesan and Gantelet are excellent horses, supple, energetic, and good-tempered. The stock of Clocher are good, but not handsome; they can stay and carry weight. The same can be said of the sons and daughters of Bra- connier, which are not so good-tempered as those of Clocher. Horses got by Castillon are slugs. Horses got by Castillon are slugs. Flavio sires good, supple and very wiry, though hot-tempered horses. It is clear that observations of this kind, which could be multiplied to infinity, are all the less exact, because the influence of the dam has not been taken into consideration. Nevertheless, they are partly true, and are consequently worthy of mention. Digitized by Microsoft® THE HACK. 179 ought to be, and if he is asked to do what may be expected from his conformation and propelling power. ought to be, and if he is asked to do what may be expected from his conformation and propelling power. * My answer to the objection that many thorough-breds are “ daisy cutters,’’ is that such animals will not make good hacks ; but this fault in a hack is a good quality in a race-horse. Although a horse which gallops high will be turned out of a training stable, he will be gladly bought by a man who wants a nice hack. * In French, the same word is used for ‘“‘ canter’ and “ gallop ’— Translator. THE HACK. The fact is that the horses which are selected for the turf are long-striding animals, which consequently “daisy cut.”* A horse which has high action is not admitted into a train- ing stable, which fact enables me to obtain in him all the gencrosity and heat which I value above all things, whether for hacking or for high school riding. People say that the thorough-bred does not make a good People say that the thorough-bred does not make a good trotter; but the reason for this statement would be difficult to give. On the contrary, the make and shape of the thorough- bred are admirably suited for all three paces. It is merely a question of education. We can train thorough-breds to the trot, as well as to the canter, and I have known thorough- breds which were wonderfully good trotters. Breaking will develop in a sound well-built horse all his innate qualities, and by its course of rational gymnastics, will partly replace absent qualities. It will make him clever, light, and well-balanced. A horse which can walk, trot, canter, turn easily, rem back and move from one side to the other, is sufficiently broken for work in the open ; but he should first be made quiet to mount, at which he should not require to be held. He should have a free long-striding walk. The trot and canter should be lengthened and shertened, according to the wish of the rider ; this is a question of breaking. At the walk a horse should be allowed to have his head free, so that he may get confidence and may be able to lengthen his stride at his ease, and his neck should be nearly horizontal. Above all things, he should not be allowed to jog. 12% Digitized by Microsoft® ORDINARY RIDING. 180 At an ordinary trot, a horse should be let as free as possible. We should keep him well in hand, with his head and neck high, so that he may be light by being well-balanced. In the fast trot, the head and neck should be nearly hori- zontal, so as to allow the animal to reach forward as far as possible. The horse ought to go freely up to the bridle, and the rider ought to keep only a light feeling on the snaffle. Digitized by Microsoft® THE HACK. There are three kinds of canter,* namely, the we/-collected canter, the hand gallop, and the full-speed gallop. In the wedd-collected canter (strong action of the legs and slight action of the hands: taking and giving), while gaining but little ground, he should carry his head and neck high. The hand gallop is a shortened gallop, which should be well-collected, if the rider is worthy of that name; but in it the horse is almost always behind his bit and extended. If we go to the Bois any morning, we shall see many of these unfortunate thorough-breds, which are worthy of a better fate, fully extended, with their hind legs dragging behind, head and neck low, and going stiffly in a mechanical style of gallop. Such horses caricature the movements of a gallop, while having a man on their back; but this inelegant combination has nothing to do with equitation. In the hand gallop we should give some liberty to the horse, and as we press him up to the hand, we should pro- portionately support him. Although the collection is not full, our legs should bring his hocks well under the body. In the full-speed or race-course gallop, the horse should In the full-speed or race-course gallop, the horse should lean well on the hands, and should stretch out his head and neck. Although he is not collected, the hocks are not less energetically brought under his body ; for speed could not be obtained without their action. Digitized by Microsoft® THE HACK. 181 The rider ought to be able to easily make his horse go from one of these paces into another. The horse ought to be able to lead equally well with either fore leg. Ability to change the leg is of great use, when riding in the open, so as to be able to readily turn to either side. It is not, however, indispensable if the rider takes care to stop at the moment of turning, and to start off on the leg of the side to which the turn is made. It is of the utmost necessity that the horse readily yields to the legs, so that we can place his haunches as we like at all paces. * A horse that stumbles, shies, pulls, or won’t go on unless spurred, soon disgusts us with riding. Digitized by Microsoft® THE HACK. When a rider passes or crosses another rider, he should be able to direct the movements of the hind quarters with his legs at the same time as he does those of the fore hand with his hands, so that he may guide the horse as a whole and not in parts. If, for instance, the hands pull the forehand to the right, and the haunches are allowed to swing round to the left, the horse will be put crossways, which is just the thing he ought not to be made to do. In this way the rider may get thrown off, or the man who passes by him may get kicked. If a horse obeys the legs properly, we can easily, at all three paces, approach or leave one or more riders. When we are all together, obedience to both legs will enable us to leave the others, and, keeping the horse in hand, to let them pass us, if such is our wish. There is extreme pleasure in riding a well-broken /oz horse.* Nothing is more agreeable than to work a horse in the cpen. Flexions, diagonal effects, two tracks, the vassembler, starting into the canter with the off fore and near fore leading, and other fine points of horsemanship, keep us on the alert, and make the time pass quickly when hacking. * A horse that stumbles, shies, pulls, or won’t go on unless spurred, soon disgusts us with riding. ORDINARY RIDING. 182 The weather is always good for riding, except when there is snow and frost.* In cold weather there is extreme pleasure in hearing the quick foot-falls on the hard ground, of a horse which is excited by keen air. If it is raining or misty, the damp ground will be excellent for the horse, who will bravely plunge his feet into puddles and mud, and will joyously splash everything in his road. When it blows hard, how pleasant it is to feel the wind striking our face, while we dash along at full speed! Behold the sun! Let us go to the woods to enjoy the freshness and deadened brilliancy of the soft light. The walk is the pace for reverie. * When the ground is slippery, we should take our feet out of the stirrups, so that one of our legs may not get under the horse, in case of a fall. Digitized by Microsoft® Digitized by Microsoft® HORSES WITH VICES. Nervous Horses—Horses which throw their heads about—Horses which run away. THE HACK. The land spreads out its panorama, of which we are a living, moving part, because we are carried away by the country in the cadence of a continual movement to the long striding walk of a thorough- bred. What pedestrian will believe that the walk of a horse can inspire his rider with the feeling of speed? Nevertheless, that is the pleasure which is reserved for us. The trot and hacking canter give us the pleasure of travel- ling without fatigue, of enjoying a harmonious movement produced by a reaction as free as it is elastic, with an energy increased a hundredfold by the generous ardour of the noble animal which is at our disposal, and which will give us all that is in it. Where does man end; where does the horse begin? We know not. The two make the most intimate, supple, living and vigorous whole which forms all the joy of life and action. What intoxication of happiness to rush at full speed into space towards the unknown! If fences come in our way, what excitement there is in flying over them! What quiet daring fills our heert! THE HACK. 183 We cannot express the infinite joy there is in the full speed of a thoroughbred. It is like the rocking of a wave, but it is so soft, so refined, that the air which we cleave annuls the feeling of weight. It is a mighty, intoxicating flight, with- out effort and without fatigue; a physical joy which puts the mind to sleep, and leaves nothing living in us, except the maddening pleasure of flying through space. I consider that the thorough-bred horse makes man perfect. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® NERVOUS HORSES. IT is usually said that a horse which is nervous will jump to one side or spin round, when the sight of some object or some noise makes him stop. Any horse can be more or less surprised, according to its degree of impressionability. Happily all horses are not timid. The impressionability of a horse can be greatly diminished and modified by breaking. Custom establishes mutual confi- dence between horse and rider. If the animal has not been beaten, or violently forced up to the object of his alarm, and if the presence of his rider reassures him, instead of frighten- ing him, he will soon become steady. It is a sound principle never to flog a horse which is frightened by some external object. We should, on the contrary, try to anticipate or remove the impression by “making much” of the animal. I have already said that a horse has but little intelligence. He cannot reason, and has only memory. If he is beaten when an object suddenly comes before him and startles him, he will connect in his mind the object and the punishment. Digitized by Microsoft® NERVOUS HORSES. 185 lf he again sees the same object, he will expect the same punishment, his fear will become increased, and he will naturally try to escape all the more violently. All horses are not equally timid. Some are more impres- sionable on one side than on the other, whether accidentally or naturally, which fact gives rise to the saying that some horses are nervous on the near side, and others on the off. We may note that wall-eyed horses are always nervous. It is often wrongly said of a horse that he is nervous, when he is only fresh or green. It would be more correct to say that he is zz the air, that he wants to use his muscles and extend himself, or, in other words, that he is in the position of a child who, having been for a long time quiet, begins to play and frisk about. In such cases punishment and caresses are equally useless. What we ought to do is to give him what he wants, namely, plenty of exercise. Send him along for two or three miles, and after that he will be per- fectly quiet. Digitized by Microsoft® NERVOUS HORSES. There are certain horses which are restless by nature, and are constantly on the alert to notice anything they may see or hear. They are afraid of all sorts of imaginary things, they make sudden starts at every moment, and are consequently very disagreeable to ride. Give them lots of work to cool them down and pats on the neck to give them confidence. The number of the different kinds of nervous horses is so large, that it is impossible to enumerate them all. There are, however, certain ones which I would like to particularise. Some are afraid of everything which goes past their head, er everything which overlooks them, such as a carriage, omnibus, a low bridge, or carriage entrance. All things of that kind frighten them. People say that these horses are afraid of their heads being hurt. They are, however, abso- lutely indifferent to things which are low. Other horses are afraid only of things on the ground, such Digitized by Microsoft® 186 HORSES WITH VICES. as shadows, rays of the sun, puddles, streams, and heaps cf stones. They pay no heed to things which are above them. There are also horses which are afraid only of things which come behind them, such as children, dogs, horses, and carriages. They seem afraid only of things which they do not see. My only advice about the management of nervous horses is to give them confidence by “making much of them.” If we see in front of us an object which we know our horse will be afraid of, we should not force him to go up to it. Better let him at first go away from it, and then gently induce him to approach it, without bullying him too much. Work him in this way for several days, as long as may be necessary. Never bring him so close up to the object in question that he will escape or spring round ; because in this case we will be obliged to punish him; not for his fear, but on account of his spinning round, which we should not tolerate at any time. In punishing him, we will confuse in his mind the fear of punishment and the fear caused by the object. In a word, with nervous horses we should use much gentleness, great patience, and no violence. Digitized by Microsoft® NERVOUS HORSES. It now remains only to speak of horses which are wrongly classed as nervous, which have all the appearance of fear, but which are only vicious. All horses are very fond of their stable, and are more lively and impetuous when they are returning to it than when leaving it. Those of whom IJ speak seem to have only the idea of returning to their stable, and of seeking every occa- sion of making a move to the rear, in the form of a spin round. We see them stop at the most trifling object which they meet, and then they wheel round sharply if the rider shows the slightest hesitation. These horses, I repeat, are not afraid; they are vicious, and nothing I have said about Digitized by Microsoft® NERVOUS HORSES. 187 nervous horses applies to them. They have to be promptly brought back, and severely punished. nervous horses applies to them. They have to be promptly brought back, and severely punished. The proof that they are actuated only by vice is that, - when returning to their stable, they will treat with absolute indifference the objects which caused them to spin round, when going out. If under these or any other conditions the horse shies, we ought to steady him and bring him back by lateral effects. If he shies to the right, the right snaffle rein ought to be strongly carried to the left, and the right leg should be vigorously applied. In other words, to correct a shy to the right, we should apply two effects from right to left. By using the left rein—which people usually have the bad habit of doing—the rider will help the horse to throw to the right his haunches, which his right leg will not be able to keep straight. Further, if there is danger to the right, the horse may throw himself unwittingly into it, because, having his head turned to the left, he will not be able to see where it is. But the right rein, when applied strongly to the neck, presses him to the left, and aids the effect produced by the right leg, which prevents the haunches from being carried to the right. If the horse shies to the right, the rider is shifted to the ieft. Digitized by Microsoft® NERVOUS HORSES. In such cases he keeps his seat in the saddle by the strong pressure of the right leg, from which he obtains safety. to one Horses which spin round, almost always do so to one particular side. If we have a mount which uses this mode of defence, we should begin by finding out the side he turns to, and by holding on that side a stick about eighteen inches long. I do not like a whip for this purpose, for it is too flexible; and if, for instance, we use it on the left, its end may bend and strike the right side, or, what is worse, may injure the animal’s eyes. ound, but Immediately the horse turns his head to spin round, but Digitized by Microsoft® HORSES WITH VICES. 188 before he can do so, strike him sharply with the stick on that side of the nose. I have never met a horse which has not been quickly made obedient by this method of correction when it has been applied with severity and precision. After a few times the mere sight of the stick will be enough.* I like to work alone without any help, not even that of a groom. Those who do not believe this can easily verify my statement. I have kept my horses at livery in several schools and I have always broken them in these places. I have never asked the help of any one at the waneges of Latry, Vincent, Quartero, or \|’Etoile. * The most disagreeable animal I ever met was a stallion which was very restive and a fiend to bite. As it is impossible to break a horse without the help of the legs and spurs, it was necessary to find a means to make this horse bear their touch. The first six weeks passed off fairly well, because I did not ask him to do much, but as soon as I tried to get him in hand by means of the spurs, he tried to bite my legs, threw himself violently on his knees, and in this position made desperate efforts to catch hold of my feet; and he had the best of the struggle, because I was not able to use the spurs, on account of having to draw back my feet out of the way of his teeth. I then put on the snaffle reins two iron rods, fifteen or sixteen inches long. Having thus put the animal in such position that he could not turn his head round, and as my legs were then safe from his teeth, I felt sure of victory. But this demon of a horse, being rendered furious by his feeling of powerlessness, continued to throw himself on his knees, and being unable to bite my feet, bit his own breast, which I thought he would stop doing on account of the pain. So far from that, he tore away strips of flesh from his breast, and I have no doubt that if he had unseated me, he would have devoured me. I then put on him a very thick leather apron, which was in shreds in three days. HORSES WHICH THROW THEIR HEADS ABOUT. There are very few horsemen who have not had the trouble and even the danger of riding animals which threw their heads about. Some forcibly lower the head, and thus, so to speak, * The most disagreeable animal I ever met was a stallion which was very restive and a fiend to bite. As it is impossible to break a horse without the help of the legs and spurs, it was necessary to find a means to make this horse bear their touch. The first six weeks passed off fairly well, because I did not ask him to do much, but as soon as I tried to get him in hand by means of the spurs, he tried to bite my legs, threw himself violently on his knees, and in this position made desperate efforts to catch hold of my feet; and he had the best of the struggle, because I was not able to use the spurs, on account of having to draw back my feet out of the way of his teeth. I then put on the snaffle reins two iron rods, fifteen or sixteen inches long. Having thus put the animal in such position that he could not turn his head round, and as my legs were then safe from his teeth, I felt sure of victory. But this demon of a horse, being rendered furious by his feeling of powerlessness, continued to throw himself on his knees, and being unable to bite my feet, bit his own breast, which I thought he would stop doing on account of the pain. So far from that, he tore away strips of flesh from his breast, and I have no doubt that if he had unseated me, he would have devoured me. I then put on him a very thick leather apron, which was in shreds in three days. I succeeded, however, in mastering him by placing under his chin a kind of half funnel made out of white metal. Being unable to bite, he soon dropped that detestable habit, and J completed his education at No. 78 Avenue Malakoff, in a place kept by the son of Mr. Gost, who was a horse dealer, and who more than once was present at these equine battles, which I fought singly as a rule. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® HORSES WHICH RUN AWAY. 189 take a spring to raise it with greater violence. Others con- tent themselves with throwing the head sharply back, with- out lowering it in the first instance. In the former case we should catch the horse with the snaffle reins (which are held in the right hand) at the exact moment when he begins to lower it. On account of the shock he will quickly raise his head, at which instant we should drive him forward by a strong pressure of the legs. The curb reins are loose, but are firmly held in the left hand in such a way that the moment when the horse, by raising his head, tightens the curb reins, he will receive a severe blow on the bars of the mouth. Therefore he has been stopped by the snaffle when he wished to lower his head in order to get his impulse, and has been punished by the curb when, in throwing up his head, he brought it too far back. When he thus throws back his head without obtaining an impulse, we should drive him forward with a pressure of the legs the moment he raises his head, and should receive him on the curb, under the same condi- tions as those first mentioned. To sum up, we make in the first case three movements of the snaffle, legs and curb. In the second, we use only the legs and the curb. Although these movements are succes- sive, they follow each other so closely that they almost unite into a single one. In both cases, if the action of the legs does not precede by ever so little that of the curb, we shall run the risk of making the horse rear, or at least by crushing the hind quarters we make him get behind his bit. HORSES WHICH RUN AWAY. I have always been fairly lucky, both as regards my pupils and myself, in avoiding accidents from run-aways. My good luck was not altogether a matter of chance. When a horse is running away, he will not listen to reason, and I do not think any one rider would be able to stop him Digitized by Microsoft® HORSES WITH VICES. 190 much better than another ; but we can do a good deal to stop him from running away. Here the real knack consists in prevention, and therefore the watchful horseman will never let his animal get out of hand. Immediately he feels that the horse is ready to break away, he will steady him and calm him down by the voice and pats on the neck. A horse often runs away because the bars of his mouth A horse often runs away because the bars of his mouth have become insensible on account of the rider pulling at him, which in this case is like pulling at a wall, and consequently the horse can bolt when he hikes. To keep the mouth fresh, the rider ought to use the snaffle and curd alternately; that is to say, he should not let the animal take a bearing on either reins. Some horses run away with their heads high, others bring the chin into the breast. We should always try to lower the heads of the former with the curb, and to raise those of the latter with the snaffle. If the horse succeeds in bringing the head so low down and in rounding the neck so much that the cheeks of the curb are in contact with the breast, the more the rider pulls at the curb reins, the less will the bit act on the mouth,* and the more will he maintain the wrong position of his head and neck. The only thing he has to do in this case is to saw the snaffle. With horses which get the chin into the chest it is well to use a gag snaffle, which acts upwards instead of backwards. It is also useful with a horse that has a heavy head and neck, which such an animal always tries to make his rider carry. Of course I offer this advice only to those who do not know how to balance their horses. Digitized by Microsoft® * Many old race horses try to carry off their rider when he turns them round, because on race courses, horses start on being turned round. We should be on the look out with such animals. Digitized by Microsoft® HORSES WHICH RUN AWAY. A horse is often said to run away when in reality he is only carrying away his rider. * By pulling on the cheeks of the curb, we fix them against the breast and cause the mouthpiece to shift upwards in the mouth, which action relieves the bars. HORSES WHICH RUN AWAY. 191 A horse that runs away is a maddened horse which does not answer to the aids, and which cannot be guided. A horse that carries away his rider is an old rascal who gets excited by a gallop, and who bolts whenever he gets out of hand. Although we cannot stop him, we can guide him. He takes care to avoid obstacles that are in his path; but the run-away gallops with his head down, his eyes are injected with blood, and he will dash himself to pieces against any- thing he meets. A horse can carry his man away at all paces. I have seen an animal do it even at a walk. Such horses pull without ceasing, they make a half rear when the rider tries to stop them, and when they have completely tired him out, they carry him off. We should therefore take care never to allow them to get out of hand. If they succeed in catching the rider unawares and carrying him off he should simply guide them into some open space. The first thing which the rider should do is to separate his reins. By sawing the snaffle, by the alternative effects of the snaffle and curb, and by loosening the jaw, he can succeed little by little in changing the pace, putting the horse on his hind quarters, and consequently mastering him. Above all things we should take care to manipulate the reins in a uniform manner. Giving and taking is the in- variable principle, and the legs ought always to remain close to the sides of the animal. On these occasions the majority of riders think that they gain strength by making a “triangle” with their legs stuck out in front, in which case the horse will always be stronger than the man. Many horses which have been raced, try to go off* with their rider, either at the trot or canter. To accomplish this HORSES WITH VICES. 192 they stretch out the neck and forcibly lower the head. Digitized by Microsoft® HORSES WHICH RUN AWAY. If the rider pulls sharply at the reins, the horse will probably drag him out of the saddle on to his neck by throwing his head down. We should “give” to the horse by bringing the hands forward without letting the reins slip through the fingers, then rather vigorously raise the neck, take up the snaffle and curb, and use the legs as soon as the neck has been straightened. When a horse is really running away, the reins should be used in the same manner as when he is carrying off his rider, but the difficulty is much greater, because it is almost im- possible to guide him. If we have a plain in front of us we may succeed, even in this case, in turning the horse to the right or to the left. To do this, supposing the reins are separated, as they ought to be, we should let go one of the reins, take hold of the other with both hands, pull with all the weight of the body, so as to produce a more or less decided lateral flexion, which will certainly turn the horse out of his course. If the rider of a run-away horse finds himself alongside a stream, he ought to force the animal into it. There is danger in everything on land, whether we meet a fence or get a fall on level ground ; but in water there is no danger, and a bath makes us quits. The only thing to avoid is a perpendicular bank. The majority of horses will stop as soon as the water comes up to their breast, and they will become perfectly quiet. Others strike the water with their fore feet, give themselves up to disordered movements, but always finish by becoming quiet in a short time. In order to correct run-aways, I have often let them gallop as fast as they could along the low banks of a river, and when they were in full swing I sent them into the water, which I was able to do very easily. Any horse which has been properly rasseméZéd can neither run away nor carry his rider away, because the use of the Digitized by Microsoft® HORSES WHICH RUN AWAY. 193 spurs, by bringing the hocks under the body, raises and lightens the forehand, and consequently the animal can be easily stopped. HORSES WHICH RUN AWAY. While riding with my pupils I have often asked them what would they do if a run-away horse came up to them either from the front or the rear? I have also often asked persons who have ridden horses all their lives the same question, but I have rarely received a satisfactory answer, although this con- tingency ought always to be provided against. We should bear in mind that a man ona run-away horse generally runs less danger than the riders who are in his road. -We often sée a run-away animal dash into a group of horses with his head down. To avoid him we must get behind the first obstacle. If we are in a forest we should get into some bushes, and will then escape with a few scratches. If we are on a road we should get behind a tree; if in a street, behind a lamp post. In fact, anything will serve to shield us from the terrible shock. Above all things we should move as quickly as we can. If in place of getting out of the way, we wish to help a man on a run-away horse, which is a praiseworthy but very difficult attempt, we should gallop at full speed in the direction he is going, while keeping a few yards in front of him. We ought to speak in a loud tone of voice and try to give the rider con- fidence. We should then slightly slacken speed, and as the run-away passes alongside of us, we should try to catch hold of his curb reins as near as possible to the mouth, and en- deavour to stop him little by little. If we do not gallop in front of the run-away nearly as fast as he is going, the jerk we will receive at the moment we catch the reins may throw us out of the saddle. As we ought to have one hand free, we should hold our reins in the other hand, so as to be able to control and guide our mount. I may add that it is almost impossible to stop a run-away 13 13 Digitized by Microsoft® HORSES WITH VICES. 194 while going on a straight line. J have never succeeded in this. HORSES WHICH RUN AWAY. If the amount of open space and the nature of the ground allows us to do so, we should at first take a very large circle, and should decrease it little by little. We should of course place ourselves on the inside of the circle, so as to be able to pull the run-away in the direction we are taking. If we are on the outside we will not be able to make him turn. It is evident that we cannot employ this means of stopping a run-away unless we are sure of our horse and ourselves. while going on a straight line. J have never succeeded in this. If the amount of open space and the nature of the ground allows us to do so, we should at first take a very large circle, and should decrease it little by little. We should of course place ourselves on the inside of the circle, so as to be able to pull the run-away in the direction we are taking. If we are on the outside we will not be able to make him turn. It is evident that we cannot employ this means of stopping a run-away unless we are sure of our horse and ourselves. I was lucky to stop two mad run-aways in the manner I have just described. The first one was at Havre, and his rider had let go the reins and had caught hold of the pommel of the saddle with both hands. The second was in the Bois at Paris, and was ridden by a young girl. In both cases, it took me from fifteen to twenty minutes to stop the horses. As I was on thoroughbreds, I had strength and speed at my disposal. With respect to the lady, I took the precaution to place myself on her off side, for her legs would have been in the way on the other side. Digitized by Microsoft® 195 JUMPING. IT is generally said that to make a horse jump, the hands should bz raised the moment he comes to the fence ; but in acting thus his natural powers are hampered and he is prevented from jumping freely. In order to jump, he should have his head and neck perfectly free, because if they are raised by the hands their freedom and spring will be spoiled, and the weight will be thrown on the hind quarters. Consequently, if he has a light mouth he will pull up in front of the fence, and if he has a hard mouth he will get the better of his rider, in which case he can only make a half-rear, and consequently his fore legs will clear the obstacle and his hind legs will catch in it. Hence, while driving him forward with the legs, we pull him back with the reins. The forehand can get the better of the curb, but only by an effort which will considerably tire the horse. There is a general theory for making horses jump, but we can quickly see that, in practice, each horse has his own particular way of leaping. In my opinion, the best way to teach a horse to jump is at first to place a log of wood on the ground, and lead him over it at the walk. When he obeys, he should be patted on the neck and have some carrots, so as to gain his confidence. This will be an affair of only two or 13* 13* Digitized by Microsoft® 13* 13* 13* Digitized by Microsoft® JUMPING. 196 three lessons of about ten minutes’ duration. When he has full confidence, we may lunge him over the log, while gradually getting further and further away from him. As soon as he will walk over the bar, while the breaker stands in the middle of the school, the bar can be raised from 12 to 16 inches, and the horse left to jump it in his own way. The chief thing is that he clears it. This plan of accustoming a horse to a fence, has been practised in circuses from time immemorial. A horse which likes jumping will generally rush, and should be calmed down, so that he may take the leap quietly. JUMPING. If, on the contrary, he stops or hesitates, he should be encouraged by the voice and should be shown the driving whip, but should not be struck with it, or frightened at starting. We should, however, make him go over the bar, and should carefully note how he jumps. The best fencers jump straight, freely, and without pausing. Some leap “stickily,” and others jump sideways. Horses which jump freely and of their own accord need only be taught to clear the fence, successively, at the walk, trot and canter. Horses which make a half halt when coming up to the obstacle, should be stimulated by the driving whip until they have lost the habit of stopping. Consequently, we should not let them jump at the walk and trot, until they will freely do so, at the canter. Things are not much more complicated with horses that jump sideways, and we have only to oppose their shoulders to their haunches. If, for instance, we are on the left hand, and the horse throws his hind quarters to the left, and con- sequently to the inside, we should pull the lunging rein, in order to bring his shoulders to the left, and at the same time we should touch the left haunch with the lash of the whip, to send the hind quarters to the right. If, on the contrary, the horse brings his haunches to the right, that is to say to the Digitized by Microsoft® JUMPING. 197 outside, we should let out the lunging rein, and at the moment the animal is taking off, we should make him carry his shoulders to the right, by threatening him with the whip under the muzzle. The bar should be kept very low for this work, and should be raised only little by little, according to the strength and cleverness of the horse. We should take great care never to raise the bar so high as to require the animal to make a great effort to clear it, especially if he is young. Although this would not be so bad with old horses, we should do all we can not to discourage them. As soon as the horse jumps freely with the lunge, he can be mounted, and made to follow the same programme, while beginning with the bar on the ground. * Tt is an absolute rule in riding that the hands ought always to remain in com- munication with the mouth. Digitized by Microsoft® JUMPING. At first, we should not trouble about the animal's style of jumping, but should let him fence in his own way, and should study it. In leaping, as in every other exercise which demands great energy on the part of the horse, the rider should take account of the natural capabilities of his mount, and should adapt himself to them. If they requue to be set right, he should gradually do it later on. I have said that the hands should make no effort to raise the horse when taking off. I repeat that the head and neck should be perfectly free, but I do not say that the reins should be let go; because the horse, when he is jumping, ought to maintain on the hands a light bearing,* which he does not take at the moment of raising himself, because he has had it when coming up to the fence ; in fact, he merely preserves it. The elasticity of the hands and even of the arms of the rider allows him to keep up this bearing on the bit, without * Tt is an absolute rule in riding that the hands ought always to remain in com- munication with the mouth. Digitized by Microsoft® Digitized by Microsoft® JUMPING. 198 increasing it. It is better for him to diminish it a little, when the horse extends his head and neck to jump. In other words, the horse, not the rider, takes this bearing. increasing it. It is better for him to diminish it a little, when the horse extends his head and neck to jump. In other words, the horse, not the rider, takes this bearing. Some horses jump best if they have a good hold of the bit, when coming up to the fence, especially if they are going fast. Others require to be ridden with a slack rein, so that they may take the necessary spring. Nevertheless, in order to be certain that a horse will jump, we should firmly close our legs and feel his mouth, for otherwise he can very easily refuse. It is of course understood that the reins should be slackened a little the moment he is taking off. If the reins are slackened too much, he will often jump stickily or refuse, and if we slacken the reins too late, we will prevent him jumping, or will hamper his movements. * T use only the snafile when jumping, and I employ this curb only to regulate the speed between the fences. Digitized by Microsoft® Digitized by Microsoft® JUMPING. The hands, therefore, ought to do three things :—1. Support the horse up to the moment he takes off. 2. Give him his head during the time he is clearing the obstacle. 3. Feel the snaffle,* so that we may lightly get him into hand the moment he lands. The legs of the rider ought to support the horse during the whole time—namely: 1. Before leaping, so as to press him up and make him jump. 2. During the leap, in order to make him get his hind legs under him, so that he may not hit the fence. 3. After the leap, in order to support the hind legs when they come down and relieve the fore legs. Finally, when the legs are thus supported they will keep the rider in a good position. The pluck and confidence of the rider plays a large part in jumping. If he comes up to the fence without having decided to clear it, the chances are that he will remain on this side of it. It is said that the horse understands the feelings of the * T use only the snafile when jumping, and I employ this curb only to regulate the speed between the fences. Digitized by Microsoft® JUMPING. 199 rider. This is not quite true, because it is evident that the animal cannot know what is in the mind of the man on his back; but he can feel that the ‘aids,’ like the will of the rider, are hesitating. To make the horse resolute we should, in the first instance, be resolute ourselves, If our pluck gives way, our “aids” will be vacillating ; but if we harden our heart, we will transmit h, yey, =a Fig. 46.—Clearing a fence. Fig. 46.—Clearing a fence. confidence to the horse by means of the “aids,” which, in this case, should act with vigour and precision. The rider who goes up to a fence for the first time, usually imagines that he will receive a terrible shock. As a rule, he instinctively stiffens himself so as not to be displaced, and when he falls off, the accident is generally due to this stiffness. Digitized by Microsoft® JUMPING. As I have already said, we should give with the hands at Digitized by Microsoft® JUMPING 200 the exact moment when the horse raises himself, and should preserve his suppleness by a stronger pressure of the legs: In this way the shock and displacement of the seat are only trifling. the exact moment when the horse raises himself, and should preserve his suppleness by a stronger pressure of the legs: In this way the shock and displacement of the seat are only trifling. I have said that all horses do not jump in the same way. With those which raise themselves well with all four feet, and Fig ays Hipae valde his forchand a good deal ven jumping. Fig ays Hipae valde his forchand a good deal ven jumping. clear the fence while keeping the body nearly horizontal, the rider has only to keep his body in a perpendicular position (Fig. 46). If the horse, when jumping, raises his forehand a great deal, as in a half-rear (Fig. 47), the rider ought to pro- portionately lean forward at the moment when the horse raises himself ; but as the horse comes down, he should bring his body back, for three reasons: First, not to be thrown Digitized by Microsoft® JUMPING. 201 forward by the propulsion’ given by the horse; second, to lighten the ‘forehand, which, on coming to the ground, will have to bear all the weight of both horse and rider; and, third, to keep his seat and support his horse in case the animal’s forelegs give way. forward by the propulsion’ given by the horse; second, to lighten the ‘forehand, which, on coming to the ground, will have to bear all the weight of both horse and rider; and, third, to keep his seat and support his horse in case the animal’s forelegs give way. When a horse brushes a fence with his forelegs, and raises Fig. 48.—Horse raising his croup when clearing a fence. Fig. 48.—Horse raising his croup when clearing a fence. his croup as if he were kicking,* we should carry the body back the moment the forehand is raised as high as the fence, so as to avoid putting weight on the forehand. When the fence has been cleared, the body will be put back in its place hy the force of propulsion. * See Fig. 48 :—If the reins were drawn tighter, this illustration would serve equally well to show the position of a rider who is about to receive his horse after a jump. Digitized by Microsoft® JUMPING. I cannot too strongly impress on my readers that in all the * See Fig. 48 :—If the reins were drawn tighter, this illustration would serve equally well to show the position of a rider who is about to receive his horse after a jump. JUMPING. 202 backward movements of the body during the leap, the arms should preserve the greatest possible elasticity, so that the tension of the reins may be in no way increased, and that the horse may jump freely. Ifthe reins are too short, we should let them slip through the fingers, and should take them up again as soon as the animal lands. I am naturally led to say a word about steeplechases and hurdle races. The ignorance on the part of jockeys about the paces of horses is inconceivable. Very few of them can tell with which leg a horse is leading in the gallop. Baron Finot, who is a master, astonished me one day by saying: “Jockeys ride by instinct, and do not take the trouble to think.” In hurdle racing a horse clears the hurdles, thanks to his enormous momentum, and the harder he pulls the better pleased is his jockey. In France, steeplechases are ridden at the same speed as hurdle races, which at first sight appears dangerous. I have spoken to many jockeys on this subject, and they have all told me that the faster the speed the less danger there is for them. This seems a paradox, but it is really true. Their reason is that if a horse which is going at a moderate speed strikes a fence and comes down, the horse will nearly always fall on the jockey, in which case the result will be very serious, if not actually fatal. But if a similar fall occurs when the horse is at full speed, the jockey is thrown a few yards to the front, and generally escapes being hurt. In such a case the jockey rolls like a ball, huddles himself up, takes care not to stretch out an arm or leg, and thus nearly always gets off with only a few bruises. In England, a steeplechase jockey slackens his pace when he gets near a fence, and thus husbands the horse’s strength, and allows him to more accurately measure his distance. Horse and man thus act in harmony. Digitized by Microsoft® Digitized by Microsoft® * In that fence there was nothing to catch a horse’s legs. To clear it, like all other wide jumps, the only thing that was required was plenty of impulse. Seeing Baudres change his leg a few yards from this fence on account of the jockey trying to raise his head, I said to his owner: ‘‘ Your horse is going to fall.’? In fact, the impulse was stopped and the fall came off. Digitized by Microsoft® JUMPING. The French system is more break-neck, and requires less Digitized by Microsoft® JUMPING. 203 knowledge, but it can help to win a race. The English system is a matter of good horsemanship, but we must admit that the French system has more chances of success if the horse does not fall. In my opinion, the jockeys Hatchet and H. Andrews are brilliant exceptions respecting the way steeplechases are generally ridden. Hatchet has a very remarkable method of riding over fences, and I always follow him with great pleasure, because his style bears out the theory which I 2m never tired of maintaining—namely, that there is only one way of riding. There is only one kind which is always good on the racecourse as well as for hacking, and without it success is either pure luck or mere knack. To see Hatchet ride a steeplechase is a great treat for a connoisseur. He remains glued to the saddle, and daylight is never seen between him and it. On coming up to a fence he does not lean back, for he knows that the impetus of the hind quarters would throw him forward. He gets well down into the saddle and rounds his back so as to keep his seat and suppleness. He holds his hands low, his arms half-extended, and the reins just sufficiently felt to keep him in constant communication with the mouth of the horse. He evidently yields his fingers at the same time as he does his arms, because he is never pulled forward at the moment when the horse stretches out his head and neck to jump. He keeps a nice feeling on the remms—more by the play of the fingers than by that of the arms—even during the wild speed of a race, which is a fact that few sportsmen will admit. Also, he keeps his position before, during, and after a jump. With him there is not the slightest shock ; everything is smooth and perfect. I often hear people say on racecourses that if a horse is going to fall, nothing will prevent him, but we can deceive ourselves. It is evident that if an animal makes a mistake and fails, he will fall; but what I want to say is that a mistake Digitized by Microsoft® JUMPING. 204 which will not happen with one jockey will occur with another jockey. JUMPING. A horse’s fall is very often due to the man who rides him. I mention the following instance of this because it is conclusive :— which will not happen with one jockey will occur with another jockey. A horse’s fall is very often due to the man who rides him. I mention the following instance of this because it is conclusive :— In one season at Auteuil, Hatchet won nine races out of eleven on Baudres, and the horse fell in the two races he lost. For personal motives, another jockey of very high reputation was put on Baudres, who, in seven races, fell four times and lost each of these four events. After that Hatchet won ten consecutive races on Baudres without a fall. Thus, Baudres fell four times out of seven with a good jockey, and fell only twice out of twenty-one races with a jockey whom I con- sider to be exceptionally good. A remarkable fact which perhaps will help the reader to remember what I have said, is that all these falls occurred at the brook in front of the stands.* ” Hatchet has a particular way of his own in taking the last turn on the Auteuil racecourse, which is on the left of the stands, and is very sharp. He takes it very short, and slackens his speed to a marked extent. The others keep up the speed, and are consequently obliged to take a wide turn. Although the difference between the two methods may appear to be of no matter, it is of great importance. As Hatcher turns very short, he loses no ground, although he slackens speed, because he has less space to cover; and as he thus allows his horse to recover his wind, he is able, in the straight run home, to get out of his animal a last and supreme effort, which the others cannot obtain, because they kept up full speed the whole time. We should not forget that in such cases, horses win races JUMPING. 208 not with their legs but with their lungs, namely, by being able to stay. I have lately mentioned the name of H. Andrews, which was a pleasure I could not resist, when finishing the descrip- tion of the particular qualities which make him unrivalled. Digitized by Microsoft® JUMPING. Any one who has not seen Andrews finishing, can have no idea of the enormous energy he possesses. I have seen him, on losing his whip, take his cap to flog his horse; and then losing his cap, he used his right arm and hand with the utmost vigour and perseverance. It is no exaggeration to say that when coming up to the winning-post, he communicates energy to his horse, and shoves him in front in a particular way of his own, without the slightest slackening off. Digitized by Microsoft® Digitized by Microsoft® 206 Digitized by Microsoft® FLAT RACING. I HAD occasion to say, that in all exercises, the ordinary horseman, riding master or jockey should know what he ought to require from his horse. It is generally but wrongly thought that this rule does not hold good with respect to flat racing, at which the most successful jockeys are those who best conform to it. In a flat race, when all the horses are on about the same level, as regards speed and staying power, the jockey who can set the pace in his own way will have the best chance, and consequently his success will depend on his knowledge of his animal’s capabilities. He will give him the exact support which is necessary, and will put him at the speed to which he has been accustomed towards the end of his training; will maintain it during the race, and will reserve his supreme effort for the finish. As the horse has not been over-ridden during the race, he can easily, at the finish, make the last rush, upon which success almost always depends. I can easily prove this fact by the case of Archiduc, whom everyone remembers. On three consecutive occasions, Archiduc took the lead at a pace which suited him, and no horse was able to get near him. In the Chantilly Derby, however, Fra Diavolo tried to take the lead. These two struggled for supremacy, Digitized by Microsoft® FLAT RACING. 207 and were at the top of their speed before a quarter of the distance had been covered. The jockey of Little Duck, who was behind, did not trouble about the others, but judged his own pace for three-quarters of the course, and thus saved his animal’s powers for the last moment, at which time he suddenly sat down and rode, passed the others, who had not an effort left in them, and won easily. The jockey of Little Duck simply put into practice the rule which I have specified. In a word, he knew how to keep something for a rainy day, which in this case was a reserve of energy to be used at the finish. * Such judgment is extremely difficult with a speed of about eleven hundred yards in a minute. Digitized by Microsoft® FLAT RACING. The greatest difficulty for a jockey who is leading, is to judge the pace, and to know how fast his horse ought to go.* If he goes beyond this speed, he will take the wind out of his horse, and will be unable to make a last rush. If he keeps back, he will necessarily do so by taking too strong a hold of the reins, and will consequently tire the horse’s loins and hocks. But it is the loins and hocks which win a race at the finish. The best jockeys win at the last moment, and as near the winning-post as possible. Their perfect knowledge of pace enables them to judge if their opponents can keep up the speed at which they are going, and to feel if they can increase their own speed. At Chantilly, I saw Watts on Louis d’Or apply this principle in a very clever and lucky manner. It was a welter race, and all of them carried 12st. 8lb. Atalante was the favourite, and was certainly the best made to carry weight. In this two-mile race, Watts let Louis d’Or go his own pace, and did not try to catch up the others. Although he was two hundred yards behind at the beginning, he did not increase his speed. The others having gone too fast, were obliged to * Such judgment is extremely difficult with a speed of about eleven hundred yards in a minute. Digitized by Microsoft® FLAT RACING. 208 slacken speed, but he maintained the same pace and beat the favourite by a few yards. We can say with confidence that he won this race by his knowledge of pace. Fred. Archer gave a grand performance by winning the Grand Prix of Paris on Paradox. He did not fail for an instant in fine horsemanship, coolness and cleverness. At first he remained behind his field, but towards the end he drew up alongside Reluisant, who had won the Chantilly Derby, and was his only formidable opponent. , He stuck close to him, and although he saw that Reluisant could go no faster, he kept with him until close to the winning- post, and then won by a neck. This victory shows us only a part of Archer's talent. The thing which was best about him and which made him a great master, was his seat. * Jockeys, of whom the majority sit badly, do not now ride in a ‘‘triangle,” ‘as people formerly said, with the reins, saddle and stirrups as their three points of support. If this style has left the turf, it is still unfortunately employed in hacking. Digitized by Microsoft® FLAT RACING. He sat well into the saddle, and rode with long stirrups. When he raised himself on his stirrups, his seat grazed the saddle. His seat had no resemblance to the strange, if not ridiculous position which many jockeys adopt, and which some of them exaggerate to such a degree that we could place a hat between them and the saddle.* Archer always sat like a horseman, with his horse enclosed between his hands and legs. In short distance races, he was unjustly said to be tricky at getting off first. At the moment when the starter lowers his flag, the majority of jockeys are content to slacken the reins and let the horses go off as they like or as they can. Archer left nothing to chance, and he ruled his horse like the true horseman that he was. Having always his legs close to the horse’s sides, he surprised the animal at the moment of starting by a vigorous pressure of the legs, and instantly put Digitized by Microsoft® FLAT RACING. 200 him on his feet before the others could get on theirs. He was therefore going fast before his opponents were out of a canter. We see by these examples that all kinds of riding resemble each other. The science of equitation is as necessary to a jockey as to any one else, and consists of judgment, hands and legs. 14 Digitized by Microsoft® Digitized by Microsoft® THE HUNTER. ALTHOUGH a hunter need not be of any particular breed, he should be chosen from the best horses. My advice to a man who wants to hunt is to select a horse which has been born, reared and trained in the country where the hunting is to take place. Horses, like men, have natural capabilities which correspond to the climate and nature of their native land. If they are sent elsewhere they will lose these qualities, without being able to acquire those of the animals which were born in the place. Let us take for instance the small horse of the Pyrenees, which has marvellous qualities. In his own country he is clever, active, very sure-footed and temperate, and in the country about Pau he makes a first-rate hunter ; but if he is taken to Rome or Vendée he will lose some of his good qualities. It is the same with all other breeds of horses. Therefore, for hunting, take a horse of the country. The English hunter is the only animal which is good in all countries, and which preserves his good qualities, no matter what is the nature of the sport. He combines almost all the desirable points. He is nearly thorough-bred, although his shape and make are not what we would look for in a race horse, which has plenty of daylight under him. The hunter Digitized by Microsoft® Digitized by Microsoft® THE HUNTER. 211 is closer to the ground, and should be particularly good about the loins and hocks.* is closer to the ground, and should be particularly good about the loins and hocks.* His breaking begins later than that of the thorough-bred. He is taken up at about three-and-a-half years old, and requires a year or eighteen months to learn his work.t In judging him, we should specially consider his style of jumping different fences, and the pace at which he takes them. If the obstacle is a hedge, he should take it quietly, on account of the small effort required to clear it. If it is high and stiff he should collect himself for a big effort, and should go very straight and steadily at it. If it is a fairly wide stream, the pace should be quite different, and the head and neck, which should not be hampered by the hands, should be extended. t His work is simply jumping, and he never fetches a high price, unless he is a free goer, very sure-footed and a big jumper. The long training which he requires is the cause of his high market value. Ordinary jumpers are ready at from three-and-a-half to four years old. 14* * The hunter is the only horse they have not tried to produce in France. This is to be regretted, because breeders would have succeeded with him, as with other horses. Frenchmen make the mistake of wanting cheap hunters. THE HUNTER. The horse should stretch himself out, should take a light but very free bearing on the hands, and jump at full speed with freedom and pleasure. The only fault an English hunter has, is that he costs a great deal of money. Usually one pays about three hundred pounds for a good animal of this sort; but in exceptional cases, the price may be five or six hundred. I repeat that this kind of horse is the only animal which is fit to hunt anywhere. In every country, except in England, it is generally thought that as accidents are very common in hunting, only cheap horses should be used for this work. All my readers know from experience that in buying a horse, they cannot be sure of getting value for their money. Therefore I think it very foolish to practise economy when buying a hunter. The English, who are very practical, and who know the value of 14* 14* Digitized by Microsoft® THE HUNTER. 212 money, are economical in the purchase of harness horses ; but price does not stop them when buying a hunter, to whom they will have to trust their life. money, are economical in the purchase of harness horses ; but price does not stop them when buying a hunter, to whom they will have to trust their life. We often hear it said that Mr. X is fortunate with all his horses, and that Mr. Y, being out of luck, cannot place his hand on a good animal. We may be certain that chance has not much to say in this matter. The fact that Mr. X has often good horses is perhaps due to his understanding their powers and working them with good judgment. If Mr. Y has only bad horses, notwithstanding the long prices he gives, it is perhaps because he is an indifferent rider who does not understand horses, and is ignorant of what he ought to require from a horse and of the manner he ought to require it. I do not hesitate to recommend those who will take my humble advice, to get the best and strongest horse they can for hunting. The English say that hacking is an art,* and hunting is pluck. In my opinion this proverb is absolutely correct. * I must say that I have always seen more hacking than art in Rotten Row. THE HUNTER. Without doubt pluck is required more with hounds than when hacking, because the speed is greater, and the fences are un- known. Nevertheless, pluck cannot replace the science of riding, which is even more necessary when crossing a country than when hacking, because the risks are greater and more frequent. To hunt, as well as to hack or ride races, it is necessary to know how to ride. Digitized by Microsoft® 213 Digitized by Microsoft® * T hope that my readers will excuse the liberty and freedom of my criticisms, on account of my absolute conviction that they are only too well founded. Be- sides, I believe that many persons who have special knowledge about these things, would willingly agree with the majority of my observations, if they had liberty of speech. * T hope that my readers will excuse the liberty and freedom of my criticisms, on account of my absolute conviction that they are only too well founded. Be- sides, I believe that many persons who have special knowledge about these things, would willingly agree with the majority of my observations, if they had liberty of speech. ¢ In my opinion, the half bred is the best animal for war. In this I am not contradicting myself, although I said at the beginning of this book, that I pre- ferred the thorough-bred to all others. He has energy and cleverness which is rarely found to the same extent in other horses; but these qualities are not the ¢ In my opinion, the half bred is the best animal for war. In this I am not contradicting myself, although I said at the beginning of this book, that I pre- ferred the thorough-bred to all others. He has energy and cleverness which is rarely found to the same extent in other horses; but these qualities are not the THE ARMY HORSE.* My intention was to discuss in this book all kinds of riding, but up to the present I have spoken only of hacking, riding for sport, and breaking. The saddle horse is not only destined for sport, and riding is not merely a luxury. At the present time the horse is an essential element of the military power of a country. He isa warlike arm which it is necessary to choose, prepare, and manage. Late wars have proved that cavalry are required to play a decisive part in military operations. In every country, this branch of the service has been increased in number and strength. Recently pamphlets, leading articles of newspapers, and reviews have shown the great attention with which the public has studied the subject. I trust I may be permitted, or at least pardoned for saying a few words about the war horse.t ¢ In my opinion, the half bred is the best animal for war. In this I am not contradicting myself, although I said at the beginning of this book, that I pre- ferred the thorough-bred to all others. He has energy and cleverness which is rarely found to the same extent in other horses; but these qualities are not the Digitized by Microsoft® 14 THE ARMY THE ARMY HORSE. 214 The troop horse, which is the constitutive element of the cavalry, ought to have certain qualities, of which the principal are soundness and hardiness, With him it is not a question of fine handling or scientific movements. He has to carry his man safely and for a long distance, and by the strength of his constitution he has to bear fatigue and all the miseries of a campaign. Also, he must not cost too much, because money is the chief sinew of war, despite progress and innovations. A horse does not cost merely the sum paid to his owner, to which has to be added all the money spent on him from the day of his purchase to the time he is really fit for work. A horse bought for forty pounds, which has to be looked after and fed for a year at a remount depot, before being sent to his regiment, will really cost eighty pounds up to the day he takes his place in the ranks. Digitized by Microsoft® THE ARMY HORSE.* I have not the presumption to discuss financial and military questions, which are entirely beyond my province. I reason only as a horseman. I say with respect to remounts, that we should consider their necessary qualifications and net cost at the same time. If I occupied myself with their qualifications without touching on the subject of price, my theory might appear useless, but I have the one firm ambition to give only practical advice. We all know how horses are bought for the army. Remount only desirable ones in an army horse. The thorough-bred will perhaps stand fatigue better than any other. In a charge he has marvellous and incomparable dash ; but how will he bear all the privations and miseries of a campaign ? In the Crimea, the English lost the majority of their thorough-breds, but the Normans, Percherons, Bretons and Auvergnans held out admirably. The war horse ought to remain serviceable under conditions of hunger, cold, rain, snow and nights without shelter or covering. I do not think that the thorough-bred is capable of this form of endurance. A breed which has not been hardened may yield capital horses for ordinary work, but they will not be hardy and enduring enough for warfare. To make use of a thoroughbred, one inust know more than ordinary cavalry men do about riding. Digitized by Microsoft® THE ARMY HORSE. 215 officers go to the different breeding centres, examine the horses from three to four years old, and make purchases. These visits are necessarily foreseen. Dealers of all nationalities, and especially foreigners, take the precaution to come prior to the arrival of the remount officers, and buy the best they can find, which is easy for them, because they give a better price. The remount people choose the best of what is left. These horses, which are considered too young for any work, are sent to the remount depots, where they are kept until they are five years old. Sometimes they are put out to grass with farmers at a moderate cost. * Technically, the horses are five years old, because they take their age from the first of January; but really they are only four years and nine months, because they are born in the spring. They go to their regiments in the first half of the October of their fourth year, and are then four years and six months old. They are put in the squadron of the depot to have the rough edge taken off them, and are accustomed to the stable, saddle, weight of a man, and external objects. Inthe first half of January, that is tosay, when they are four years and nine months old, they are sent to their respective regiments, and their breaking commences. Digitized by Microsoft® THE ARMY HORSE.* I set aside all details to arrive at the principal point, namely, the age at which remounts should be bought; taking for granted that they are not fit for work until five years old.* Only at that age they are begun to be exercised and are put to more or less appropriate regular work, in other words, they are broken in. This system is a tradition and a principle ; but at the risk of running against all accepted ideas, I say that the tradition is an error, the principle false and the system bad. It is a loss of precious time to keep a horse until he is five years old before exercising, breaking and training him for military purposes. Besides, this delay doubles his first cost and deteriorates his physical organs, which suffer atrophy, on account of insufficient feeding and work. At three years and a half, a well-shaped horset which has * Technically, the horses are five years old, because they take their age from the first of January; but really they are only four years and nine months, because they are born in the spring. They go to their regiments in the first half of the October of their fourth year, and are then four years and six months old. They are put in the squadron of the depot to have the rough edge taken off them, and are accustomed to the stable, saddle, weight of a man, and external objects. Inthe first half of January, that is tosay, when they are four years and nine months old, they are sent to their respective regiments, and their breaking commences. ¢ I mean a French horse, from whatever part he may come. Digitized by Microsoft® THE ARMY HORSE. 216 been properly fed and exercised, is sufficiently developed and strong to bear the gradual work which precedes and facilitates breaking. By the age of four years he could be properly broken and rendered fit for military service, after a few months of which work, his training is complete, and he has the strength and endurance that are pre-eminently neces- sary in an army horse. * Tf the objection is made that some (not many) of the animals may suffer, I answer that I propose that only horses three years and six months and even three years and nine months old should be broken, in which case they will have a preparation of three months. Also, I would not require from them such severe work as similarly bred animals which race, have to do, and which are trained at two-and-a-half years old at the latest. Another objection is made about the great difference between the weights carried by a race horse and troop horse ; but the latter is a year older than the former, and his stronger build enables him to carry weight better. He is hardier, he carries his full weight only on exceptional occasions, and his work is much slower. If we go on gradually, as is done with race horses, we shall succeed without any difficulty in developing his weight-carrying power. To the pure theorists who wish (o wait for the complete ossification of the Digitized by Microsoft® THE ARMY HORSE.* If we wish to discard routine, and to adopt a system of liberal feeding and rational breaking, we will obtain a troop horse which, at four and a half years, will be worth as much or more than the six-year old horse, after he has been broken. Whence comes the deeply-rooted idea that a horse cannot be used in the army before he is five years old? How is it that many eminent remount officers have perpetuated this principle? I suppose that, having ascertained that the five- year old horses they procured were weak and undeveloped, they concluded that it would have been impossible to work them earlier. They were content with the fact, but did not try to find out the cause. Nevertheless it has been long and repeatedly proved that a three-year old horse, which has been well fed and well exercised, can do very hard work, and maintain a high rate of speed.* * Tf the objection is made that some (not many) of the animals may suffer, I answer that I propose that only horses three years and six months and even three years and nine months old should be broken, in which case they will have a preparation of three months. Also, I would not require from them such severe work as similarly bred animals which race, have to do, and which are trained at two-and-a-half years old at the latest. Another objection is made about the great difference between the weights carried by a race horse and troop horse ; but the latter is a year older than the former, and his stronger build enables him to carry weight better. He is hardier, he carries his full weight only on exceptional occasions, and his work is much slower. If we go on gradually, as is done with race horses, we shall succeed without any difficulty in developing his weight-carrying power. To the pure theorists who wish (o wait for the complete ossification of the * Tf the objection is made that some (not many) of the animals may suffer, I answer that I propose that only horses three years and six months and even three years and nine months old should be broken, in which case they will have a preparation of three months. cartilages before putting a horse into work, I reply by advising them to buy five- and-a-half year old horses, so as to have them ready at six. They will tell me that such animals cannot be found. Why? Because trade gives them to the buyer at a much earlier age. Therefore the universal practice is to work them at an earlier age. Q. &. D. The four year old should, of course, be treated with care. Here the important thing is to prove that at that age he is fit for work. THE ARMY HORSE.* Also, I would not require from them such severe work as similarly bred animals which race, have to do, and which are trained at two-and-a-half years old at the latest. Another objection is made about the great difference between the weights carried by a race horse and troop horse ; but the latter is a year older than the former, and his stronger build enables him to carry weight better. He is hardier, he carries his full weight only on exceptional occasions, and his work is much slower. If we go on gradually, as is done with race horses, we shall succeed without any difficulty in developing his weight-carrying power. To the pure theorists who wish (o wait for the complete ossification of the To the pure theorists who wish (o wait for the complete ossification of the Digitized by Microsoft® THE ARMY HORSE. 217 Let us take as an example the numerous half-bred horses of Normandy. They are supposed to be the least precocious of all the horses produced in France, and it is generally admitted that they cannot be got ready much earlier than six years old. Norman breeders naturally class their colts, at first, accord- ing to their origin, and afterwards by their make and shape. The young animals are put into the three following classes :— 1. Match trotters. 2. Horses for fashi 2. Horses for fashionable purposes* and for trade work. 2. Horses for fashionable purposes* and for trade work. 3. Remounts. 3. Remounts. Trotters begin Digitized by Microsoft® t It is interesting to note that half-bred trotters stand racing better than thorough-breds. Thorough-breds ‘‘click”’ (‘‘ forge”) when running or when being trained, far 3. Remounts. Trotters begin 3. Remounts. Trotters begin Trotters begin to be gradually exercised and trained at two years old, or at two and a half at latest. They are generally full grown at three, are in good form and compete in races. The best trotting prizes are reserved for three-year olds. They can actually trot a distance of two miles and a half in about six minutes and a half. The distance is often over three miles, to successfully cover which the trotter requires speed and staying power. During his training, and during the time he is racing, he has to undergo the severest exertion of which a horse is capable.t A horse for fashionable purposes, which is intended for a dealer who can pay a good price for him, is almost a foal at * This class comprises horses which have been intended to act as stallions in studs, but as they did not fulfil the expectations of their owners, they were cut and used for commercial purposes. t It is interesting to note that half-bred trotters stand racing better than thorough-breds. Thorough-breds ‘‘click”’ (‘‘ forge”) when running or when being trained, far more frequently than half-breds. Digitized by Microsoft® Digitized by Microsoft® THE ARMY HORSE. 218 three years old. His growth is incomplete, his body slender, he is soft, and he ought not to be worked. He approaches his full height, puts on muscle, and becomes capable of moderate work only when he is rising four, or is four off ; and we then find him in the hands of the dealer, fat, glossy, and ready for the purchaser who is in no hurry and who does not want much from him. The same remarks may be made about the commercial horse, who differs from the fashionable one only as regards price. The three-year-old remount is of no value. He is generally fat, at from three to four years old, when he is shown to the purchasing officer. A few feeds of oats at the last moment, the whip, and ginger, give him an appearance of strength ; but in reality his muscles are weak, and he has neither good paces nor staying power. Left to himself he gives way, goes on his shoulders, and is not fit for any kind of work. Such are the horses which are nearly of the same breed. Digitized by Microsoft® 3. Remounts. Trotters begin Some are fully furnished at three years of age, and can stand training and racing, which are the hardest kind of work. Others at four-and-a-half years old are hardly ready for even very moderate work. It is therefore necessary to take great care of them, to let them acquire strength, and to give them the muscular development of which they are deficient. Whence comes this difference? Does Nature make some more precocious than others, all being of the same origin? Is a horse’s muscular development in inverse proportion to his good looks and speed? In no way! This great difference results solely from the method employed in feeding and educating different classes of horses. If a uniform system were used, all would have similar precocity, development, and power. Horses differ in shape, action, and usefulness, but if they were all treated properly up to the age of three years, they would all be sufficiently hardy for any kind of work. The half-bred trotter which 1s trained and run at three years Digitized by Microsoft® THE ARMY HORSE. 219 old is a proof of this. His work is extremely hard, and is out of all proportion as compared to that required from a fashionable horse, or from a troop horse, and he can stand it as well or better than any other kind of animal without becoming unsound. He is, however, well fed from the first, and is methodically exercised from an early age. In fact, he is brought up like a thorough-bred. old is a proof of this. His work is extremely hard, and is out of all proportion as compared to that required from a fashionable horse, or from a troop horse, and he can stand it as well or better than any other kind of animal without becoming unsound. He is, however, well fed from the first, and is methodically exercised from an early age. In fact, he is brought up like a thorough-bred. All race horses are not thorough-breds, but all have been brought up with a view to racing. From their birth they receive the care and feeding which is appropriate to the work they will have to do. They begin to eat a little oats during the first days of their hfe, and when six or eight months old they get five quarts of oats a day. Digitized by Microsoft® Digitized by Microsoft® 3. Remounts. Trotters begin At eighteen months they are stabled, highly fed, and trained. At two years old they take part in special races. A similar, though somewhat slower method, is employed with trotters, which do not run until they are three years old. In both cases nature is helped by feeding, and by gradual and constant exercise. The breeder of other horses hinders nature by insufficient feeding. A horse grows fast, and his skeleton and muscular system are large. In order that he may grow quickly, and acquire the necessary strength, his food should be abundant, and his exercise should be favourable for the development of his strength and speed. The only young horses which are well fed and exercised, are those which are intended for racing or for stud purposes. Other animals are kept at grass, because that is the most economical method of bringing them up. Under the influence of this watery and innutritious food and want of exercise, the horse’s development is retarded in every way, his bones ossify slowly, and his muscles do not stand out. The older he grows, the more debilitated does he become. At three years he is only backward, but between four and five years of age he suffers greatly fror: anaemia, Digitized by Microsoft® THE ARMY HORSE. 220 which is a poverty-stricken disease that needs repair. I feel certain that success in its treatment can only be incomplete, and that a horse recovering from it can never be as good as he would have been, had he been brought up differently. The longer we wait to restore the young horse, the longer and more unsuccessful will be the treatment. If the animal is only a three-year-old, a few months may be sufficient, but if he is four years, and still more if he is five, he will require a year or longer. This undeniable fact is easily explained, because the longer the animal suffers from anzmia, the more severe does the disease become, and the greater is the general wasting. which is a poverty-stricken disease that needs repair. I feel certain that success in its treatment can only be incomplete, and that a horse recovering from it can never be as good as he would have been, had he been brought up differently. The longer we wait to restore the young horse, the longer and more unsuccessful will be the treatment. 3. Remounts. Trotters begin If the animal is only a three-year-old, a few months may be sufficient, but if he is four years, and still more if he is five, he will require a year or longer. This undeniable fact is easily explained, because the longer the animal suffers from anzmia, the more severe does the disease become, and the greater is the general wasting. The numerous cases of mortality among young horses are to be attributed to this state of impoverishment (anzmia). The debility which results from insufficient food during the period of growth, makes them unable to resist the slightest illness or the most trifling accident. It is also the cause of many diseases, strangles, for instance, and obscure lamenesses which are generally put down to youth A young horse is vigorous and healthy, if he is brought up under good conditions; but is weak and sickly if he has a lymphatic temperament, or is anzemic from want of food or exercise. Norman horses are generally lymphatic, soft and backward, because Normandy produces less oats than other breeding districts, and it is the custom in that country to give it only to trotters. The breed feels the bad effects of this, and soon degenerates, unless the stallions are chosen from trotters which, having been always kept in good condition, are able partly to correct the lymphatic temperament of the dam which has suffered from anemia in her youth. It 1s not correct to say that trotters acquire their magnif- cent development and strength on account of their being exceptional animals. Besides the brilliant instances which Digitized by Microsoft® THE ARMY HORSE. 221 have been mentioned, there are many trotters which compete in local events, and after leaving the racecourse do excellent work. Often they have nothing remarkable about them, except their pace. They are not naturally precocious and have advanced by care, good feeding, and education, while others have remained behind. At five years old the French horse could have nearly all his height and strength and be ready for breaking. If he is otherwise, the state of his development will be propor- tionate to the amount and nature of the food and exercise he has received. Formerly it was the rule among riding masters not to break- in a horse for high-school work until he was at least seven years old. * Without doubt the objection will be made that a great number break down intraining. To this I reply that they begin their training at the age of eighteen months, which is a very dangerous proceeding. I finally propose to begin breaking at three-and-a-half years; the amount of exertion which I require being far less than that incurred during training. Digitized by Microsoft® * The precocity of the thoroughbred will of course be put forward as an objection to what I say ; but we are not concerned with two-year-olds. Besides this, the precocity of feeding and exercise has always appeared to me to have a more decisive effect than the precocity of breed. Digitized by Microsoft® 3. Remounts. Trotters begin For many years I thought and acted like the others, and to make my school horses, I took animals of from seven to eight years old; but little by little, 1 became im- pressed by what I saw on racecourses. At Epsom, Chantilly, Auteuil, and Longchamp, the longest and severest races were reserved for three-year olds.* I observed that when breaking in a school horse I did not require from him a tenth of the exertion which he would have to do, if he was trained or raced. Starting on the principle that if a horse can do mucn he can do httle, \| came to the conclusion that a three-year- old which could stand training and racing, would much more easily stand school breaking. Although this reasoning ap- pears to me to-day to be very simple, I was not able to act on it without extreme timidity, so great is the imfluence of preconceived ideas. Whenever I spoke about it to horsemen and authorities of that time, they shrugged their shoulders, and regarded it as nonsensical. Since then I have learned THE ARMY HORSE. THE ARMY HORSE. 222 that every attempted innovation runs a great chance of being received in this manner. Nevertheless I decided, not without great apprehension, to make an experiment, and I took in hand a four-year-old which had been well looked after. I found that he had quite as much strength, and much more suppleness, than my other pupils which were seven years old. Encouraged by this result, I tried a three-year-old with the same success. Since then I have broken-in for high-school riding a score of thoroughbreds of this age, and have always obtained better results than with older horses. Finally, I pushed the experiment further, in order to find out the greatest amount of exertion which could be demanded from a very young horse without distressing him. I broke Viscope, a thoroughbred mare by Vermouth out of Vinai- grette,* when she was two years old. At three years her education was complete as a hack, jumper, and school horse, and she received a medal at the horse show. She is now seven years old, perfectly sound, unblemished, and had not even a windgall when \| disposed of her. I could cite many other examples. I only wished to ex- plain how experience led me to the conclusion that to take a horse at three-and-a-half years old in order to break and train him for any particular work, is to begin at an age when all horses which have been properly brought up and fed, can stand work. Further, it is to take him at an age when progressive muscular exercise is indispensable to him. This exercise, so far from wearing him out and making him un- sound, strengthens his limbs and body, and quickly puts him in full possession of all his powers. THE ARMY HORSE. 223 A three-and-a-half year old horse is more supple and can be more easily educated than a five-year-old. He has not been able to acquire the faults of temper which we find in him later on, and which are almost always the result of injudicious or cruel treatment by people about him. He has not the bad habit—which all horses left to themselves contract—of going on his shoulders, on which account the hind quarters become less developed than the forehand. Digitized by Microsoft® THE ARMY HORSE. We can then easily make him rely on himself, and put equal weight on all four limbs, which is of-great importance ; be- cause true equilibrium of the body is the one thing of all others which we should try to obtain in breaking and riding. The horse ought to be neither on his shoulders nor on his haunches ; but should have his own weight and that of his tider equally distributed on both ends. Only on this con- dition can he be light, active, and sure-footed, and capable of doing long and severe work without injuring himself. When a horse has been addicted for a long time to the habit of standing and moving in a state of bad equilibrium, his con- formation becomes distorted and can rarely recover from its effects. If, as is the most frequent instance, the horse is on his shoulders, his hind quarters are soft and drag themselves along the ground, on account of want of exercise and develop- ment; but the forehand works too much and becomes fatigued. Being overloaded and heavy, it has difficulty in changing its place, and the slightest mistake or weakness will involve the entire body. The limbs which are overworked become soon worn out. This is the only cause of the pre- mature reductions which each year decimate the effective strength of cavalry squadrons, and put many horses out of work at an age when they ought to be in their prime. The most essential part of breaking a horse is to teach him to balance himself properly. No one will deny that a horse can be more easily educated at three-and-a-half than at five THE ARMY HORSE. 224 years, consequently it is most important to begin with a horse at the former age. The entire question is whether a three-and-a-half years old horse is or is not sufficiently developed and strong to be broken and worked. In this respect theory and argument are useless ; for facts are only of value, and in this case are undeniable and invariable. Half-breds can be broken and trained for racing at two-and-a-half, and they become so fit and well, that they can run at three. Digitized by Microsoft® THE ARMY HORSE. Their work during the six months of training and during their races is twenty times, a hundred times more severe than that which a troop horse undergoes during his very moderate and restricted course of breaking. We are therefore right in concluding that remounts ought to be bought at three years old; and by taking this as the average age, we give a chance to backward breeds, and also to the ordinary methods of breeding, which, being as economical as possible, have a retarding influence in spite of everything. Remounts at four-and-a-half and five years of age are not really what they appear to be, because they have not been properly fed and exercised. [I am not afraid of adding that they are worth more at three years, because they have suffered for a shorter time, and because the watery and debilitating food which their breeders give them, though insufficient for their requirements during the first three years, becomes most detrimental to them during their fourth and fifth year. Instead of improving from three to five years by means of the food of his breeders, the young horse deteriorates. Whatever one does, whatever price or premium one gives to the breeder, he will not feed his stock properly between the age of three and five, because it is not his interest to do so, Whatever one does, whatever price or premium one gives to the breeder, he will not feed his stock properly between the age of three and five, because it is not his interest to do so, But we can easily get him to have his animal in good condition at three years of age. Precisely because breeding is a trade, the breeder’s chief object is to get rid of his stock, But we can easily get him to have his animal in good condition at three years of age. Precisely because breeding is a trade, the breeder’s chief object is to get rid of his stock, THE ARMY HORSE. 225 as early as possible. Knowing that every three-year-old which is in good condition and is well developed will be taken, he does his best to promote the development of the animal. THE ARMY HORSE. With a horse which he sells at three years of age, at a price even lower than what he would obtain for him between four and five, he can defray the cost of feeding, which he could not continue much longer. He is forced to make this expenditure, under pain of not being able to sell his stock, because with a three-year-old there is but little trickery. Above all things the horse must have height, which he will not have unless he has been well fed and well looked after. Finally, the State by buying young horses would no longer have to compete against dealers of all countries, because it can do what they cannot do. In fact, a dealer buys only to sell again, with the shortest possible delay; but the three- year-old, when he leaves his breeder’s hands, is not fit, even for fashionable work. He requires to be fed on corn and broken during a fairly long time, say, for about a year. When the remount authorities buy a horse between three and four years of age, they put him out to a farmer, whose interest is to have him poorly fed, or he is sent to a remount depot. Either in the former or latter case, the horse is under better conditions than he was when with his breeder, although the conditions are not perfect. He does not improve as much as he ought to do, and he takes eighteen months before he is ready. I consider that these young horses should be taken in hand at once, and put on food which will complete their develop- ment, will give them the desirable strength and docility, and will make them ready for work at the end of six months. In a word, they ought to be fed oh corn and broken in, from the first day, but gradually and methodically. A remount depot, instead of being an equine sheep-fold, as it was recently defined by a general officer, ought to be a true 15 15 Digitized by Microsoft® THE ARMY HORSE. 226 breaking school, strictly supervised and managed by a competent man, who should have well-instructed horsemen under him. Young horses are not got into condition by stuffing them with oats, nor broken in by giving them lots of exercise and quickening their paces. THE ARMY HORSE. When the horse is between three and four,* he leaves his breeder and is put into a stable. I beg my readers to note the progressive conditions of the following programme, which I propose, for feeding and work during the first three months. Digitized by Microsoft® FIRST MONTH. First fortnight. Six litres (54% quarts) of oats a day,f 2 in the morning, 2 at noon, and 2in the evening. Leading by the hand for an hour in the school, or better still, in the open, if the weather is fine. Second fortnight. Seven litres (6 quarts) of oats; 2 in the morning, 2 at noon, and 3 in the evening. The same leading by hand. Lunge the horses for five minutes daily, half at the walk, and half at a slow trot. * Colts ought to be cut at least six months before any work ; at two-and-a-half years at latest. Horses which are ridden too soon after the operation always become weak in the loins, and exhibit a characteristic rocking of the croup. t I take for granted that the animal has never eaten oats. If he has been fed on oats, he can get at first 8 litres (7 quarts) ; 2 in the morning, 3 at noon, and 3 in the evening. When I speak of oats, I mean French oats, and not that horrible stuff which is given even now in the army, under the name of white Russian oats. * Colts ought to be cut at least six months before any work ; at two-and-a-half years at latest. Horses which are ridden too soon after the operation always become weak in the loins, and exhibit a characteristic rocking of the croup. SECOND MONTH. First fortnight. Eight litres (7 quarts) of oats; 2 in the morning, 3 at noon, and 3 in the evening. * Colts ought to be cut at least six months before any work ; at two-and-a-half years at latest. Horses which are ridden too soon after the operation always become weak in the loins, and exhibit a characteristic rocking of the croup. t t I take for granted that the animal has never eaten oats. If he has been fed on oats, he can get at first 8 litres (7 quarts) ; 2 in the morning, 3 at noon, and 3 in the evening. When I speak of oats, I mean French oats, and not that horrible stuff which is given even now in the army, under the name of white Russian oats. THE ARMY HORSE. 227 The time for leading by hand can be increased by half an hour. The lunging should be very gradually increased, up to making the horse trot on each hand for five minutes without stopping. A few minutes’ interval should come between the changes of hand. Second fortnight. Nine litres (8 quarts) of oats; 3 in the morning, 3 at noon, and 3 in the evening. The same leading and lunging. Usually, a horse is given three feeds during the twelve hours between six in the morning and six in the evening, and he is therefore left without food for twelve hours, which I think is a bad method of distribution. The first feed ought to be given at five in the morning, and the last at eight in the evening. Digitized by Microsoft® THIRD MONTH. First fortnight. Ten litres (834 quarts) of oats; 3 in the morning, 3 at noon, and 4 in the evening. Twelve litres (10% quarts) of oats should be given to heavy cavalry horses. This ration and the same leading by hand should be con- tinued. The speed of the trot should be increased during five minutes towards the end of the lunging on each side, and the horses ridden at the walk in clothing in a plain snaffle for five minutes, the men having neither whip nor spurs. The horses should be quietly saddled in the stable, and loosely girthed up. At first the saddles should be left on for only a few minutes, and the time gradually increased. dling Second fortnight. The same work, saddling and bridling the horses in the stable and putting them on the pillar reins. Bridling after lunging, and direct flexions of the jaw. Lunging at the trot when saddled and bridled, with the stirrups hanging down. As lunging at a canter puts a horse too much on his shoulders, it should not be practised until the 15* 15* 15* Digitized by Microsoft® THE ARMY HORSE. 228 animal is broken and well balanced. No cavesson should be used in lunging, only a snaffle bridle, and any light rope, a forage cord, for instance. When the horses are ridden, some men should be kept in reserve to catch hold of the bridles of any of them which do not want to follow the track. This is not a matter of mouthing, but is merely to strengthen the animal’s loins and to accustom him to carry a man. After the three months’ feeding which I have advised, the horses will have had enough corn put into them to bear more work. As they have developed their muscles at the trot, learned to carry a man, have been bridled and saddled, and have had their jaws loosened by flexions; they are, I think, ready for mouthing, which is well under way. In fact only the A, B, C of breaking is required from the troop horse, with- out any elaboration. It will be well if he needs rather strong indications, for we should take precautions against heavy hands and rough legs. FIRST MONTH. FIRST MONTH. First fortnight. Begin by lunging the saddled and bridled horse at a trot. Gradually increase the speed, by means of the driving whip, so as to make him get his hind legs under him. Press him up to his full speed for a few minutes, while taking great care not to let him over exert himself, because, in that case, he would put too much weight on his shoulders, or break into a canter. This work should continue for five minutes to each side. Ride the horse quietly at a walk for a quarter of an hour, so as to accustom his back to the saddle, without hurting it. Lead the horse for ten minutes ; practise the direct flexion ; make him turn on his forehand; and rein him back. Lead him for an hour in the school, or, preferably, in the open if the weather is fine.* Second fortnight. Similar lunging. Begin the changes of direction by voltes, and diagonal changes of hand, and going down the centre. Teach the horse to yield to the heel. At the beginning, be content with two or three steps. Finish with work by the hand. Digitized by Microsoft® THIRD MONTH. The horse’s breaking is finished when he freely goes forward under the action of the legs, while taking hold of the snaffle ; and when he readily jumps, turns, and reins back. Here we require only horizontal equilibrium. Without doubt, an attempt should also be made to get the animal in hand, although this condition should not be insisted upon. It will be sufficient if the rider knows when to place his horse’s head in the necessary position for obtaining equilibrium. The horse should be accustomed to objects and noises, so that he may not be afraid of them. An army horse should be bold and ready to pass anything. To teach him to do so, all sorts of objects should be placed under his feet in the school. He can be taught best in the stable, at the time of feeding, to stand the noise of tambours, clarions, clashing of arms, firing, etc. I consider that three months will suffice to properly Digitized by Microsoft® THE ARMY HORSE. 229 finish this elementary and special breaking with any kind of horse. finish this elementary and special breaking with any kind of horse. After these three months of preparatory work, the properly called breaking has to be undertaken. * Up to this, it should only be asked for. + The shoulder to the wall, which is much more easy, should be done only when mounted. { What should a rider do, when he is in water, trying to cross a river ? We must not think that a horse which is not accustomed to the water will swim with ease the moment he loses his feet. The only idea which such an animal has, is to keep his head out of the water and to raise his neck as high as SECOND MONTH. Lunge at the trot for two minutes on each side. Ride. Repeat the preceding exercises. Divide the horses into twos and fours. Accustom them to cross each other in every direction. Frequently pull them up, so as to teach them * This remark applies to the entire period of breaking. THE ARMY HORSE. 230 to stand quietly. Do at a slow trot the same movements which had just been taught at a walk. to stand quietly. Do at a slow trot the same movements which had just been taught at a walk. Increase the number of side steps in rotation. Rein back mounted. Work with the hand. Second fortnight. Lunge in a similar manner. Confirm the preceding work. Do the ordinary changes of direction at the trot, with all the horses in twos and fours, and make them cross in every direction. Do the demi-volte and finish it up by a few side steps. Continue the work with the hand, while requiring the direct flexion* with the head high. Begin the lateral flexions, side steps and shoulder in.t Digitized by Microsoft® * Up to this, it should only be asked for. + The shoulder to the wall, which is much more easy, should be done only when mounted. { What should a rider do, when he is in water, trying to cross a river ? We must not think that a horse which is not accustomed to the water will swim with ease the moment he loses his feet. The only idea which such an * Up to this, it should only be asked for. + The shoulder to the wall, which is much more easy, should be done only when mounted. * Up to this, it should only be asked for. { What should a rider do, when he is in water, trying to cross a river ? We must not think that a horse which is not accustomed to the water will swim with ease the moment he loses his feet. The only idea which such an animal has, is to keep his head out of the water and to raise his neck as high as Digitized by Microsoft® possible. Therefore, the croup will be pushed down, and the position of the horse will be a half-rear; that is to say, he will be three-quarters upright (Fig. 49). This position will prevent him from advancing, and if his rider is inexperienced, the chances are ten to one that both will be drowned. If the rider pulls ever so little on the snaffle, or simply leans back, when he is in the THIRD MONTH. Furst fortnight. Similar lunging. Increase the speed of the trot in all the changes of direction, and in all the move- ments in which all the horses are working together. Lengthen the trot as much as possible in a straight line. “ Two tracks.” Begin starting into the canter on the right leg, and as soon as the horse will do this readilymake him lead off with the left leg. When the horse has become proficient at the canter, make When the horse has become proficient at the canter, make him do at this pace all the movements he has learned at the walk and trot. Increase and improve the suppling exercises by the hand. At the end of the lesson teach the horses to jump (p. 195 ef seg.). Second fortnight. Similar exercises. Confirm and improve the preceding ones. Accustom the horses to flags, sabres, lances, firearms, explosions, passing through fire, swimming,t etc. THE ARMY HORSE. 231 By this kind of breaking, which ought to be continued for three months, the horse will have learned enough for all cavalry movements, and will only require to be accustomed to outside work in the manceuvres, and to become hardened, so as to be able to stand long marches and bad weather. Fig. 49.—-Horse and rider being drowned. Fig. 49.—-Horse and rider being drowned. To recapitulate: The horse attains three years of age in the spring. If the remount authorities buy between April and September, he will be on an average three-and-a-half when he arrives at the depot, where he will get three months’ prepa- possible. Therefore, the croup will be pushed down, and the position of the horse will be a half-rear; that is to say, he will be three-quarters upright (Fig. 49). This position will prevent him from advancing, and if his rider is inexperienced, the chances are ten to one that both will be drowned. If the rider pulls ever so little on the snaffle, or simply leans back, when he is in the 232 THE ARMY HORSE. THE ARMY HORSE. 232 ae ratory work and three months’ breaking. At four years he will be broken and perfectly ready for work. Although it may be said that this four-year-old work would prematurely wear him out, T assert, and experience proves Fig. 50.—Rider making his horse swim according to Mr. James Fillis’s method Fig. THIRD MONTH. 50.—Rider making his horse swim according to Mr. James Fillis’s method that, under the conditions I have laid down, it will develop and strengthen him. position just described, the horse’s croup will become depressed more and more, the animal will become upright, will turn over backwards, while beating the water with his fore feet, and will end by sinking. The moment the horse is carried off his feet, the rider should take hold of a good handful of the mane, and bend forward on the neck ; but he should on no account touch the head of the horse. He should firmly close his knees, for if he does not do so, the water will instantly separate him from the horse. This is the only position which enables the man to remain in the saddle and the horse to swim. ‘The rider ought to hold one of the snaffle reins in each hand, and when he wishes to change the direction to the right or left, he should, for the moment, bring his hand away from the shoulder, so as to act on the animal’s mouth. But, as I have just explained, he should, above all things, avoid pulling the reins in a backward direction (Fig. 50). I presume that similar instructions are given to the cavalry of all nations. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® THE ARMY HORSE. 233 In reality,an army horse is taken up when he is five years old, and a year is spent at his breaking, which is regarded as imperfect by competent men; the reason being that he is put into movement before he is given equilibrium, which is the mainspring of correct and useful propulsion. This was the fault that Baucher found with old-time equita- tion, which knocked up horses by movement, whatever it was. Its teachers tried to obtain equilibrium, whatever it might be worth, by putting the horses into movement, so that they might get it for themselves. The actual change made by Baucher was to secure equilibrium at first, in order to obtain from it correct and useful movements. The only fault of the great riding master was wishing to perfect and refine this equilibrium, by rendering it unstable, before requiring forward movement. Although I disagree with him, in that I work my horses during propulsion, 1 do not the less begin, like he does, by getting my horse into equilibrium before putting him into movement, as previously explained. The difference between us is that, in place of demanding efforts from the horse while he is in a confined equilibrium, which is akin to immobility and getting behind the bit, I try from the first to make him obedient to the aids while he is in an equilibrium which is maintained during propulsion.* Persons who trot and canter horses before teaching them to start with their weight equally distributed on all four legs, follow antiquated methods which spoil the breaking. We cannot advantageously modify a horse’s equilibrium by keeping up or increasing his speed at fast paces. Such a proceeding would accentuate his defects and would knock him up, without teaching him anything. The great majority of horses are heavy in front, particularly * The horse which is on his shoulders can move forward, but will drag his hind legs; and he has no power of propulsion, which results from the hind legs being well under the body, in which case the necessary equilibrium is obtained. THE ARMY HORSE. 234 when one begins to vide them. By making them go fast, without having previously balanced them, one succeeds only in putting their weight more and more forward, and in pre- maturely spoiling them. Digitized by Microsoft® Digitized by Microsoft® Such animals carry the head low and the neck on a level with the withers, they are ugly, every effort in turning or stopping is painful to them, and they readily fall. Being able to place the head and neck as they like, they can effectively resist the aids. In fact the neck must be high and the head almost perpendicular, in order that the curb may act with all its power. The curb loses the greater part of its action on the bars, when the head is low, in which case it is not much more severe than a snaffle, and the horse gets out of hand. It is acknow- ledzed that a very large percentage of troop horses refuse to leave the ranks, which is a fact that proves that they are badly broken. A horse which will not obey is not broken. What a poor result is obtained by a year or more of irrational work! The military regulations ought to insist that the breaking should be conducted in such a manner that the remounts would be fit to go into the ranks on the Ist April, in case of mobilisation, which would entail a breaking of only three months’ duration. But in time of peace this period of break- ing is zztentionally prolonged for an entire year, and the remounts are not sent into the ranks until the following year. To justify this practice, it is alleged that those regiments which put remounts into the ranks after three months’ breaking, are those which use up most horses. This may be possible ; but I have my doubts. The truth is that remounts which are badly prepared by insufficient food and exercise, are prematurely broken down by the mere breaking. Rational breaking is, on the contrary, much quicker, and so far from fatiguing them develops them Digitized by Microsoft® THE ARMY HORSE. 235 and gets them into hard condition, which is a truth that I have demonstrated by incontestable facts. and gets them into hard condition, which is a truth that I have demonstrated by incontestable facts. I imagine that men would adapt themselves much more readily to the kind of breaking I have described than to the violent exercises which they have to go through, and which discourages them, because they do not know the reason. Digitized by Microsoft® Instead of interesting them in riding and inspiring them with love for horses, which should be the master feeling of a rider, they are often made to suffer through the horses, and the horses through them. People may say that my breaking is too elaborate. How is it, then, that it is not found to be too elaborate by Germans, whose dominant quality is certainly not elaboration? Why do they work troop horses zuzdi- vidually much more than they do in France? Why do they begin by balancing them? Why do they adopt rational breaking, instead of knocking up their horses? Why do their horses last for a long time? Alas! the reason for this is that they have profited by the teaching which has come to them from France; but in this classic country of riding we are kept back by routine. Digitized by Microsoft® 236 CHAPTER VIE. HIGH-SCHOOL RIDING. Digitized by Microsoft® HIGH-SCHOOL RIDING. The Spanish walk—Reversed pirouette on three legs—Reversed and ordinary pirouettes with the feet crossed—Reining back without reins—Rocking the forehand—Rocking the haunches—Spanish trot—Shoulder-in at the canter— Pirouettes at the canter—Changes of leg at each stride—Changes of leg without gaining ground—Piaffers and fassages—Differences between the passage and Spanish trot—Serpentine at the trot—Canter on three legs— Passage to the rear—See-saw piaffer—Canter without gaining ground and canter to the rear-~New school movements—School horse for ladies. THE exercises which we are going to consider are the sequel and perfected product of those which have been described. These high-school exercises, as, for instance, the Spanish trot, are more difficult, less used, and are artificial ; but the others, like the canter, are natural. I have often heard it said that high-school work ruins horses and prematurely makes them unsound. It is constantly said: “ How is it possible for an animal to do such severe work without knocking himself to pieces?” The answer is very simple: Professional gymnasts also go through extremely severe exertion. Are they in bad condi- tion? Are their arms, legs and shoulders spoiled? On the contrary, they are in a state of grand condition which distin- guishes them from everyone else. Their muscles stand out and are as hard as steel, and their general health is perfect. No doubt, if they began their work abruptly—I speak of Digitized by Microsoft® THE SPANISH WALK. 237 horses as well as of men—they would not be able to stand it, and would break down. But if the effort required for the gradual exercises is proportionate to the increase of muscular power, the work—no matter how severe it may be—will be comparatively easy, and even healthy.* It is true that many horses have been ruined by high-school work ; but only because the work was badly carried out, or because the preliminary training was insufficient. Riding, like other sciences, has its charlatans and its empirics. When a horse has been methodically broken, the practice of all the exercises through which he has gone, so far from ruining him, will only strengthen him. With respect to horsemen who turn up their noses at high- schoo] riding, and speak of it with contempt, I content myself with reminding them of the fable of the fox and the grapes. * My horses never get even the slightest blemish from my breaking, although I begin to break them much earlier than any of my predecessors have done. Digitized by Microsoft® ft It has never been known why this movement is called the Spanish walk. There is nothing Spanish in it, except the name, and it by no means resembles the walk of the Andalusian animal. When the Spanish horse walks, he bends his knees and brings the lower part of the legs inwards, which peculiarity is called e/ pasode campafia (the walk of the bell) on the other side of the Pyrenees. It would be more appropriately termed the ‘“‘ recruits’ balance step,’’ because there is a great similarity between the two, * My horses never get even the slightest blemish from my breaking, although I begin to break them much earlier than any of my predecessors have done. ft It has never been known why this movement is called the Spanish walk. There is nothing Spanish in it, except the name, and it by no means resembles the walk of the Andalusian animal. When the Spanish horse walks, he bends his knees and brings the lower part of the legs inwards, which peculiarity is called e/ pasode campafia (the walk of the bell) on the other side of the Pyrenees. It would be more appropriately termed the ‘“‘ recruits’ balance step,’’ because there is a great similarity between the two, THE SPANISH WALK. A horse is said to do the Spanish walk,+ when he raises his fore legs one after another, by carrying them forward and ex- tending them. The principal thing to observe is the way in which a horse puts his foot on the ground ; for although it is easy to make him raise his legs, it is very difficult to make him put them down properly. The breaker ought to begin this work on foot, while standing at the left shoulder of the horse and on the left track, in which ft It has never been known why this movement is called the Spanish walk. There is nothing Spanish in it, except the name, and it by no means resembles the walk of the Andalusian animal. When the Spanish horse walks, he bends his knees and brings the lower part of the legs inwards, which peculiarity is called e/ pasode campafia (the walk of the bell) on the other side of the Pyrenees. It would be more appropriately termed the ‘“‘ recruits’ balance step,’’ because there is a great similarity between the two, Digitized by Microsoft® Digitized by Microsoft® HIGH-SCHOOL RIDING. 238 case the wall will prevent him escaping to the right. He should hold the head of the animal rather high, while pushing him to the right, in order to throw the greater part of the weight of the forehand on the right fore leg, and thus to allow the left leg to be easily raised. He should then very lightly touch the left fore leg of the horse with the end of the cutting whip; but it is somewhat difficult to determine the exact spot where the leg should be touched. The sensitive spot, which varies in each horse, lies somewhere between the elbow and the pastern. The horse’s first impression is one of surprise, because he does not understand what is required of him, and he generally tries to escape. Nevertheless, he cannot go to the right, on account of the wall, and if he swerves to the left, he ought at once to be straightened by the whip. If he runs back, the breaker ought to immediately bring him forward by touching him with the whip behind the girths. After a moment, he almost always shows his impatience, by pawing the ground with the leg which has been touched. Digitized by Microsoft® THE SPANISH WALK. As soon as he raises this leg, he ought to be patted on the neck, in order to make him understand that he has done what was required. After repeating this exercise for several days, the horse, in order to avoid being touched, raises his leg the moment he sees the whip approaching it. When he well understands, and does this work with the left leg, the right leg should be submitted to the same practice by placing him to the right hand, and taking care to raise his head and carry it to the left. As the repeated touching of the whip irritates a horse, we should at first be satisfied with the slightest sign of obedience, and will thus take the best means of not disgusting the animal. He will usually raise his legs during the first lesson; but the way in which he raises them will be far from satisfactory, because he does not extend them, and contents himself by making impatient movements, pawing the ground, etc. Never- Digitized by Microsoft® THE SPANISH WALK. 239 theless, as I have just said, we ought to be satisfied, at the beginning, by the semblance which he makes of raising his legs. It would be wrong to insist, or even to strike the animal, when we can do nothing with him, beyond astonishing him. theless, as I have just said, we ought to be satisfied, at the beginning, by the semblance which he makes of raising his legs. It would be wrong to insist, or even to strike the animal, when we can do nothing with him, beyond astonishing him. Nevertheless we should gradually obtain the extension of the legs, which will not be satisfactory, unless the legs are completely and horizontally extended at the height of the shoulders. In my opinion, it cannot be said that a horse does the Spanish walk, unless this height and extension are obtained. As soon as the horse understands what he is required to do, and performs it correctly, that is to say, when he fully extends his legs horizontally, he ought to be taught to place them on the ground without the slightest bending of the knees, which is the most important condition in this movement. THE SPANISH WALK. To succeed in this, we should, as soon as the legs are fully extended, draw the horse forward by the snaffle, so that he will place the foot well in front, the leg remaining straight until the foot touches the ground. If he bends the knee, he can take only a short step ; and, besides, as the knees will be bent unequally, the steps will not be of the same length. Finally, if the horse is allowed to bend his knees, it will be impossible to prevent him pawing the ground, which would necessarily make the steps unequal. We should therefore take the greatest care to have the leg perfectly straight, until the foot touches the ground. When the legs are thus extended, the steps cannot fail to be equal in length. As soon as we get the horse to take one correct step with each leg, we should not continue this work on foot, although the progress would certainly be more rapid. The Spanish walk is much more graceful, brilliant and regular, if the breaker teaches it when he is in the saddle. In fact, when he is on foot, he has to draw the horse forward, and he cannot then get the animal in hand, because the for- Digitized by Microsoft® HIGH-SCHOOL RIDING. 240 ward puli causes the neck to be straightened. The head is consequently placed in a wrong and ungraceful position. Instead of pulling the horse forward, it is far better to press him up with the legs. Therefore we can teach the Spanish walk only when mounted. Having obtained the first two steps on foot, we should mount, and try to do the work I have just explained. My plan is as follows: I hold the curb reins and the left rein of the snaffle in my left hand, and the right rein of the snaffle and the whip in my right hand. Having stopped the horse, I take a sufficient hold of the snaffle to keep the head and neck high, and being on the left hand, I try to get the first step with the right leg. Digitized by Microsoft® THE SPANISH WALK. Having the wall on my right, I am certain that the horse cannot place himself sideways when I try with my left leg, to make him raise his right leg ; but if I were to try to make him raise his left leg, my right leg would force his croup round to the left. I carry my hands to the left, and I strongly close my left leg, which is helped later on by the spur. The right rein of the snaffle is drawn tighter than the left rein, and both legs are kept close to the girths, so as to prevent the horse reining back; the left leg having a much stronger pressure than the right. The effect of the reins is to put almost all the weight of the forehand on the left shoulder. The left leg of the rider should now make the horse raise his right leg, for which object, having the whip pointed down- wards, I lightly touch him several times on the right shoulder, while continuing to keep up his head with the right snaffle rein. Being ignorant of what I want him to do, the horse will always become impatient; therefore, we should make our demands on him in a very gentle manner. Nevertheless, we should keep on at him until he raises his leg. As soon as he makes the slightest movement with his right leg, we should Digitized by Microsoft® TILE SPANISH WALK. 241 stop and pat him on the neck, and walk him round the school before recommencing. stop and pat him on the neck, and walk him round the school before recommencing. In a week, the horse will extend both legs. It goes without saying that I do similar work with his left leg, but in a reversed way. From this period, I combine the respective actions of my legs and whip, and in proportion as I obtain the desired effect, I diminish the action of the whip, and increase that of the legs, until I obtain obedience solely by the legs. After the horse answers to the indications of the legs, and, if need be, to those of the spurs, by complete extension, I drop the whip, and it is then easy for me to press him up to the bridle, which it would be impossible for me to do on foot. Digitized by Microsoft® THE SPANISH WALK. I shall now describe in detail the action of the “ aids” during the entire movement: the action of the left lee and right rein makes the horse raise his right leg. At the moment he is about to place it on the ground, I press him forward with both legs, the action of which performs the double office of keeping him straight and of obliging him to put down his right leg, completely extended, and in front of his left leg. I keep an equal feeling on both reins during the forward step, up to the time he places his foot on the ground. Then my right leg and left rein, in their turn, make him raise the left lez. The connection between one step and the next one should be made in this manner, and it should be done with great care, so as to obtain perfect regularity of movement. We have then had recourse, as we should always do, to a diagonal effect. The Spanish walk can also be practised when reining back, in which case the reins draw the animal back, the moment the extension of the leg is obtained. The difficulty in this back- ward movement is to prevent the hind quarters from turning to the right or left, each time we bring the leg which is in the air, back in rear of the one which is on the ground. It often happens that the horse, by going back too quickly, 16 It often happens that the horse, by going back too quickly, 16 16 16 Digitized by Microsoft® HIGH-SCHOOL RIDING, 242 gets out of hand, and only about half extends his fore legs. In this case we should always recommence the work by send- ing him forward and requiring a complete extension. We must not think that we are always certain to succeed even by exactly following the directions I have given. It depends on the intelligence of the rider to use all the means in his power to make the horse understand what is required of him. This is a question of tact. Besides, when a riding master breaks his first horse he is not sure of himself, and he is, therefore, obliged to feel his way. Only after breaking three or four horses, he will be able to tell if he has done well or if he has failed. Digitized by Microsoft® THE SPANISH WALK. A horse ought not to be asked to do the Spanish walk unless he is very supple, and, above all things, unless he is perfectly obedient to the aids. In fact, we should be always certain of making him go forward. But in this work we require him to stand the spur without hardly going forward. As we are obliged to have the hands high in order to raise the neck, we thus throw a great portion of the weight on the hind quarters, and consequently risk making the animal get behind his bit. It may thus happen that instead of raising one fore leg, he will, by raising two, make a half-rear, which is not a formidable defence, because it is a forward move- ment. Or he may rear, which is more dangerous, especially if the reins are pulled. It frequently happens in breaking, that in order to make the horse go forward, we are obliged, for a time, to give up the attempt of obtaining a movement which we are teaching. If we sometimes tolerate, even a little, a tendency on the part of the horse to get behind his bit, we will soon be powerless to make him go forward. He will quickly understand that he can get out of hand by getting behind his bit, to which expedient he will continually have recourse. All stationary work, the object of which is to teach the Digitized by Microsoft® THE SPANISH WALK. 243 horse to bear the touch of the spurs without hardly advancing, is always dangerous. It often makes a horse restive, in which case the method is almost always blamed, although it would be safer to blame the manner of its application. Besides, it is also difficult for a riding master who is breaking his first horse or his first horses, to avoid making them get behind their bit. Very often a horse has this tendency without the breaker perceiving it. As a general rule, if a horse does not go up to his bridle, he has this fault. * It is well to note that after each stormy lesson, if the man is the victor, the horse’s docility will be well marked in the following lessons. There is no exception to this rule. 16* Digitized by Microsoft® THE SPANISH WALK. Hence my motto 1s “forward, always forward, and again forward!” I admit that we can never obtain a concession from a horse without a more or less lively struggle, but we must not forget that a man’s authority over a horse is dependent on this struggle.* If we require a difficult thing from a horse without having prepared him sufficiently, not only will there be a struggle, but—which is a much more serious thing-— the horse will win. We should, therefore, know the precise instant when each demand can be made. This is a question of tact. If we put a horse to some work for which he has been badly prepared, we may be certain that we shall not be successful. Without a proper preparation the horse will certainly resist. The same work can be demanded later on without any trouble. It is better to try to break a horse in a year than in six months. The more difficult a horse is, the more time should be devoted to his education. If it is necessary, we may take six months to balance him well in his three natural paces, and to make him to rein back correctly before teaching him high-school work and artificial movements. I would attach no importance to the Spanish walk, if I were 16* Digitized by Microsoft® HIGH-SCHOOL RIDING. 244 not obliged to teach it to the horse, in order, later on, to complete his high school education by pirouettes on three legs, the Spanish trot, and the canter on three legs. For this, it is of the utmost necessity that the breaker can obtain, at will, extension of the legs, and especially by the spurs, which is the only means of keeping a horse in equilibrium, of teaching him to go up to his bit, and of making him hold his legs as high and as extended as he ought to do. There is, alas! another way for teaching a horse the Spanish walk, and I hasten to say that I never use this method of which I am obliged to speak, because it is greatly in vogue among the new school. THE SPANISH WALK. By this term I designate the riding masters who have sprung up since the death of Baucher, and who have substituted for the school movements he executed most brilliantly, a series of strange contortions obtained by a host of accessories, the employment of which necessitates the assistance of a large number of men. To teach the Spanish walk according to the method of the new school, it is necessary to employ an entire squad of breakers—four privates and a corporal: never less, and often more. They begin by placing on each fore leg a hobble, to which is attached a cord, each cord being held by a man. A third person holds the bridle and a cutting whip. The corporal mounts the animal, and a fifth tormenter has the noble mission of flourishing a driving whip. We will now see the collective operations which the squad performs on the unfortunate animal. No. 3, who has the cutting whip, taps the left leg of the horse with it. No. 1, who holds the cord of that leg, pulls the leg straight, and the corporal, who is on the back of the animal, presses his right leg against the horse’s side so as to accustom him to extend his leg on being touched by the spur. Digitized by Microsoft® REVERSED PIROUETTE ON THREE LEGS. 245 245 At the same moment No. 5, who wields the driving whip, touches the horse on the croup to make him go forward. Thus, a step with the left leg is obtained. No.2, who holds the cord of the hobble, which is on the pastern of the right leg, comes on the scene only when the squad proceeds to manipulate the right leg in the way I have just described for the left leg. This double manceuvre continues until the horse has learned the Spanish walk. This is a case which demands the intervention of the S.P.C.A. I am at a loss for a word to express this kind of proceeding. It is certainly not breaking, and has nothing in common with horsemanship. REVERSED PIROUETTE CN THREE LEGS. The reversed pirouette on three legs, the fourth being held in the air, during the rotation of the croup round the shoulders, is the easiest movement to teach the horse, of course supposing that he is well balanced ; because it is an affair of uniting, in a single movement, the rotation of the croup and the extension of the legs. Having ridden the horse into the middle of the school, we make him take a few steps in the rotation of his croup, while stopping him at every three or four steps to make him extend his leg. As we use the same leg for both movements it ought to be kept close to the girths, the other leg being the one which stops the rotation. For example, in the rotation of the croup round the shoulders from left to right, my left leg prompts the rotation of the croup, and also the extension of the horse’s right leg. When the horse has described about a quarter of the circle of rotation, I stop him by the pressure of my right leg, and I touch him with the left spur, which I keep close to the girths, while at the same time I lightly feel the right rein of the snaffle, so as to oblige the animal to extend his right leg. Digitized by Microsoft® HIGH-SCHOOL RIDING. 246 By frequent repetition we can soon make a horse take a step or two in the rotation of his croup, without putting his right fore on the ground, and little by little we can make him do the complete pirouette in this manner. We ought, during the entire time, to maintain a fairly strong pressure of the opposite leg, so as to keep the horse up to his bridle and prevent him getting behind his bit To be ina good position, the horse should have his head and neck high, and his neck slightly bent to the right. The action of the right snaffle rein is necessary to make this bend, and to help in keeping the right leg in the air. We use the reversed means to obtain the pirouette from right to left, with extension of the left leg. REVERSED PIROUETTE CN THREE LEGS. The difficulty is to keep the right leg extended; the equi- librium on the three legs being sufficiently secure to allow the horse to maintain this position, and to move his hind quarters round the left fore foot, which forms a pivot and does not shift out of its place. In the pirouette from left to right, we should carry the hands to the left, while lightly feeling the right reins, so as to put the weight of the forehand on the left shoulder. In the pirouette from right to left, the right foot is the pivot, and consequently we should carry the hands to the right, and feel the left reins, so that the right leg, in its turn, will bear the weight of the forehand, and thus allow the left leg to be raised, Digitized by Microsoft® REVERSED AND ORDINARY PIROUETTES WITH THE FEET CROSSED. The reversed pirouette with the fore feet crossed is done in the same manner as the reversed pirouette on three legs, except that the fore legs, instead of being successively ex- tended in the air, rest on the ground and become crossed while the horse turns. Digitized by Microsoft® REINING BACK WITHOUT REINS. 247 In the ordinary pirouette with the feet crossed, the forehand turns, and the hind feet become crossed. The former is very simple, and the latter very difficult. REINING BACK WITHOUT REINS. This movement is not very difficult to teach; but it is not without annoyance to the horse and danger to the rider, because the legs and spurs are the only agents employed to make the animal go back, with the consequent risk of putting him behind his bit or making him rear. I would not advise young and inexperienced breakers to try I would not advise young and inexperienced breakers to try this exercise before they have broken several horses. Until they have done so, they will not be able to exercise the necessary judgment. We may safely try it on impetuous horses, which are always ready to go forward; but it would be a mistake to teach a soft or sluggish animal, because the vigorous use of the legs is the only way to make him go up to his bridle. In any case, we ought not to teach it to a horse without being certain that he will not take advantage of the instruction, by reining back in opposition to the wishes of his rider ; in other words, by becoming restive. Therefore, I do not begin this exercise until the horse is perfectly obedient, and, especially, until I am sure of being able to make him go forward under all circumstances. In teaching it, I begin in the same way as I have described for the rein back (p. 119 e¢ seg.), namely, I employ both legs and hands. By degrees I diminish the action of the reins, while indicating to the horse by means of the legs, which is the great difficulty, that the hind quarters should begin the move- ment. It is necessary in some sort of a way that the horse should be pulled back by our thighs and seat, and that he receives a backward propulsion from his forehand. At first, I encounter some hesitation on the part of the Digitized by Microsoft® HIGH-SCHOOL RIDING. 248 horse, which is all the greater because up to this time I have always trained him to go forward at the slightest pressure of the legs. We should keep him as quiet as possible and not insist too strongly, because the less a horse understands what is required of him, the more irritable does he become. REINING BACK WITHOUT REINS. We ought to be content with two or three steps to the rear, and immediately he has taken them, we should make him take the same number of steps forward, by relaxing the pressure of the thighs, and by touching him with the heel or spur. We should, above all things, avoid letting him rein back quicker than we wish him to do. horse, which is all the greater because up to this time I have always trained him to go forward at the slightest pressure of the legs. We should keep him as quiet as possible and not insist too strongly, because the less a horse understands what is required of him, the more irritable does he become. We ought to be content with two or three steps to the I sum up by saying that at first I close the legs and feel the reins sufficiently to bring about the rein back. Having obtained the retrograde movement, I hasten to pat the horse on the neck, and I repeat the work, while each time increasing the pressure of the legs, and diminishing the tension of the reins. Finally, when the horse has learned, little by little, what is required of him, I leave go the reins. ROCKING THE FOREHAND. In rocking the forehand, the horse raises his fore legs successively, without extending them, and rocks his forehand from one leg to the other, while separating them as widely as possible from each other, at the moment when they touch the ground. Having taught the horse to extend his legs as already described, it is easy by this means to make him balance the forehand. With this object, his head and neck should not be raised too high, and his legs should only be half straightened, which I get him to doin the following way. The horse being halted, I make him raise the right fore, as if to extend it, but as soon as he raises it, and before he has completely extended it, I carry my hands to the right, and, consequently, all the weight of the forehand which was on the left leg is suddenly Digitized by Microsoft® ROCKING THE HAUNCHES. 249 transferred to the right leg, and the horse naturally comes down on that side, while separating the right leg from the left. Having obtained this first step, I demand a second one from the left leg, by employing similar means. I have then one time for one leg, and another time for the other leg, and to connect them I have got only to bring them together and to put them into harmony. The separation of the legs will naturally be in proportion to the action of the hands. The action of the legs of the rider is the same as that for the extension of the animal's legs, but it ought to be simul- taneous on both sides, so as to prevent the rocking of the croup. In a short time we will obtain the alternate rocking of the forehand to the right and to the left. We ought not to require a great separation of the fore legs until the rocking is very regular. The wider the separation, the slower, softer and more graceful is the movement. We can easily succeed in getting a separation of forty inches, and sometimes of five feet. ROCKING THE FOREHAND. The rocking of the forehand is made on one spot, and it can also be made while advancing, in which case it is more grace- ful, and has the advantage of not prompting a horse to get behind his bit; but it is much more difficult, because we must add forward propulsion to the other actions I have just described. Digitized by Microsoft® ROCKING THE HAUNCHES. This movement requires more tact, as regards the seat, than the preceding one. To obtain it we should feel the reins very lightly, and should make the horse take a light bearing on the snaffle while pressing him down, so as to carry his weight on his shoulders. As the haunches become lightened, their mobility will be increased. We should take care to keep the horse stationary and very Digitized by Microsoft® HIGH-SCHOOL RIDING. 250 quiet ; for we are going to require from him two times, one after the other ; because one time will not be a school move- ment, and will not give him any indication. I exert a very light pressure of the right leg, which causes the horse to raise his right hind leg, as if to make a side step to the left, but at the moment this leg comes close to the left hind, and before it is placed on the ground, a pressure of my left leg pushes it back into its place, and at the same time makes the horse raise his left hind, which he places by the side of the right hind, as if he were taking a side step to the right. If the rider does not catch with precision the moment when the horse is going to put down his right leg, this leg will touch the ground without the rider having pushed it back to the right by the action of his left leg. Under these conditions, the right leg of the horse will be placed quite close to his left leg, and they will not be sufficiently separated for him to make a small rocking movement. The rider should be content with these two first steps until the horse does them very steadily, and then he should demand them the reverse way, namely, from left to right. He should wait until later on to do four, and should increase the number gradually. I recommend the breaker not to try to obtain a great separation of the feet before the horse rocks in a very regular manner. The spur has to be used, in order to obtain as great a sepa- ration as possible, so to give more vigour to the movement. * With the head low a horse is never graceful and light. All horses which are broken by means of the cutting whip adopt the faulty position of having the croup high, the reason being that by striking the croup with the cutting whip, we Digitized by Microsoft® ROCKING THE HAUNCHES. When rocking the haunches, the horse has a natural tendency to carry his head low, which at first I allow him to do to a certain extent, becaus2 he will thus lighten the hind quarters. As soon as he knows the work, I make him hold his head in the position which a school horse should always do.* SPANISH TROT. 251 By increasing the pressure of the legs, and accentuating it a little by a touch of the spurs, we can easily obtain a small piaffer (p. 275) of the forehand, which accompanies the rock- ing of the haunches. If we press the horse forward, while making him take very short steps, we will obtain rocking of the haunches with passage (p. 275) of the forehand. The movement is less graceful when done without gaining ground, than when going forward, because there is a want of propulsion, and it is less easy to keep the neck high. Finally, as it is necessary to obtain a certain elevation of all the legs, we should avoid putting more weight in front than behind. On the contrary, we should carefully try to keep him in equilibrium.* Digitized by Microsoft® cause this portion of the body to become raised, as a result of stimulating the part. On this account I condemn the use of the cutting whip in high-school work. Horses which have been ‘‘routined,’’ I cannot say ‘‘ broken,” by the cutting cause this portion of the body to become raised, as a result of stimulating the part. On this account I condemn the use of the cutting whip in high-school work. Horses which have been ‘‘routined,’’ I cannot say ‘‘ broken,” by the cutting whip, are always ugly and badly balanced. Having the neck on a level with the withers and the croup high, the forehand is depressed, and is consequently in a position the direct opposite of that of a true school horse (Fig. 62), which has always his neck high and his hocks well bent under the body. * I believe I was the first to make a horse do the rocking of the haunches with SPANISH TROT. OF all artificial movements, the Spanish trot is the easiest and most brilliant. With a horse which knows the Spanish walk perfectly, nothing is easier than to make him do it at the trot. It is enough to increase the action of the “aids” to give a horse more energy. At first we ride the horse at the Spanish walk, and when we have gone a few steps, we should seize the moment when the animal’s leg is raised, to touch him more sharply with the spur. I have never had horses which took a long time to do this Horses which have been ‘‘routined,’’ I cannot say ‘‘ broken,” by the cutting whip, are always ugly and badly balanced. Having the neck on a level with the withers and the croup high, the forehand is depressed, and is consequently in a position the direct opposite of that of a true school horse (Fig. 62), which has always his neck high and his hocks well bent under the body. * I believe I was the first to make a horse do the rocking of the haunches with passage of the forehand in 1880, at Paris, with the thorough-bred Amour. HIGH-SCHOOL RIDING. 252 movement. But, as I have often said, I content myself with a little at the beginning of a new work. movement. But, as I have often said, I content myself with a little at the beginning of a new work. Let us suppose, for example, that at the moment when the right leg is extended, I touch the horse sharply with the left spur, he will make a small leap forward: that is the first period. I content myself with this, and pat him on the neck. Then I begin again. When I am sure of my first period, I do not demand a second, but I begin, quite simply, with the left leg, in the same manner as I did with the right. When I have obtained from my horse a single time of the trot on each leg, I demand two, but only when such particular time is correct in length and height, and is done with ease. * See Fig. 51. The thorough-bred Markir (by the thorough-bred Anglo- Arab Cyrus, out of the thorough-bred Thérésine), at the Spanish trot: right diagonal in the air ; perfectly in hand ; head a little beyond the vertical; and jaw bent. Fig. 52, Germinal at the Spanish trot: left diagonal in the air; and in hand. The cutting whip would never give extensions of the legs like those shown in these two illustrations, which were entirely an affair of the spurs. We can see in these two cases, how the hock which is in the air is brought well under the body, which is the whole secret of the elevation of the forehand. The expenditure of energy in Fig. 51 is so great that the fetlocks almost touch the ground, Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® SPANISH TROT. 257 which we should not take undue advantage; because if we wish to obtain twenty or thirty steps of this movement in a short time, it is certain that the last will not be as brilliant as the first, and the horse will acquire the bad habit of not extending his legs with energy. which we should not take undue advantage; because if we wish to obtain twenty or thirty steps of this movement in a short time, it is certain that the last will not be as brilliant as the first, and the horse will acquire the bad habit of not extending his legs with energy. If, on the contrary, we know how to be contented with little, the more modest our demand, the greater will be the energy which we can obtain in its execution. Before requiring the horse to keep up the Spanish trot for a long time, we should wait until he can do it without effort. When he is very familiar with this movement, it will not fatigue him, and we can then make him do it three or four times round the school, but we should not go beyond that. I have already said that during breaking, we should not allow the horse to take the initiative in any movement, for if we do so, he will take advantage of our leniency, and will not obey our orders. He will frequently do what he finds easy, and what pleases him; but not what will please us. Thus, while we are teaching him the changes of the leg at the canter, he will often do more than we demand. If we allow him to do this, we will not be able to make him do the changes regularly, because he will do them of his own accord, without our being able to regulate him. When a horse takes the initiative in a movement which we have taught him, we should correct him, but not harshly. If we leave him to go his own way, he will quickly take advan- tage of our slackness, and our authority will be lost. If I say that in this case the punishment should never be very severe, J assume that in taking the initiative in a movement which he has been taught, he gives a proof of good will. Nevertheless, I repeat, it should not be tolerated. SPANISH TROT. If one leg is lazier than the other leg, which is almost If one leg is lazier than the other leg, which is almost always the case, I work only that leg, which is the best means of impressing the memory of the horse, and of making him understand, by means of my indications, that he uses that leg too slackly. Having established harmony, I connect the two first periods and afterwards demand four; but no more for a long time, not even when the horse tries to do more of his own accord, It is better to be contented with four times well done, than to try to quickly obtain a greater number which would be doubtful. In the Spanish trot, the horse displays great energy,* of * See Fig. 51. The thorough-bred Markir (by the thorough-bred Anglo- Arab Cyrus, out of the thorough-bred Thérésine), at the Spanish trot: right diagonal in the air ; perfectly in hand ; head a little beyond the vertical; and jaw bent. Fig. 52, Germinal at the Spanish trot: left diagonal in the air; and in hand. The cutting whip would never give extensions of the legs like those shown in these two illustrations, which were entirely an affair of the spurs. We can see in these two cases, how the hock which is in the air is brought well under the body, which is the whole secret of the elevation of the forehand. The expenditure of energy in Fig. 51 is so great that the fetlocks almost touch the ground, Digitized by Microsoft® Digitized by Microsoft® Fi 51.—Spa trot ; right diagonal in the air; perfectly in hand ; head a little beyond the vertical ; and jaw bent. F 51.—Sp trot ; right diagonal in the air; perfectly in hand ; head a little beyond the vertical ; and jaw bent. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Fig. 52.—Spanish trot ; left diagonal in the air ; and in hand. Fi 52.—Spa trot ; left diagonal in the air ; and in hand. d by Microsoft® itize ti igi Digitized by Microsoft® Digitized by Microsoft® SPANISH TROT. Digitized by Microsoft® * When a horse who knows how to do a thing very well, becomes obstinate on some particular occasion, he should be taken in hand very resolutely and reduced to obedience by every possible means. I advise the rider never to yield ; for if he does so, the animal will quickly understand that if he wants to be Digitized by Microsoft® SPANISH TROT. Each time he tries to do a movement for himself, we should 17 17 Digitized by Microsoft® HIGH-SCHOOL RIDING 258 replace him in the position he previously occupied, and keep him in it until he remains in it without trying to get out of it. I have often seen horses which had been taught the Spanish walk use it subsequently as a defence. To every demand they replied by extending their legs without moving on. This was caused by their being taught artificial azrs too soon. Before coming to them, the horse should be very supple and should be absolutely obedient in all natural movements. Young breakers generally wish to go too fast, and it amuses them to see their horse extend his legs. When he does it, even of his own initiative, they pat him on the neck, and are astonished later on at not being able to get him to do something else which they ask him. It is easy to understand that a horse will use the Spanish walk as a defence, when the order of the breaking is faulty. In fact, when we teach him this movement we force him to bear the spur, while remaining stationary. This is the great danger in all breaking, because, if a horse has not been at first accustomed to go freely forward on being touched with the spur, he extends the leg, or leans on the spur. Before teaching a horse to bear the spur without moving forward, we ought always be sure of being able to make him go forward by means of the spur when we wish. It very often happens that a horse, through softness, uses as a defence an azy to which he is accustomed, but will never use an azy which requires great energy. AsI have just said, we should of course put him back into his old position, and should prevent him from getting out of it.* SPANISH TROT. 259 Besides, the horse ought to be so intently occupied with his rider, during the entire course of a lesson, that he should think of nothing else ; and the rider should mentally act ina similar manner towards the horse. master, he has only to ‘‘show fight.’’ The rider ought, nevertheless, to keep cool, and never lose his temper, or at least, not to give way to anger, beyond allowing it to stimulate his energy, and to make him forget the dangers of the struggle. By such faults, the breaking of a horse, instead of being finished, becomes per- verted. The slightest fault which is tolerated on one day, becomes aggravated on the morrow, and is converted into a vice which cannot be cured later on, except by the most severe battles. A broken horse is nota machine which requires only to be wound up, but is a living creature who continually tries to escape from the ‘“‘aids’’ of his rider, and who requires to be constantly kept in the discipline of work. Digitized by Microsoft® SPANISH TROT. The man, so to speak, ought to take entire possession of the animal, and should make him so attentive to his orders that the horse should not think of anything except what he is asked to do. As regards myself, I take such possession of the horse I ride, that the only idea I leave in his head is: “What is he going to ask me to do?” Certain authors say : “It requires so much time to do this, so much to do that, at the end of so many lessons we will be able to obtain this thing or that thing,” etc. This is entirely wrong. One never knows how much time will be required to succeed in making a horse do any particular work. Some horses learn very quickly what others acquire with great difficulty. On the other hand, the former are often very unruly when an attempt is made to teach them a move- ment which the latter will learn at once. In breaking, we should therefore pay great attention to the peculiarities of each horse, and should not be discouraged if we do not obtain a result in the time laid down in a book. Thus, I have had horses which learned the Spanish walk in a week, and others which required three months with the same method of instruction. master, he has only to ‘‘show fight.’’ The rider ought, nevertheless, to keep cool, and never lose his temper, or at least, not to give way to anger, beyond allowing it to stimulate his energy, and to make him forget the dangers of the struggle. By such faults, the breaking of a horse, instead of being finished, becomes per- verted. The slightest fault which is tolerated on one day, becomes aggravated on the morrow, and is converted into a vice which cannot be cured later on, except by the most severe battles. A broken horse is nota machine which requires only to be wound up, but is a living creature who continually tries to escape from the ‘“‘aids’’ of his rider, and who requires to be constantly kept in the discipline of work. By such faults, the breaking of a horse, instead of being finished, becomes per- verted. SPANISH TROT. The slightest fault which is tolerated on one day, becomes aggravated on the morrow, and is converted into a vice which cannot be cured later on, except by the most severe battles. A broken horse is nota machine which requires only to be wound up, but is a living creature who continually tries to escape from the ‘“‘aids’’ of his rider, and who requires to be constantly kept in the discipline of work. 17* Digitized by Microsoft® 260 HIGH-SCHOO 260 HIGH-SCHOOL HIGH-SCHOOL RIDING. 260 With certain light and well-shaped horses, I have obtained changes of the leg at the canter almost immediately ; although the majority of horses generally require six months to learn to do it properly. The same can be said of everything in riding. SHOULDER-IN AT THE CANTER. I have put demi-voltes at the canter into ordinary riding, because one might require, when hacking, to take a few steps at the canter on two tracks, in a more or less correct manner, in order to get out of somebody’s way. 1 will now take up the study of the canter in high-school riding. When the horse does demi-voltes correctly at the canter, I try by shoulder-in work at the canter, to bring his shoulders away from the wall. For this, the horse has been admirably prepared by work on two tracks at the school walk, and further by demi-voltes at the canter. I start my horse at a canter on the near fore, and being on the right track, I keep him on it for a few strides. I then carry my hands to the right, in order to bring his shoulders away from the wall, but only about a yard. I work my right leg strongly, in order to carry the haunches from the right to the left ; whilst my left leg receives, so to speak, the haunches, which my right leg has just sent it, and throws the impulse on the hand. In fact, the left leg presses the horse forward, and thus prevents him from getting behind his bridle. It also regulates the pace by preventing the animal from sidling off to the left. If there is nothing to check the speed of the pace to the side, whether at the walk, trot, or canter, the horse will fatally regulate his pace, and will rush to the side in order to get out of hand, that is to say, he will no longer keep on two tracks. It is therefore necessary, in order for the movement to be regular, that the rider can regulate the cadence at all paces, and that he always keeps his legs close to the sides of Digitized by Microsoft® SHOULDER-IN AT THE CANTER. 261 the horse, in order to keep him in hand, and to be able to make him go forward at any moment.* I have said that I carry the hands to the right in order to bring the shoulders away from the wall. SHOULDER-IN AT THE CANTER. The pressure of the left rein on the neck should be the only means employed to bring the shoulders to the right, and it should cease the moment the shoulders leave the wall, under pain of stopping the shoulders. We should instantly carry the hands to the left ; the left rein acting directly to its own side, and the right rein pressing, in its turn, on the neck to push the shoulders to the left. This is an effect of the left diagonal. The left rein, by lightly drawing the forehand to the left,f prevents the hind quarters from going in advance of the fore hand. In “two tracks,” the haunches are always inclined to go in front of Fig. 54.—Germinal at the canter: shoulder-in; going from left to right. Left diagonal in support. , Fig. 55.—Germinal shoulder-in at the canter; going from left to right in complete vassembler. We will note that the horse gains less ground, an especially, that the canter of the vassemb/er is in four time, namely, near hind, off hind, near fore, off fore. In Fig. 55, the off hind has just been put down after the near hind, and the near fore—which, in the ordinary canter, would be put down at the same time as the off hind, so as to make the third time, namely, the left diagonal in support—has not yet been put down. This canter is there- fore in four time. It should be noted that there is a moment when the off hind and near fore, which form the left diagonal, are in support at the same time ; but what makes the four time is the fact that the off hind precedes a little the support of the near fore. + For a long time, like all riding masters since the time of Baucher, I bent the horse’s neck to the side he was going ; but I found out that this was a mistake, because nothing is more liable to stop propulsion. I now restrict myself to lightly zzzc/?nzng the head of the animal to the side he is going by only very slightly bending the neck. * Fig. §3.—Germinal at the canter: shoulder-in; going from right to left. This is the second period of the canter, and the right diagonal is in support. The right hock is about to be extended, and the near fore has not yet come down. Fig. 54.—Germinal at the canter: shoulder-in; going from left to right. Left diagonal in support. , Digitized by Microsoft® * T do not allude to the shoulder-to-the-wall ; because I regard it as a bad exercise in which the horse is really held by the wall, so that he is no longer under the guidance of the rider. SHOULDER-IN AT THE CANTER. The rein of the side opposite to which he is moving can then have its full effect to push the fore hand, to keep the horse straight, and to secure the maximum amount of propulsion, by its combination with the other rein and with the legs. Digitized by Microsoft® HIGH-SCHOOL RIDING. 262 the shoulders, especially in demi-voltes, which is a fault we should carefully check ; because the moment the haunches go before the shoulders in an oblique line, the horse is behind the bit. Immediately after I do similar work for the shoulder-in at the canter, from left to right.* Digitized by Microsoft® Digitized by Microsoft® PIROUETTES AT THE CANTER. In the pirouette at the canter, the horse’s hind legs ought, so to speak, to“ mark time” at that pace, while pivoting round, in order that the haunches may remain on the same line as the shoulders, which describe a circle round them. A _ horse should on no account rest continuously on one of his hind legs, as some authors maintain, for if he does so, he will stop cantering. Before pirouettes are required at the canter, they should be taught at the walk, with the horse very well vassembléd. With this object, I place my horse in the centre of the school. IfI wish to make a pirouette, with the shoulders turning round the croup from left to right, I carry my hands to the right, the right rein drawing the head to the right, and the left rein pressing both the neck and the shoulders to the right. Both legs are firmly closed, the left leg being carried a little back to fix the croup and to prevent it turning, whilst the right leg presses the horse up to his bridle, so as to prevent him getting behind his bit. This work requires great delicacy and light- ness of hand. If the hands are too strongly used from left to right, their action will cause derangement of the shoulders, but not a pirouette. If the hands feel the reins too strongly from front to rear, the horse will get behind his bit. When the horse fully understands this work and does it Digitized by Microsoft® Fig. 53.—Shoulder-in at the canter ; going from right to left. Fig. 53.—Shoul at the canter ; going from right to left. Fig. 53.—Shou at the canter ; going from right to left. Fi 53.—Sho at the canter ; going from right to left. d by Microsoft® ize ti igi Digitized by Microsoft® Digitized by Microsoft® Fig. 54.—Should at the canter ; going from left to right ; left diagonal in support. Fig. 54.—Should at the canter ; going from left to right ; left diagonal in support. Fig. 54.—Shoul at the canter ; going from left to right ; left diagonal in support. Fig. 54.—Shou at the canter ; going from left to right ; ft nal at the canter ; going from left to right ; left diagonal in support. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Fig. Digitized by Microsoft® PIROUETTES AT THE CANTER. 55.—Shoulder at the canter ; going from left to right in complete sasseméler. Fig. 55.—Should at the canter ; going from left to right in complete sasseméler. Fig. 55.—Shoul at the canter ; going from left to right in complete sasseméler. at the canter ; going from left to right in complete sasseméler. d by Microsoft® ize ti igi Digitized by Microsoft® Digitized by Microsoft® PIROUETTES AT THE CANTER. 269 easily at the walk, I canter him on a rather larger circle, and gradually shorten his stride; not from front to rear, but by the shoulders, while gaining ground to the side ; and I increase the action of my legs, in order to keep up the speed. The hind legs ought to “ mark time” at the canter ; but, as I have just said, the horse ought never to turn by continuously resting on one hind leg. The pirouette ought to be done as slowly as possible. The great difficulty consists in obtaining a very slow canter, without losing that pace. J employ reversed means for the pirouette from right to left. We can also do at the canter reversed pirouettes, which consist in making the croup turn round the shoulders. This is an excellent exercise at the walk, but I never teach it to my horses at the canter, because the great difficulty in all work is to make the shoulders move. In reversed pirouettes, on the contrary, we teach the animal to keep his shoulders stationary, which is an absurdity in riding. Besides, this movement is ungraceful, because the fore legs, not being able to gain ground, remain stiff while stamping on one spot. On the other hand, ordinary and reversed voltes are graceful and useful. We obtain them by means of the same “aids” by which we get pirouettes, only we make the horse describe a large circle instead of turning him on one spot. When we begin a volte, it is difficult to previously determine the exact diameter of the circle which we are going to make; but, nevertheless, this will be the only means for the rider to know if his horse is right. To find this out, it is sufficient to trace a circle at the centre of the school. If a horse follows it exactly, he does so, because his position is good, and he is well-balanced in propulsion. * “ Opposite leg’? means the leg opposite to that with which the horse changes. If I am going to the right, and the horse is cantering with the off fore, I use my right leg to make him change and the left leg to keep him straight. If I do not receive the horse on my left leg, the croup will inevitably swerve towards the wall. PIROUETTES AT THE CANTER. When a horse does correctly at the canter all the different exercises just described, we can ask him to do any movement at the canter. Having been made supple and obedient by all Digitized by Microsoft® HIGH-SCHOOL RIDING. 270 the preceding exercises, he will never refuse to obey the orders of his rider, provided that the man is patient and tries his best to make the animal understand. Digitized by Microsoft® * “ Opposite leg’? means the leg opposite to that with which the horse changes. If I am going to the right, and the horse is cantering with the off fore, I use my right leg to make him change and the left leg to keep him straight. If I do not receive the horse on my left leg, the croup will inevitably swerve towards the wall. t It is always very difficult to make the horse change correctly when turning. { I have previously explained that I never do, properly speaking, the shoulder- to-the-wall in the sense that I will not allow my horse to be guided by the wall. I use this expression because it is the popular one, but as I do this work always away from the wall in voltes, it would be more correct to call it ‘‘ shoulder-out.’? { I have previously explained that I never do, properly speaking, the shoulder- to-the-wall in the sense that I will not allow my horse to be guided by the wall. I use this expression because it is the popular one, but as I do this work always away from the wall in voltes, it would be more correct to call it ‘‘ shoulder-out.’? t It is always very difficult to make the horse change correctly when turning. CHANGES OF LEG AT EACH STRIDE. I have discussed, in the hacking canter, the changes from the outward to the inward leg upon a straight line. I shall now speak of the changes of leg in the school canter. I teach the horse the changes of leg from within to outside, only when he does them from outside to within very easily, and of course I use similar means. In the beginning, we should carefully avoid getting the horse to do them in the corners of the school, the outward side of the horse having, by reason of the turning movement, more ground to cover, which fact requires a stronger support of the rider’s opposite leg* to prevent the horse going sideways, and to press him forward on to the hand. We should not require changes of the leg from within outwards in the corners of the school until he does them easily in a straight line.t I never require the shoulder-to-the-wallf at the canter before obtaining the changes of leg in the air—that is to say, at the canter without stopping, because when we require the horse to change from the outward leg to the inward one, he almost always tries to bring his haunches to the inside, as he does in the shoulder-to-the-wall, since the same “aids” are CHANGES OF LEG AT EACH STRIDE. 271 used. If the rider’s inward leg does not instantly receive the change of leg which his outward leg sends it, the horse wiil naturally put his croup sideways. Under these conditions, the changes of leg are never regular or well done. And, as it always takes two or three strides to straighten a horse, it will be impossible to do these changes with short intervals between them. On the outside the evil is less, because the wall prevents the haunches from being brought too much round. When the changes are correctly done with both legs to the right and to the left, and especially in the corners of the school, I only then begin to regulate them, which it is impossible to do before being certain that the horse will change each time, and at the moment I want him. At first I content myself with getting him to change in every eight or ten strides of the canter, and I demand only a single change on each leg. CHANGES OF LEG AT EACH STRIDE. If he becomes excited, he should be stopped and practised again at starting. To be on good terms with the horse, it is necessary to count the strides of the canter. For example, when going to the right, with the horse cantering with the off fore, I count I, 2, 3, 4, 5, 6, each number marking a stride. At the sixth I make him change, and then I make him do the same work with the near fore leading. If the horse does correctly the two changes I have asked, I stop him and pat him on the neck. I then begin again by requiring him to do only a single change on each leg. At the end of a few days, when I feel that the horse does this work freely, I demand four and then six changes, always at the sixth stride. As long as the horse does this work in a tranquil frame of mind, I gradually increase the number of changes at the sixth stride, or, as it is said, at the sixth “time,” but without over-doing it. If the horse gets excited I stop him, but I do not pat him, Digitized by Microsoft® HIGH-SCHOOL RIDING. 272 for, if I did so, he might think that I was encouraging him to get excited. I walk him, well in hand, and then I begin again the lesson, which I do not finish until he has done the changes steadily. , The pitfall in the changes of leg exist entirely in the pre- parations which the breaker generally makes to obtain them. Of course, I do not allude to the preparation of the horse, which is indispensable, but only to the movements of the breaker before the exact moment when he wants the horse to change. It isextremely important that he remains quiet while the horse is taking his strides, during which the animal should not change. Thus, during the first five strides he should not make the slightest movement. He ought not to change his “aids” until the sixth time, and should then change them together and with great precision. If at the fourth or fifth time he prepares to make the horse change, he will be taking a wrong course, because the horse, being very sensitive to the “aids” and attentive to the demands of his rider, will become disunited. Digitized by Microsoft® CHANGES OF LEG AT EACH STRIDE. If he touches the reins, the forehand will change, and if he alters the position of his legs, the hind quarters will change. But as there will be no harmony between the “ aids,” the horse will not change his legs. It is wrong to blame the horse for becoming disunited, because in such cases the rider almost always provokes him to do so, by not remaining perfectly quiet until the decisive moment. It also happens that the horse, thinking that he is going on all right, does not attend to his rider's demand, and anticipates the order, or what he thinks is the order. Therefore I cannot too strongly advise the rider to keep absolutely quiet during the first five strides of the canter. It happens with all horses, that when they have learned to do the changes easily, they will do them of their own accord, and without an indication from the rider. In this case the Digitized by Microsoft® CHANGES OF LEG AT EACH STRIDE, 273 horse should be punished, because if we let him take the initiative, we can get no regularity in the changes. When I say that he ought to be punished, I mean that the fault ought to checked. Thus, if when the horse is canter- ing on the off fore, he changes before we ask him to do so, he should simply be touched more sharply than usual with the left spur, in order to oblige him to rest on the right leg. Of course we should use the right spur in a similar way, if the horse has started with the left leg leading. Having checked the fault in the manner I have indicated, we should avoid again requiring a change of leg in the air during the same lesson, as it might confuse the horse. It is better to go back to the starts, while keeping him for a long time on the same leg. If we have checked the same fault several times and have taken care to pat the animal when he changes only when we ask him, he will soon understand that he ought not to do anything without being asked. But, I repeat, we should do the changes only when we have not been obliged to punish the animal. CHANGES OF LEG AT EACH STRIDE. It is better to devote several lessons to checking the fault, in which case the horse will understand much better, will be quieter, and his breaking will be more rapid. I make the horse change at shorter intervals only when he fully understands what I want him to do. Instead of be- ginning at the sixth time, I demand the change at the fourth time, by using always the same means. For the first few times I content myself with a single change with each leg, and during the following days I gradually require a greater number of changes. We can see that the shorter the interval between the changes, the greater will be the effort required from the horse, who is consequently apt to become irritable and con- fused, in which case he should be halted, walked for some 18 18 18 Digitized by Microsoft® HIGH-SCHOOL RIDING. 274 time, but not patted on the neck. After that, we should begin again starting at the canter on each leg with the horse well in hand, without asking him to change, and should not finish the lesson until he has become perfectly quiet. Each time that I find any difficulty in the changes of leg, I go back to starting at the canter, which I find very profitable. The starts are for the horse, what scales are for pianists, and steps for dancers. From four times I do three, then two, and finally one, in which each stride of the canter ought to be done on a different leg. I leave a sufficiently long time to elapse be- tween each of my demands—from four to three, from three to two, and from two to one. If we go too fast we will not succeed, for the horse cannot help makinz mistakes, because he has not had the chance of understanding the difference between the changes of leg demanded at different times. By going quietly in riding, we shall be certain of suc- ceeding quickly. Besides, the horse is the one who indicates to us the number of changes and the interval between them without spoiling his equilibrium, strength, or lightness. Digitized by Microsoft® PIAFFERS AND “ PASSAGES.” 275 CHANGES OF LEG WITHOUT GAINING GROUND. To do the changes of leg at the canter without gaining ground, we should proceed in the same manner as in the changes at each stride, with only the difference that the horse should be better in hand. It might appear that in this movement we should greatly increase the feeling on the reins, and diminish the pressure of the legs, in order to make the horse go slower. This would be a mistake; for in that case the hands would bring the hocks away from the centre of the body. We should, on the contrary, act vigorously with both legs, to keep up pro- pulsion, without which the canter will be lost. The hand ought to take possession of this propulsion in a very light manner, and ought to drive it back to the horse’s centre of gravity, which is close to the rider’s legs, when the animal is well balanced. Changes which are made without gaining any ground are hardly perceptible to the eye, on account of the very short extension of the fore and hind legs. Hence we have to be very close to the horse to see these changes. We owe the invention of this difficult and complicated exercise to Baucher, who did it brilliantly on Turban; but not on Partisan, as many assert. CHANGES OF LEG AT EACH STRIDE. Only when he is quiet, light in hand, and when he does the changes with ease, do I obtain the changes in two time, and then in each stride, during two or three turns round the school on both hands; and I make him do the same work in two times, in voltes and changes of hand. When I am satisfied with the results obtained, I make him do the changes at each stride in these movements. The rider who succeeds in doing the voltes and changes of hand, while making the horse change very steadily at each stride, can be satisfied with himself and with his horse, because he has overcome the great difficulty in riding. Digitized by Microsoft® PIAFFERS AND “ PASSAGES.” Digitized by Microsoft® PIAFFERS AND “PASSAGES.” The natural passage is a very well collected, short and high trot, which is regularly cadenced from one diagonal to the other. But to fully merit the title of passage, the fore legs, being raised high, but gently, should pause for a moment in the air, with the knees and feet bent. The hind legs are raised and the hocks and pasterns are bent and carried 1e* 1e* 1e* Digitized by Microsoft® HIGH-SCHOOL RIDING. 276 under the centre. To sum up: all four legs should be raised high and in cadence, so that the horse is suspended in the air from one diagonal to the other. This natural passage, which is graceful and brilliant, ought also to be slow, and as high as possible.* We should bear in mind that no horse will do the passage of his own accord ; for it can be obtained only by teaching. Some horses can be very easily taught the passage, especially those which have slow paces and bend their knees ; that is to say, those which have high action before and behind, and bend their knees and hocks—in a word, slow and high action. A horse which goes stiffly, without bending the knees or hocks, is not fit for this movement. I will not go so far as to say that he cannot be taught it, but supposing that he is made to do it, he moves ungracefully, because he looks stiff, on account of dragging his feet. It is extremely difficult to explain how the natural passage is obtained. The first thing is to vassembler the horse, without doing which the passage is not possible ; but collecting him is not enough, for he has also to be taught to passage. Having perfectly collected him, we ought to use the spur, in order to obtain the raising of the legs, which gives the passage. While riding the horse at a well-balanced walk, I make him trot, by strongly closing my legs, while keeping him well in * Fig. 56.—Germinal at the passage: right diagonal in the air. Fig. 57.—Markir at the fassage: left diagonal in the air. In these two illustrations, we will note the elevation of the legs, the fore one being raised much higher than the hind. The spur is the only means for obtaining this result. Digitized by Microsoft® * Fig. 56.—Germinal at the passage: right diagonal in the air. Fig. 57.—Markir at the fassage: left diagonal in the air. In these two illustrations, we will note the elevation of the legs, the fore one being raised much higher than the hind. The spur is the only means for obtaining this result. Thecontrary takes place with horses broken by the cutting whip, which, by being applied to the croup, fatally produces a predominant elevation of the hind quarters. * Fig. 56.—Germinal at the passage: right diagonal in the air. Fig. 57.—Markir at the fassage: left diagonal in the air. In these two illustrations, we will note the elevation of the legs, the fore one being raised much higher than the hind. The spur is the only means for obtaining this result. Thecontrary takes place with horses broken by the cutting whip, which, by being applied to the croup, fatally produces a predominant elevation of the hind quarters. In Fig. 57, the fetlock being extended almost to the point of touching the ground, is preparing the energetic spring which will give height ; but in Fig. 56, the forward movement is the more accentuated one of the two. Digitized by Microsoft® Digitized by Microsoft® PIAFFERS AND “PASSAGES.” Thecontrary takes place with horses broken by the cutting whip, which, by being applied to the croup, fatally produces a predominant elevation of the hind quarters. In Fig. 57, the fetlock being extended almost to the point of touching the ground, is preparing the energetic spring which will give height ; but in Fig. 56, the forward movement is the more accentuated one of the two. Digitized by Microsoft® Fig. 56.— Passage ; right diagonal in the air. Fig. 56.— Passage ; right diagonal in the air. Fi 56.— Passage ; right diagonal in the air. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Fig. 57.—Fassage ; left diagonal in the air. Fig 57.—Fass ; left diagonal in the air. Fi 57.—Fas ; left diagonal in the air. ; left diagonal in the air. d by Microsoft® igitize Digitized by Microsoft® Digitized by Microsoft® PIAFFERS AND “PASSAGES.” 281 hand, to prevent him extending himself. Not being able to extend his legs, he raises them, and thus gains in height what he loses in length. At the same time, with the spur helping, he makes, so to speak, little jumps from one diagonal to the other, which are the beginning of the passage. But the cadence is not yet correct, because he always becomes irri- tated at first, as he does not know what we want him to do. During the first few days we should take care not to keep at him too long, if we wish to avoid making him play-up. The horse, being irritated by the spurs, and not under- standing why they are used on him in this work, which is strange to him, sometimes gets into a state of desperation, through fear of the spurs, which are always close to his sides.* If, at this time, we try to make the horse do what we want, we shall be wrong, and shall probably lose the battle ; because, when the quietest horse becomes maddened, he will fight ike a demon. We should keep calm, and at the slightest sign of cadence, that is to say, at the first, or later on, at the second time, we should stop using the “aids,” pat the horse’s neck, give him time to become quiet, and begin again. A horseman who has great delicacy and tact, will stop the anunal at the first time and pat him. * It generally happens during the breaking, that one day, or during several ways, the irritated horse makes up his mind, plays his last card, and acts in the most furious manner. When the breaker, by means of tact and energy, has over- come his final resistance, the animal is submissive, but is not broken. Digitized by Microsoft® Fig. 59.—Markir at the fassage on ‘* two tracks ’’ from left to right, with the right diagonal in the air. Here the movement on “two tracks’ is well marked The near hind, which gains but little ground, is well raised. The off fore, after having been raised, like the off fore in Fig. 58, is carried away from the near fore, in order to gain ground to the right. * Fig. 58.—Markir at the fassage on ‘‘ two tracks’’ from left to right, with the left diagonal in the air. The position is that of ‘‘two tracks,” but the action is that of the passage. Fig. 59.—Markir at the fassage on ‘* two tracks ’’ from left to right, with the Digitized by Microsoft® * Fig. 58.—Markir at the fassage on ‘‘ two tracks’’ from left to right, with the left diagonal in the air. The position is that of ‘‘two tracks,” but the action is that of the passage. Fig. 59.—Markir at the fassage on ‘* two tracks ’’ from left to right, with the right diagonal in the air. Here the movement on “two tracks’ is well marked The near hind, which gains but little ground, is well raised. The off fore, after having been raised, like the off fore in Fig. 58, is carried away from the near fore, in order to gain ground to the right. Digitized by Microsoft® PIAFFERS AND “PASSAGES.” But the less tact he has, the less capable is he of judging if the time is in cadence. Such a man will continue in his attempts to catch the cadence, and will succeed only in upsetting the nerves of his horse. These remarks explain the fact that a clever and tactful horse- man will obtain all he wants from his mount, without making him either vicious or unsound. Being able to recognise the Digitized by Microsoft® HIGH-SCHOOL RIDING. 282 slightest sign of obedience, he immediately stops the work, in order to make the horse understand, by pats on the neck, that he has done well. The quickness with which he perceives the slightest signs, saves him from overtaxing and disgusting the horse, and provoking him to battle, which will wear them both out. The unskilful rider, who is slow in catching the cadence, will continue to use the spur, in order to obtain several cadenced times, and to be sure he is right, and will thus punish the horse, who, not knowing why he is punished, will defend himself, while the rider is spurring him. The result will be, that when he wants the horse to again do the passage, the animal will think he is going to be punished, and will become mad at the approach of the spurs. Hence, the important point is to recognise the slightest signs of good will, and to be content with little. If the horse does not fear the approach of the legs, and if we are not too exacting, a time will always arrive when he will take up the cadence of the passage with ease and pleasure. We may then ask more; because, as the horse understands us, there will be no fear of over-exciting his nervous system. All the work on “two tracks” can be made at the passage but it is difficult to do it correctly.* The artificial passage is another form, which is only an adjunct of the Spanish walk. It is an uncollected passage, and consequently it is opposed to all the principles of equitation. I practised it in my youth, when I had not Digitized by Microsoft® Fig. §8.--Passage on ‘two tracks ” from left to right ; left diagonal in the air. Fig §8.--Pas on ‘two tracks ” from left to right ; left diagonal in the air. Digitized by Microsoft® I could make similar observations respecting certain artificial ars which I have long given up with horses which I break for myself ; not because they are opposed to the principles of equitation, but because I do not find them sufficiently scientific, as for instance, the Spanish walk, reversed pirouettes on three legs, ordinary and reversed pirouettes with the legs crossed, reining back without reins, and rocking the fore and hind quarters. } Fig. 60.—Markir at the piaffer: right diagonal in the air. Fig. 61.—Markir at the piaffer: left diagonal in the air. In these two illustrations, we will note the energy which is displayed and which is marked by the fact that the fetlocks come down to the ground, and by the great elevation of the fore legs, which are raised higher than the hind ones; contrary to what takes place in the piaffer taught by the cutting whip. Digitized by Microsoft® PIAFFERS AND “PASSAGES.” Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Fig. 59.—Passage on ‘two tracks’? from left to right ; right diagonal in the air. Fig. 59.—Passa on ‘two tracks’? from left to right ; right diagonal in the air. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® PIAFFERS AND “PASSAGES.” 287 enough tact to obtain the natural passage. I never teach it nowadays. enough tact to obtain the natural passage. I never teach it nowadays.* The piaffer is only a natural passage without gaining ground. There are two kinds of piaffers: one, which is slow, high and difficult, and which some horses will not stand ; the other which is hurried and close to the ground, and which almost all horses can learn. The high and slow piaffer differs from the natural passage, only by its being done entirely without gaining ground. We can obtain it by shortening the passage step by step, until it is done on one spot. The piaffer therefore requires more rassembler, and therefore more use of the legs and more delicacy of the hand, than the passage.t When a horse has not been taught the passage, the piaffer will be a consequence of the walk. A vigorous and impatient horse can easily be taught the hurried piaffer, which will not be regular unless he has been instructed in accordance with the rules of equitation. It is not enough for the horse to be lively and vigorous, in order to make him do the piaffer by exciting him with the cutting whip, legs, or clicks of the tongue. By these means we will no doubt succeed in making him impatient, but not in teaching him the manner in which he should put his feet on the ground; and we may be certain that if he puts them } Fig. 60.—Markir at the piaffer: right diagonal in the air. Fig. 61.—Markir at the piaffer: left diagonal in the air. In these two illustrations, we will note the energy which is displayed and which is marked by the fact that the fetlocks come down to the ground, and by the great elevation of the fore legs, which are raised higher than the hind ones; contrary to what takes place in the piaffer taught by the cutting whip. Digitized by Microsoft® HIGH-SCHOOL RIDING. 288 down regularly, he will do so by mere chance. PIAFFERS AND “PASSAGES.” Almost always, the hind legs will be raised and put down at the same time, and consequently the movement will not be a pace. We should teach the horse the piaffer by calming him down, because he will not understand what is wanted of him, unless he is in a quiet state of mind. To obtain the piaffer, we ought to collect the horse, by closing the legs, until we touch him with the spurs. It is dificult to explain in a book the exact moment when the spur should reinforce the action of the leg; in fact, this is evidently a question of tact. It is easier to show this on the ground, because one can then see if the rider uses the spurs too much or too little. As I have already said, the office of the spur is to make the horse yield to the pressure of the leg, when that pressure is not sufficient by itself. Besides, it is necessary for the horse to feel the spurs in order to remain rassembléd.* Also, to make a horse piaffer, we must pre- vent him going forward, and if we do not use the spurs, he will infallibly throw himself to the right or left by forcing the respective leg. Therefore, we should use the legs sufficiently to steady the horse. Nevertheless, to hold him in check, the spurs should brush his coat. To get the piaffert we should use light pricks of the spurs ;{ and, as in all other cases, we should at the begin- ning be contented with two times. * The question has often been discussed, whether the spurs are an ‘“‘aid”’ or a punishment. As I have already said, they are sometimes one, sometimes the other, according toneed. During breaking,they ought always to be an “ aid,’’ and ought not to become a punishment, unless the animal openly rebels, in which case we should try to find out if the disobedience is due to pure caprice on the part of the horse or to our fault. If caprice is the cause, the spurs ought to punish him. We should remember in all cases that the English very rightly call the spurs ‘‘ persuaders.”” t+ The hurried piaffer and the slow piaffer are obtained by the same means. I practise only the slow piaffer. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® * In fact, if I exclusively attack one side without immediately receiving the horse on the opposite leg, I will succeed only in sending his haunches to the side. t If I allow an interval between them, there will be an alternate separation of the legs to the right and to the left, as in a kind of rocking. t It follows, from what I have said, that these two first times are necessarily those of a hurried piaffer. When we begin the piaffer, we cannot foretell what will happen. As a rule, the horse will try to hurry it, on account of the spurs making him impatient. Besides, the hurried piaffer being lower than the higher plaffer, will require less exertion. Having obtained the piaffer of any kind, it needs regulating, which will test the skill of the breaker. The difficulty is to prevent the hurried piaffer occurring, or, rather, to chang? it into the slow piatfer, by giving it height brought on by the rassemdler. The more the rassemdb/er, the greater the height. The slow and high piaffer is obtained by energetic action of the legs, lightness of hand, and above all things, delicate tact in combining the “‘aids.’? In the hurried piaffer, the legs are kept stiff, on account of their being raised but little off the ground. In the high piaffer, the biped, which is in the air, is greatly bent, while gaining height, and the fetlocks of the biped which is on the ground almost touch it (Fig. 61), so as to be able, in their turn, to raise the body by their spring. Digitized by Microsoft® * In fact, if I exclusively attack one side without immediately receiving the horse on the opposite leg, I will succeed only in sending his haunches to the side. PIAFFERS AND “PASSAGES.” {This is what La Guérinitre admirably calls ‘‘the delicate pinch of the spurs.”” Digitized by Microsoft® “ITB JY] UL [BUOSeIp YSU f rayery— og “Bq “ITB JY] UL [BUOSeIp YSU f rayery— og “Bq “ITB JY] UL [BUO YSU f ray og “Bq 19 Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® ‘Te ay} Ut [eucSerp yay ‘aye J—'19 “Bry ‘Te ay} Ut [eucS yay ‘ay J—' “Bry Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® PIAFFERS AND “PASSAGES” 293 1 touch my horse on the right and on the left, but almost simultaneously.* My right spur brings the off hind under the animal’s body, which causes the near fore to be raised ; and my left spur, acting in its turn at the exact moment when the left diagonal is in the air, produces a similar effect on the right diagonal. I thus obtain the two first times of the piaffer ; but at the beginning these two times ought to follow each other so closely as to form, so to speak, a single time,f exactly like in fencing, when the swordsman makes “ one, two” very close together. It often happens that the horse will plunge on feeling the first hurried touches of the spurs. If he bounds forward, not much harm will result, and we will learn that we have used the spurs too strongly. We should therefore employ them more lightly when we begin again. If at this period of the breaking we often repeat these two first times of the piaffer, the horse will quickly understand t It follows, from what I have said, that these two first times are necessarily those of a hurried piaffer. When we begin the piaffer, we cannot foretell what will happen. As a rule, the horse will try to hurry it, on account of the spurs making him impatient. Besides, the hurried piaffer being lower than the higher plaffer, will require less exertion. Having obtained the piaffer of any kind, it needs regulating, which will test the skill of the breaker. The difficulty is to prevent the hurried piaffer occurring, or, rather, to chang? it into the slow piatfer, by giving it height brought on by the rassemdler. The more the rassemdb/er, the greater the height. PIAFFERS AND “PASSAGES.” The slow and high piaffer is obtained by energetic action of the legs, lightness of hand, and above all things, delicate tact in combining the “‘aids.’? In the hurried piaffer, the legs are kept stiff, on account of their being raised but little off the ground. In the high piaffer, the biped, which is in the air, is greatly bent, while gaining height, and the fetlocks of the biped which is on the ground almost touch it (Fig. 61), so as to be able, in their turn, to raise the body by their spring. When we go from the fassage to the piaffer by shortening the Jassage, we can egulate the times of the piaffer more easily on account of the cadence already obtained ; but the tact of the rider is not the less necessary. Digitized by Microsoft® HIGH-SCHOOL RIDING, 294 what we want him to do. As soon as he correctly marks the first two times, without showing any wish to escape from the spurs, we may try to get him to do four, six or more, and we can continue to increase the number, as long as we hold him easily in this position ; but we should not overdo it. what we want him to do. As soon as he correctly marks the first two times, without showing any wish to escape from the spurs, we may try to get him to do four, six or more, and we can continue to increase the number, as long as we hold him easily in this position ; but we should not overdo it. We should, of course, stop the piaffer and send the horse forward, well in hand, the moment we feel he can escape from us, whether by bringing his haunches sideways, doing a half-rear or reining back, which is still worse. In the first case, we should straighten the haunches by a sharp touch of the spur, given on the side to which he throws himself and by way of punishment. If he makes a half-rear, we are forced to slacken the reins \| to avoid an accident; but the spurs should be kept close to the sides (Fig. 65); because if that were not done, he would constantly rear. Digitized by Microsoft® PIAFFERS AND “PASSAGES.” If the horse finds out that he can escape work by running back, he will become the master of his rider; and every time the rider uses the spurs, no matter for what movement, the horse will run back. If the man does not inflict exemp- lary punishment, zv/c/e the animal is running back, he had best give up the breaking, and—which is still wiser—the horse. We ought to remain cool, however energetically we may act. If we give way to anger, we will become more quickly exhausted than the horse. With this proviso, punish the horse coolly and severely until he learns that the more he goes back, the more pain he will suffer, and consequently he will go forward, in which case we should immediately pat him on the neck, get off, and stop the lesson. If the horse finds out that he can escape work by running back, he will become the master of his rider; and every time the rider uses the spurs, no matter for what movement, the horse will run back. If the man does not inflict exemp- lary punishment, zv/c/e the animal is running back, he had best give up the breaking, and—which is still wiser—the horse. We ought to remain cool, however energetically we may act. If we give way to anger, we will become more quickly exhausted than the horse. With this proviso, punish the horse coolly and severely until he learns that the more he goes back, the more pain he will suffer, and consequently he will go forward, in which case we should immediately pat him on the neck, get off, and stop the lesson. It does not matter whether the struggle takes place at the beginning or middle of the lesson, but as soon as the horse yields we should stop it, because, if we recommence the work, we cannot be sure of obtaining a second concession in the same lesson, especially if the horse has been obstinate for a long time. Besides, we are both tired, and the animal can offer only passive resistance, which is the most terrible of all. We may kill him by blows, but he will not stir. We have gone beyond his strength, and by persisting we will succeed only in disgusting him for ever. Digitized by Microsoft® PIAFFERS AND “PASSAGES.” The action of the spurs ought to be increased by small touches until the horse has become quiet in hand, in which case he has “ given in.” Running back is the most difficult and dangerous fault to overcome. If, on being lightly touched, he violently runs back, the reins should be left quite loose, and the spurs should be used more vigorously. Repeated touches of the spurs close to the girths are a means which the horse cannot resist, if they are energetically continued. Besides, I have already said that we should never spur a horse which is stationary, until we have taught him to go freely forward on being touched by the spurs. Therefore, I do not incur the liability of combatting the defence which I have described. If, how- ever, I had to do so, I would easily settle the matter by repeated attacks of the spurs, which act like the roll of a drum. But if, on account of insufficient breaking, we have to deal with this defence, and if we cannot continue the repeated attacks of the spurs behind the girths, we should take a cutting whip or cane, or get an assistant armed with a driving Digitized by Microsoft® PIAFFERS AND “PASSAGES.” 295 whip. In any case, we must make the animal go forward at all cost. Here we are in a pitched battle, and we have got to know whether the man or the horse is to be master. We should on no account yield. Any means will do, because we must win the battle, otherwise the horse will be restive. Of course, I do not advise violent spurring or other severe measures, unless the horse obstinately runs back, in which case the only alternative left to the breaker is not to be beaten. whip. In any case, we must make the animal go forward at all cost. Here we are in a pitched battle, and we have got to know whether the man or the horse is to be master. We should on no account yield. Any means will do, because we must win the battle, otherwise the horse will be restive. Of course, I do not advise violent spurring or other severe measures, unless the horse obstinately runs back, in which case the only alternative left to the breaker is not to be beaten. + There, and only there, will the touch of the spur give impulsion. Its touch further back will cause forward movement, but without impulsion, and further, it will tickle the horse, on account of the far greater sensibility of that part. Only a touch, as near as possible to the girths, brings the hock under the centre of the body ; hence the impulsion. I am always tearing my girths to ribbons. * In fact, it is the pain which is renewed at each moment that makes the horse fly from the spur. If it is kept against his side he will rest on it, and will inevitably jib. Digitized by Microsoft® * In fact, it is the pain which is renewed at each moment that makes the horse fly from the spur. If it is kept against his side he will rest on it, and will inevitably jib. + There, and only there, will the touch of the spur give impulsion. Its touch further back will cause forward movement, but without impulsion, and further, it will tickle the horse, on account of the far greater sensibility of that part. Only a touch, as near as possible to the girths, brings the hock under the centre of the body ; hence the impulsion. I am always tearing my girths to ribbons. PIAFFERS AND “PASSAGES.” We should therefore profit by the smallest concession, and should, above all things, avoid prolonging the punishment when Digitized by Microsoft® HIGH-SCHOOL RIDING. 296 the horse goes forward. Otherwise, he will not know when he does wrong or when he does right, because he gets beaten whether he goes forward or back. I therefore particularly advise the breaker never to lose his temper, although retaining the utmost energy. I once had a black thorough-bred, called Negro, which I rode in public for four or five years. He never failed me in work, although he was peevish and screamed when touched with the spur. When I began to break him, he ran back immediately I closed my legs. For two months he did not stop running back under me during a lesson of twenty minutes each day. I was in despair. All the breakers of my acquaintance said that I would never succeed in making him go forward, yet he gave in at last. He even became? an excellent lady’s horse, and never relapsed into the troublesome vice, of which I had great trouble to cure him. To succeed in this, I used the spurs only by touches. They should never be kept stuck in the animal’s sides.* The spurs which I used were very sharp, and I applied them, as I always do, as close as possible to the girths.+ I, of course, took care to pat the animal on the neck the moment he went forward. I must, however, admit that after a struggle during twenty minutes without ceasing, I was completely exhausted, and so was my horse. To break such an animal, one must be young, have a very strong seat and good loins, because they are the first to get tired. The reason for ceasing to keep the horse in the same place PIAFFERS AND “PASSAGES.” 297 during the three defences I have mentioned, is to remain master ofhim. If he resists the attacks of the spurs, when he is stationary, I make him go forward at all hazards, I then clo3e my legs, and begin again, until he yields. The point upon which I insist is, that we are always master of a horse when we can make him go forward. These observations, of cours3, do not apply only to the piaffer and passage. PIAFFERS AND “PASSAGES.” My reason for having dwelt on this subject so long is that the means I have mentioned are always the same which I use for combatting defences—always the same which the horse adopts when he is touched by the spur, while he is kept in one place.* Men of the new school, of whom I had occasion to speak in the preceding chapter, employ, in order to teach the piaffer, means not less strange, though more brutal, than that which they use for teaching the Spanish walk. They open the door of the school, and place the horse, with his head turned to the side of the stable, close to the opening of this door, to make him impatient. One man is on his back, another holds the cavesson, and a third stands behind the animal, with a driving whip in his hand.+ They then put on each pastern a leather hobble, to which is attached a piece of wood in the shape of an egg. When the horse moves a foot, or rather when he puts it down, * Many breakers, when alluding to horses which have got the better of them, say, in order to excuse themselves, that nothing can be done with animals which kick, rear, or run back each time they feel the spur. The truth is that their method is faulty. Sometimes they boast that they punished their horse so severely that he could not get up for several days, a statement which only proves their brutality. In the worst battle with a horse, I have never ill-treated him to such an extent as to bring him down. I have never even fatigued him so much as to make him unable to resume his work on the following day. t These gentlemen only work all together—always several tormentors for one victim. Besides, they are so convinced that they cannot do otherwise, that they scoff at those who say that a breaker ought to ride his horse and break him without the help of anyone, a fact which appears to them to be simply impossible. * Many breakers, when alluding to horses which have got the better of them, say, in order to excuse themselves, that nothing can be done with animals which kick, rear, or run back each time they feel the spur. The truth is that their method is faulty. Digitized by Microsoft® PIAFFERS AND “PASSAGES.” Sometimes they boast that they punished their horse so severely that he could not get up for several days, a statement which only proves their brutality. In the worst battle with a horse, I have never ill-treated him to such an extent as to bring him down. I have never even fatigued him so much as to make him unable to resume his work on the following day. t These gentlemen only work all together—always several tormentors for one victim. Besides, they are so convinced that they cannot do otherwise, that they scoff at those who say that a breaker ought to ride his horse and break him without the help of anyone, a fact which appears to them to be simply impossible. Digitized by Microsoft® HIGH-SCHOOL RIDING. 2098 the egg strikes the coronet, and makes him raise the foot. The man in the saddle uses the spurs, the one who is in rear uses the driving whip, and the operator who holds the cavesson, and is in front of the animal’s head, prevents him advancing by giving him blows on the nose with the cavesson. The horse, thus imprisoned, shows great impatience, and as the egg strikes his coronet, and makes him raise his foot each time he puts it down, he performs a kind of hurried piaffer, which is neither graceful nor regular. Besides, by this method they teach a horse to piaffer in front of the stable door, which no doubt is very ingenious, and does not require much skill. It has, however, the drawback that the moment the horse’s head is turned away from the stable, his impatience vanishes, and he stops the piaffer. This is a machine-made horse. I shall not go further in this description, for my intention is only to give a sketch of the system of breaking used by the new school, who use the same means for every kind of work. I do not undertake to describe these means. The horse which has been taught the piaffer, only by the combination of reins and legs, will do the piaffer anywhere, at the wish of his rider, no matter when or where. It is no less true that the system of the new school is practised almost everywhere. Horses are broken now with- out being ridden, and by means analogous to those by which one would break in monkeys and nanny goats. * In the same category we should place the recommendation made by certain authors, to hum a tune in two time toa horse which is being taught the piaffer or passage. Digitized by Microsoft® I have never broken a horse with any music. My only orchestra is the spurs. When my horses hear music for the first time it puts them out, but they soon take to it. * In the same category we should place the recommendation made by certain authors, to hum a tune in two time toa horse which is being taught the piaffer or passage. I have never broken a horse with any music. My only orchestra is the spurs. When my horses hear music for the first time it puts them out, but they soon take to it. PIAFFERS AND “PASSAGES.” In Vienna this system is sarcastically called “ Pudeldressirung ” (poodle breaking).* It is easy to understand how the piaffer, which is learned under these conditions, is always bad. At first, progress is I have never broken a horse with any music. My only orchestra is the spurs. When my horses hear music for the first time it puts them out, but they soon take to it. Digitized by Microsoft® PIAFFERS AND “PASSAGES.” 299 impossible. Afterwards, as the action of the cutting whip or driving whip causes the croup to be raised, the hind legs are raised higher than the fore ones, and the neck is necessarily depressed, But zu the piaffer, as in the passage, the fore legs ought always have the loftier action, which is the case when these movements are taught only by the help of the hands impossible. Afterwards, as the action of the cutting whip or driving whip causes the croup to be raised, the hind legs are raised higher than the fore ones, and the neck is necessarily depressed, But zu the piaffer, as in the passage, the fore legs ought always have the loftier action, which is the case when these movements are taught only by the help of the hands Fig. 62.—Teaching the piaffer by means of a cutting whip. Fig. 62.—Teaching the piaffer by means of a cutting whip. and legs; because the hocks are then brought well under the body, and the neck and forehand are consequently raised.* We can understand that the horses of the new school are never broken in the true sense of the word. At best they are machines and automatons which the first comer can make * Compare Figs. 60 and 61 with Fig. 62, which is copied from a photograph in a recent work that describes the method of teaching the piaffer by means of acutting whip. In Fig. 62 the position of the head and neck is faulty, and the forehand is so over-loaded that the animal cannot raise the fore leg, which ought to be in the air, as in Figs. 60 and 61. In Fig. 62, on the contrary, the horse simply bends the knee, and the toe touches the ground. He does the piaffer only with his hind quarters, which are higher than the forehand. Digitized by Microsoft® PIAFFERS AND “PASSAGES.” Contrary to the intention of the author, all the vices of the piaffer, @ /a cutting whip, are displayed in a striking manner by this illustration. Digitized by Microsoft® Digitized by Microsoft® HIGH-SCHOOL RIDING. 300 do this or that work, if he is only told how, and at what moment or place he should touch certain parts of the animal’s body. The riding masters of the new school take good care not to break thorough-breds, because they know by experience that wiry, energetic and strong horses will not let themselves be tortured. In order to excuse themselves, they maintain that thorough-breds are wanting in suppleness ; the fact being that thorough-breds are the most supple of all horses, but the least patient under pain. Besides, we owe to this new school all those ladies who, not being able or not wishing to ride, find it much more easy to buy a machine-made horse and work him by touching him with a cutting whip on the legs, head, croup, and a little everywhere else, in a most ungraceful manner. True lovers of horsemanship would not engage in this kind of equitation, which is made up of contortions, and which con- sists almost always of making the horse drag himself on his knees, stretch out his head on the ground like a calf which is waiting to have its throat cut, walk on balustrades, etc. The public, knowing little of equitation, applauds in any case, but it is sad that the art of equitation should have fallen so low. In order to know if the rider really possesses horse know- ledge, it is enough to look at the horse. If the animal is li¢ht, well placed, full of impulsion, and does all his move- ments with so much spirit that he appears to work with pleasure, we may be certain that he is not a pupil of the new school, and is still less a machine-made horse, to which I have already alluded. I may add that a man should ride without a whip, and that a lady should use it only to supply the want of the right leg; that is to say, to strike on the right side and only a little behind the girths. Digitized by Microsoft® THE “PASSAGE” AND SPANISH TROT. PIAFFERS AND “PASSAGES.” 301 There is no doubt that the method adopted by the new school is the one which those who hate work would like to follow, because it is within the reach of anyone who would like to practise it after a few days’ study. On the contrary, we may see a true horseman work for years without learning much. A horseman uses only his hands and Jegs, with such delicacy and with such slight movements that the spectator can hardly see them. To sum up, the new school breaks a horse by means of tricks, on which account the first comer who knows these tricks can obtain certain results. The old school is to the new school what the piano is to the barrel organ. Long study and great perseverance are required to be able to play well on the former instrument, and even then success is not always obtained; but all that is wanted to grind the barrel organ is an arm sufficiently strong to turn the handle. In the former case, one can become an artist by work and patience; but, in the latter, one has only to make a noise. Digitized by Microsoft® SERPENTINE AT THE TROT. This movement consists in making a horse take four side steps to the left and four to the right at a well-collected trot. During the whole time the horse ought to go on two tracks, the forehand gaining a little more ground at the side to which the animal is proceeding, so as to prevent the haunches from going in front of the shoulders. This movement appears simple, but it is very difficult to do correctly. We can quickly see that there is only a very short interval to put the horse back from one diagonal to the other gently and smoothly. All the merit and all the difficulty of this exercise consist in taking exactly four steps to each side. If the animal sometimes takes three, and sometimes five, there is no difficulty, merit, or interest. DIFFERENCES BETWEEN THE “ PASSAGE” AND SPANISH TROT. Qa Even among those who ride well, there are many who confuse the passage with the Spanish trot. In the passage, the knees and fetlocks are bent; in the Spanish trot, they are extended. In the passage, the less ground is covered, the better is the movement, because the time of suspension is better marked, and the horse appears to raise himself perpendicularly. In the Spanish trot, on the contrary, we should try to cover In the Spanish trot, on the contrary, we should try to cover as much ground as possible in each stride. To be brilliant, the pace should be high and long, but not hurried ; because, if it is too fast, the fore legs will not be able to mark the time of suspension. Digitized by Microsoft® 302 HIGH-SCHOOL HIGH-SCHOOL RIDING. 302 There is another trot which is called the “swimmer,” in which the horse ought to trot as fast as he can, while indicating with his fore legs a time of suspension in the air. But, as the speed is great, the period of suspension 1s much less marked than in the Spanish trot. I do not think that this is a school movement. <A horse which trots well with his shoulders and knees will do it naturally. I content myself with merely mentioning it. CANTER ON THREE LEGS. We can make a horse canter on three legs, in which case the three first times of the canter are done in a rassemblé'd canter (in four times), and the fourth time is done in the air. If, for example, we are cantering to the right, the off fore should be held up, without touching the ground. Few horses can do in a brilliant manner the canter on three legs. Many can succeed in doing it ; but only a very energetic animal can hold his leg well extended in the air, without its touching the ground, during the whole period of the movement, Digitized by Microsoft® CANTER ON THREE LEGS. 303 only under which condition is this exercise interesting and brilliant. only under which condition is this exercise interesting and brilliant. If the reader has attentively followed my explanations, he will know that accustoming the horse to the pressure of the legs prepares him for this school movement, which is very difficult for the animal to understand and do. In fact, up to this, he has learned only to raise, extend, and place his legs on the ground, one after the other; but now he is required to extend only one leg, and to keep it in that position. The canter with the off fore in the air has only three beats on the ground—the first made by the near hind, the second by the off hind, and the third by the near fore. The fourth time is marked in the air by the off fore, which is fully extended and kept as high as possible. Before putting a horse to this work, we should wait until he is perfectly broken, and until he will offer no resistance. At this time, I have at my disposal two movements which have been separately learned, namely, the ordinary canter to the right, and the extension of the off fore. From the combina- tion of these two exercises, in which my left leg is my chief helper, I obtain the canter to the right on three legs. It seems, in theory, that I ought to easily obtain the canter to the right on three legs, by vigorously using the left spur. 304 HIGH-SC As the momentum is no halt, the horse who expec anticipates the halt, and canter with his leg in the him, pat him on the neck, We can see that these separate, are beginning t not sufficient. When the horse does thi we ought to demand the halt, by simply taking ca raise the forehand. We long time should be conte which the leg is complete sustained. If we wish to stimulate will hold his leg in the air, energy ; because, not only 304 HIGH-SCHOOL RIDING. As the momentum is not entirely checked during this half- halt, the horse who expects to raise his leg during the halt, anticipates the halt, and almost always does a time of the canter with his leg in the air, in which case we should halt him, pat him on the neck, and stop the lesson. We can see that these two movements, which at first were HIGH-SCHOOL RIDING. 304 As the momentum is not entirely checked during this half- halt, the horse who expects to raise his leg during the halt, anticipates the halt, and almost always does a time of the canter with his leg in the air, in which case we should halt him, pat him on the neck, and stop the lesson. We can see that these two movements, which at first were separate, are beginning to be formed into one; but that is not sufficient. When the horse does this work easily, and without stiffness, we ought to demand the extension of the leg without a half- halt, by simply taking care to decrease the speed, and to raise the forehand. We should hasten slowly, and for a long time should be content with two or three strides during which the leg is completely extended, and above all things sustained. If we wish to stimulate the horse to such an extent that he will hold his leg in the air, we should, ourselves, display great energy ; because, not only is the office of our left leg to make the horse keep his off fore in the air, but it also, in combina- tion with the right leg, ought to keep him at the canter, by bringing his hind legs under him. In other words, we main- tain the canter by a strong pressure of both legs, and at the same time we make the animal hold his off fore in the aur, by small and repeated touches of the left spur. We ought to use the right leg as vigorously as the left, in order to prevent the horse carrying his hind quarters to the right. Digitized by Microsoft® CANTER ON THREE LEGS. But, in practice, this is not so simple; because, having put my horse into the canter, the use of the left spur will do no good ; for it will make him bring his haunches to the right, or get away, but it will not make him extend his off fore. We should therefore canter the horse to the right, and, having halted him, make him extend his off fore. When he has done this, we should pat him on the neck, and then begin again several times. When the horse extends the leg without difficulty during the halt, we should make him go through the same performance, during a shorter halt. Digitized by Microsoft® 04 HIGH-SCHOOL Fig. 64. Germinal at the canter to the right on three legs ; rassembléd. We can see how much the action of the spur, which is necessary for the rassembler, increases the extension of the leg in the air, and increases the height of the action, by bringing the hind quarters under the body. Fig. 65 represents Germinal at the half-rear with the off fore extended, at the moment when I demanded the canter to the right on three legs in the rassemzbler. The horse had begun by a lazy extension of the leg ; but a sharper touch of the spur has made him do this half-rear, while at the same time he extends his off fore perfectly. In fact, this is a case of absolute obedience ; and the horse, immediately after the half-rear, starts into the canter to the right on three legs, in the rassembler. The photograph shown in Fig. 64 was taken immediately after this half-rear. * Fig. 63. Germinal at the canter to the left on three legs; in hand, but not rassembléd. The canter isin four time, although there is no rassembler, because the energetic uplifting of the forehand keeps in the air, even the leg which is not extended. 304 HIGH-SC As the momentum is no halt, the horse who expec anticipates the halt, and canter with his leg in the him, pat him on the neck, We can see that these separate, are beginning t not sufficient. When the horse does thi we ought to demand the halt, by simply taking ca raise the forehand. We long time should be conte which the leg is complete sustained. If we wish to stimulate will hold his leg in the air, energy ; because, not only If he goes sideways, we should stop him and put him straight, before recommencing the movement ; because, when he places himself obliquely, he will be deficient in propulsion, and in this position we shall be able to obtain only the exten- sion of the leg or the canter; but not both together. It is therefore indispensable to keep the animal very straight, and to give him a point of support on the right snaffle rein, in order to facilitate the upholding of the right leg. Digitized by Microsoft® “PASSAGE” TO THE REAR. 305 The canter on three legs may be done either to the right or to the left.* I do not know if others taught this exercise before me. In any case, I have never seen it done by anyone else, and I have never met with its description in any treatise on equitation. * Fig. 63. Germinal at the canter to the left on three legs; in hand, but not rassembléd. The canter isin four time, although there is no rassembler, because the energetic uplifting of the forehand keeps in the air, even the leg which is not extended. Fig. 64. Germinal at the canter to the right on three legs ; rassembléd. We can see how much the action of the spur, which is necessary for the rassembler, increases the extension of the leg in the air, and increases the height of the action, by bringing the hind quarters under the body. Fig. 65 represents Germinal at the half-rear with the off fore extended, at the moment when I demanded the canter to the right on three legs in the rassemzbler. The horse had begun by a lazy extension of the leg ; but a sharper touch of the spur has made him do this half-rear, while at the same time he extends his off fore perfectly. In fact, this is a case of absolute obedience ; and the horse, immediately after the half-rear, starts into the canter to the right on three legs, in the rassembler. The photograph shown in Fig. 64 was taken immediately after this half-rear. We can see, agreeably to what I have advised, that the spurs are applied to the sides during the half-rear, and the reins siackened. In fact, the reins are much less tight in Fig. 65 than in Fig. 64, in which the hands feel them ; but in Fig. 65 the hands are carried forward on the neck. “PASSAGE” TO THE REAR. This is a very difficult exercise to do; because ground has to be gained to the rear, while maintaining the diagonal steps in a well-cadenced manner, and preserving the same elevation of the hind legs, as in the forward movement. In every retrograde movement, the hind feet have a tendency to keep as close to the ground as possible. Unless we are absolutely sure of ourselves—as we ought to be, when we face such difficulties—we often produce, against our will, too strong an effect from front to rear, and thus transfer the weight from the forehand to the hind quarters, which consequently becomes lowered. To obviate this fault, we should make the horse Fig. 65 represents Germinal at the half-rear with the off fore extended, at the moment when I demanded the canter to the right on three legs in the rassemzbler. The horse had begun by a lazy extension of the leg ; but a sharper touch of the spur has made him do this half-rear, while at the same time he extends his off fore perfectly. In fact, this is a case of absolute obedience ; and the horse, immediately after the half-rear, starts into the canter to the right on three legs, in the rassembler. The photograph shown in Fig. 64 was taken immediately after this half-rear. We can see, agreeably to what I have advised, that the spurs are applied to the sides during the half-rear, and the reins siackened. In fact, the reins are much less tight in Fig. 65 than in Fig. 64, in which the hands feel them ; but in Fig. 65 the hands are carried forward on the neck. 20 Digitized by Microsoft® HIGH-SCHOOL RIDING. 306 rein back by the seat, as I have described (p. 247), and not by the reins. It is a mistake to think that we can easily go from the forward passage to the backward fassage, to obtain which it is necessary to make the transition through the natural, slow and high piaffer, which is only the passage without gaining ground. It is only when the horse piaffes correctly, that we can try to gain a few inches of ground to the rear at each step. With this object I increase the pressure of the legs. “PASSAGE” TO THE REAR. I try to rassembler the horse without pulling him back by the reins, and I sit well down into the saddle, while leaning strongly on each footfall. On account of the rassembler being carried to its maximum, the equilibrium and mobility of the horse are such that the slightest displacement of the body of the rider can draw the horse in any direction, as for instance, to the rear, which 1s the point we are considering. This means of obtaining the passage to the rear is long and difficult; but it is the only one which gives a good result. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® SEE-SAW PIAFFER. In the see-saw piaffer, the off legs ought to be raised and put down in the same place, while the near ones make a beat forward and a beat backward, while always coming back to the same spot. The near legs ought not only to mark the going and coming from front to rear, but the near hind quarter and near shoulder ought also to accentuate this movement with- out any participation on the part of the off legs, which should only piaffe regularly without gaining ground. Baucher did this piaffer with great éc/a¢ on a mare called Stades. I have taught this school movement to four horses, Digitized by Microsoft® Fig. 63.—Cantering to the left on three legs, in hand, but not rassemélé'd. Fig 63.—Can to the left on three legs, in hand, but not rassemélé'd. to the left on three legs, in hand, but not rassemélé'd. h but not rassemélé'd. 20* d by Microsoft® igitize d by Microsoft® igitize Digitized by Microsoft® Digitized by Microsoft® Fig. 64.—Cantering to the right on three legs; rassemblé'd. Fig. 64.—Canteri to the right on three legs; rassemblé'd. Fig 64.—Can to the right on three legs; rassemblé'd. d by Microsoft® igitize Digitized by Microsoft® Digitized by Microsoft® Fig. 65.—Half-rear (gozn¢e or Jancade)) with off fore extended, Fig 65.—Hal (gozn¢e or Jancade)) with off fore extended, d by Microsoft® ize ti igi Digitized by Microsoft® Digitized by Microsoft® SEE-SAW PIAFFER. 313 which did it more or less brilliantly, according to their degrees of energy. which did it more or less brilliantly, according to their degrees of energy. It is very difficult to explain how to teach the see-saw plaffer, but I will try to do so. It will at first be seen that this movement should not be required from any horse, unless he can do the slow and high piaffer. As such an animal, when doing this exercise, has extreme mobility, the slightest feeling on the reins will be sufficient to make him bring a leg a little back, in the same way that the smallest increase in the pressure of the spur will cause him to bring a leg a little forward. Being at the piaffer, we ought, by means of the legs, to make the horse bring his near hind under him where he puts it down. * On comparing Fig. 67 with Fig. 66, we will see that in the movement to the rear, the snaffle reins are drawn a little tighter than those of the curb. The angle of the cheek of the curb is very little more open, and the seat presses the horse back. Digitized by Microsoft® SEE-SAW PIAFFER. The right diagonal being in support, the near fore— which, under the same action of the legs of the rider, has been carried forward at the moment when the near hind is placed under the animal’s body—remains held up in the air, the knee bent, and the off hind also in the air (Fig. 66). Immedi- ately the hand ought to act, in order to bring back the near fore, which is about to be placed behind the off fore, and which forms, along with the off hind, the left diagonal sup- port; whilst the near hind, in its turn, does a time in the air, and the off fore performs its movement in the piaffer.* (Fig. 67.) We can see that in the forward movement the near hind is placed in front of the off hind (Fig. 66), and that in the retrograde movement the near fore is placed behind the off fore (Fig. 67). This is the cause of the see-saw action from the rear to the front. If the reader understands my explanations he will observe HIGH-SCHOOL RIDING. 314 that this piaffer is done, like the ordinary piaffer, by the diagonal, notwithstanding the see-saw lateral. It is very difficult to indicate, with mathematical precision, the exact moment when the rider ought to use any particular rein or leg ; the action of the two reins and of two legs being made by a succession of movements which follow each other so closely that it is almost continuous. Here equestrian tact, which is the supreme quality of a rider, displays itself in all its beauty. I can only say that I increase the action of the right spur to obtain the forward movement of the near hind, and to help to keep the near fore in the air. But at the same time I strongly use my left leg to prevent the near hind from going to the left, because it gets its stimulus from my right leg. For the backward movement I feel both reins, the near rein a little more than the off, at the moment when the near fore has to be brought back. During the whole time the rider ought to freely use his legs and even the spurs, but the off rein hardly at all, and the near rein very little. Digitized by Microsoft® Digitized by Microsoft® CANTER WITHOUT GAINING GROUND AND CANTER TO THE REAR. The canter without gaining ground is a canter which is shortened as much as possible. It is similar in every way to the forward canter, except that the legs do not gain ground. It is very near to the backward canter, which is a regular pace of four time, exactly like the forward canter in the vassembler. It is the most difficult and most complicated school exercise. My advice is that it should be tried only at the extreme end of training, and with selected horses, whose loins and hocks should be exceptionally good to bear the vassembler at its maximum, without which it is impossible to obtain this movement. Digitized by Microsoft® Fig. 66.—See-saw piaffer. Fig. 66.—See-saw piaffer. Fi 66.—Se piaffer. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Fig. 67.—See-saw piaffer. Fi 67.—Se piaffer. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® CANTER WITHOUT GAINING GROUND. CANTER WITHOUT GAINING GROUND. 319 The thoroughbred Gant, by Gantelet out of Mlle. de Romanerie, did the canter to the rear as if it were play, and after he had gone round the school at this pace he was as fresh as before. Baucher did not know or was not able to describe the canter to the rear. The following, which is his definition of it in the fourteenth edition of his “ Method of Equitation,” p. 155, evidently corresponds to the way he did it :—“In the rein back at the canter the times are the same as those of the ordinary canter; but the fore legs, instead of gaining ground when raised, are carried back, in order that the hind legs may do the same retrograde movement immediately the fore legs are placed on the ground.” How could Baucher make such a description of a canter to the rear, after having said that it was like the canter to the front? No doubt it is like the canter to the front, and in the vassembler it is in four time; but it is precisely for this reason that we cannot give the name of canter to a pace in which the hind legs do not make their retrograde movement before the fore legs are placed on the ground. What, then, is this pace in which the two fore feet and the two hind feet respectively come to the ground at the same time. Under these conditions where is the canter? There is no need to be a great horseman to understand that Baucher alludes to a pace of two time, the first time being made by the hind quarters, the second by the forehand. But that is not a canter; it 1s plainly little jumps to the rear. There cannot be a canter unless the fore legs and the hind legs are respectively put down one after another; and further, when one of the hind legs is on the ground at the same time as the opposite fore leg, they form the left diagonal when the horse is cantering to the right, and the right diagonal when he is cantering to the left. Baucher invented the expression, “canter to the rear,” but Digitized by Microsoft® HIGH-SCHOOL RIDING. CANTER WITHOUT GAINING GROUND. 320 he entirely misunderstood this pace, the proof being that he never said with which leg the horse led or ought to lead. Having broken Gant for a lady, I taught him the canter to the rear on the right leg so that the lady might be able to keep him at this pace with her spur, which is to the left. I have done more than this, for I succeeded in teaching the thorough-bred Germinal, by Flavio, out of Pascale, to canter to the rear either on one foot or on the other, according as I wished. I claim the honour to be the first to obtain this result. Whether a horse canters to the rear or to the front, the rider ought always to be able to say on which leg he is. The canter to the rear with the near fore leading—although it is a pace of four time—ought to be made in the same way as if the horse was cantering to the front im three time— namely, first time, off hind; second time, right diagonal ; third time, near fore. The great difficulty is to prevent the respective fore and hind legs being placed on the same transverse line. The off hind should be placed in rear of the near hind in order to preserve almost the same longitudinal distance between the two legs, as in the ordinary canter. I purposely say “ nearly the same distance,” because the strides are shorter. I need not add that the off fore should be behind the near fore, as in the forward canter. We have seen that a stride of the canter consists of three times on the ground—namely, hind leg support, diagonal support, and fore leg support, as we see done with great precision in a good hunting canter, or what we call a hand gallop. But in the full speed gallop and in the canter without gaining ground, or the canter to the rear, which are the two extremes, the stride is in four time. In the canter to the left, for instance, the two legs which form the mght diagonal, instead of touching the ground simultaneously, come down Digitized by Microsoft® CANTER WITHOUT GAINING GROUND. 321 one after another, the succession of steps being as follows: off hind, near hind, off fore, near fore. * Fig. 68.—Germinal cantering to the rear ; secondtime. This photograph was taken at the moment when the right diagonal was about to be used in support. The near hind is already on the ground, but the off fore has not yet come down. Hence the four times, the right diagonal making two beats instead of one. We should note that even in this extreme rassemdler the head remains a little beyond the vertical. We can therefore see that the reining back is done by the seat, and not by the reins, which are not drawn tightly. Digitized by Microsoft® CANTER WITHOUT GAINING GROUND. In the full speed gallop this fact is well marked, but the speed of the pace prevents it being apparent. In the stationary canter, and still more in the canter to the rear, it is much less marked, and for this reason it is almost imperceptible. In fact, at this pace the feet only graze the ground, and are drawn back for only a few inches. Instruments of precision or instantaneous photography would be required to prove this in both cases, but the fact remains. We can therefore say that in the full speed gallop and in the canter without gaining ground and to the rear, there are four successive impulsions. Let us note, however, that in the canter without gaining ground, and in the canter to the rear, the diagonal acts in the same way as in the ordinary canter, with the single difference —which is a point I wish to bring to ight—that the support of the hind leg precedes the support of the fore leg of the diagonal by an extremely short though actual interval.* To obtain the canter without gaining ground, I begin by shortening the canter every day while maintaining the pro- pulsion—that is to say, by pressing the horse with the legs up to his bit, the play of which should be fine in proportion to the extent of the rassembler. By gradually decreasing every day the length of the strides, I obtain the canter without gaining ground with impulse, but not with the horse getting behind his bit. When the animal is behind his bit in the canter, it is impossible to keep him up to his bit, and he necessarily gets away from his rider by reining back. 21 Digitized by Microsoft® HIGH-SCHOOL RIDING. 322 We have now to ccnvert the canter without gaining ground, into the canter to the rear, for which purpose, when my horse is cantering on one spot, with such ease and lightness that I have no need for the reins, I try to bring him back an inch or two by my seat and legs, and not by the reins. While my legs are raising the horse, I seize the moment when he is in the air to carry my seat back. * This is the moment we have taken for changing the leg between the last time of one stride and the first time of the next stride. It can be seized only with difficulty, and then we can get everything out of a horse, because he is in the air. A puff of wind can displace him. CANTER WITHOUT GAINING GROUND. I change the position of my seat, and not that of the upper part of my body, by taking nearly all my weight off the stirrups to put it on my buttocks. The mobility of the horse is so great, at a moment when he is in suspension,* that a movement of the rider is sufficient to make him gain a little ground to the rear, which is enough to begin the canter to the rear. By repeating and gradually increasing these effects every day, we succeed in obtaining the canter to the rear as I have described. If we try to get the backward movement by the reins, the rassembler will be immediately lost, because the action of the reins will send the hocks a long way to the rear; their duty, on the contrary, being to remain under the centre of the body. When they are to the rear, the hind quarters being over-loaded lose the mobility which enables them to gain ground to the rear. Digitized by Microsoft® Digitized by Microsoft® NEW SCHOOL MOVEMENTS. Ordinary voltes and pirouettes at the canter on thre 4. “ Two tracks” at the canter on three legs to both sides. 5. Ordinary voltes and pirouettes at the canter on three 5. Ordinary voltes and pirouettes at the canter on three legs, to the right and to the left. 5. Ordinary voltes and pirouettes at the canter on three legs, to the right and to the left. 6. Spanish trot in two beats on each leg. Up to the present the Spanish trot has been done only in one time on each leg; that is to say, one time on the right and one on the left. In the Spanish trot in two times, I make the horse successively do two beats with the off fore, with the near fore extended, and immediately afterwards two beats with the near fore, with the off fore extended. The rider can continue this movement as he likes. The horse naturally advances a little less in the second time than in the first. 6. Spanish trot in two beats on each leg. Up to the present the Spanish trot has been done only in one time on each leg; that is to say, one time on the right and one on the left. In the Spanish trot in two times, I make the horse successively do two beats with the off fore, with the near fore extended, and immediately afterwards two beats with the near fore, with the off fore extended. The rider can continue this movement as he likes. The horse naturally advances a little less in the second time than in the first. 7. The Spanish trot in one and two times alternately, as follows, the word leg being understood: right, left; might, right; left, right; left, left. I prolong this movement as I wish during one or two turns round the school. I believe \| am correct in stating that this is the most complicated move- ment that has been obtained in equitation. 7. The Spanish trot in one and two times alternately, as follows, the word leg being understood: right, left; might, right; left, right; left, left. I prolong this movement as I wish during one or two turns round the school. I believe \| am correct in stating that this is the most complicated move- ment that has been obtained in equitation. 8. Digitized by Microsoft® NEW SCHOOL MOVEMENTS. I have invented a certain number of school exercises, and will content myself by enumerating them. They are done “aids” ¢ according to the same principles and by means of the which have been used for the teaching of the preceding movements :— 1. New Spanish walk, which consists in making a step Digitized by Microsoft® Digitized by Microsoft® 68.—Cantering to the rear 3 second time, 68.—Cant to the rear 3 second time, 68.—Can to the rear 3 second time, 68.—Can to the rear 3 second time, to the rear 3 second time, 21* d by Microsoft® igitize d by Microsoft® igitize Digitized by Microsoft® Digitized by Microsoft® NEW SCHOOL MOVEMENTS. 325 forward and a step to the rear. The near fore, for instance, extends itself and takes a step forward. Then the off fore extends itself and also takes a pace to the front, but returns by taking a step to the rear. Here the left diagonal advances and the right diagonal goes back. I continue this movement as long as I like. I then change the diagonal. The off leg, in its turn, makes a step forward, and the near leg, after having been extended to the front, takes a step to the rear, in which case the left diagonal goes back. We can change the diagonals as we wish. forward and a step to the rear. The near fore, for instance, extends itself and takes a step forward. Then the off fore extends itself and also takes a pace to the front, but returns by taking a step to the rear. Here the left diagonal advances and the right diagonal goes back. I continue this movement as long as I like. I then change the diagonal. The off leg, in its turn, makes a step forward, and the near leg, after having been extended to the front, takes a step to the rear, in which case the left diagonal goes back. We can change the diagonals as we wish. 2. Serpentine at the passage. This is the same movement as the serpentine at the trot; but it is evidently much more difficult to do. 3. Canter on three legs, to the right and to the left. 4. “ Two tracks” at the canter on three legs to both si 4. “ Two tracks” at the canter on three legs to both sides. 5. * Figs. 69 and 70.—Germinal at the canter to the right on three legs to the rear. Fig. 69, second time. The off hind has just come down after the near hind. For obtaining the extension of the off fore, the spur gives the impulse that pre- serves the seat, which makes the horse rein back. We can see that the horse’s head is a little beyond the perpendicular, and that the reins are slack. NEW SCHOOL MOVEMENTS. The canter without gaining ground, and the canter to the rear, on three legs. The great difficulty of this exercise is to HIGH-SCHOOL RIDING. 326 obtain and preserve forward impulsion for the extended leg, while the other three are going back.* It is, of course, understood that all these movements are done by means of the “aids” I have described. A repetition of this would be useless to anyone who has attentively read the preceding chapters. * Figs. 69 and 70.—Germinal at the canter to the right on three legs to the rear. Fig. 69, second time. The off hind has just come down after the near hind. For obtaining the extension of the off fore, the spur gives the impulse that pre- serves the seat, which makes the horse rein back. We can see that the horse’s head is a little beyond the perpendicular, and that the reins are slack. Fig. 70, third time. The near fore has just been put down behind its point of departure, and the off fore, which would be in support during the fourth time, remains in the air. The bringing together of the three legs in support shows the extent of ground which has been gained to the rear. The seat is continuously drawn back. The reins, especially the off snaffle rein, are drawn up a little, in order to keep the off fore in the air. These illustrations enable us to appreciate the delicacy of the “‘aids.’’ Digitized by Microsoft® Fig. 70, third time. The near fore has just been put down behind its point of departure, and the off fore, which would be in support during the fourth time, remains in the air. The bringing together of the three legs in support shows the extent of ground which has been gained to the rear. The seat is continuously drawn back. The reins, especially the off snaffle rein, are drawn up a little, in order to keep the Digitized by Microsoft® Digitized by Microsoft® SCHOOL HORSE FOR LADIES. A high-school horse which is required for a lady, ought to be particularly supple, and should work from right to left with more ease than from left to right, because a lady rider does the movements from left to right as easily as a man, for she has on the left side the same “aids” as he has, namely, leg and spur. But in the movements from right to left, her whip, which is far less powerful than a leg armed with a spur, re- places the right leg of a horseman. If the horse is not very clever in movements from right to left, the action of the whip will be insufficient. This fact holds good in all kinds of work. Especially in the “ two tracks,” the lady’s horse does not hold himself so well, and is not so completely in hand from right to left as from left to right. The changes of leg are also more difficult from right to left. In the Spanish walk, the near leg is not raised so high, and is not so well extended as the off leg. Increased severity in the application of the whip will cause Fig. 70, third time. The near fore has just been put down behind its point of departure, and the off fore, which would be in support during the fourth time, remains in the air. The bringing together of the three legs in support shows the extent of ground which has been gained to the rear. The seat is continuously drawn back. The reins, especially the off snaffle rein, are drawn up a little, in order to keep the off fore in the air. These illustrations enable us to appreciate the delicacy of the “‘aids.’’ Digitized by Microsoft® “ig. 69.—Cantering to the right on three legs to the rear; second time. “ig. 69.—Cantering to the right on three legs to the rear; second time. “ig. 69.—Canter to the right on three legs to the rear; second time. “ig. 69.—Cant to the right on three legs to the rear; second time. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® 70.—Cantering to the right on three legs to the rear; third time. 70.—Cantering to the right on three legs to the rear; third time. 70.—Cantering to the right on three legs to the rear; third time. Digitized by Microsoft® * As regards myself, I claim to be a follower of Baucher. My teacher, Frangois Caron, was his pupil. I have thoroughly studied the method of Baucher in all its parts. Without Baucher I would not know as much as I do of riding. I would be very ungrateful to mention the name of Baucher without at the I would be very ungrateful to mention the name of Baucher without at the same time rendering well-merited homage to his rival, Victor Franconi, from whom I have received many excellent lessons. By his pluck, strength of seat, and by the impulse he gave to his horses, his style of equitation resembled much more that of Count d’Aure than that of Baucher. The names of the masters to whom the horsemen of to-day are beholden would SCHOOL HORSE FOR LADIES. d by Microsoft® igitize Digitized by Microsoft® Digitized by Microsoft® SCHOOL HORSE FOR LADIES, 331 the horse to make a sudden start. Besides, when the lady uses her right arm energetically, to give a cut with the whip, the left hand will almost always move, and will consequently give a jerk to the mouth, which will make the animal assume a wrong position. Almost all ladies bring their horse’s hind quarters round to the right, because they make too much use of their powerful “ aid” on the left side; their “aid” on the right side being too weak to keep the animal straight. To obviate these inconveniences, a lady’s school horse ought to work very easily from right to left. A lady’s hack ought to be the same on both sides. Digitized by Microsoft® Digitized by Microsoft® 332 * As regards myself, I claim to be a follower of Baucher. My teacher, Frangois Caron, was his pupil. I have thoroughly studied the method of Baucher in all its parts. Without Baucher I would not know as much as I do of riding. I would be very ungrateful to mention the name of Baucher without at the same time rendering well-merited homage to his rival, Victor Franconi, from whom I have received many excellent lessons. By his pluck, strength of seat, and by the impulse he gave to his horses, his style of equitation resembled much more that of Count d’Aure than that of Baucher. The names of the masters to whom the horsemen of to-day are beholden would form a long list. France can boast of an admirable host of great horsemen. If Italy can name Pignatelli; England, Newcastle; and Germany, Count von Schweppe, France can put forward hundreds of illustrious names, at the head of which shine—to speak only of bygone celebrities—such horsemen as Dupaty de Clam, La Guériniere, the Chevalier d’Abzac, the Marquis of Bigne, and others. The school. of Hanover, which is the most celebrated of foreign schools, is the direct offspring of the great school of Versailles. Beyond all dispute, France is the classic country of equitation. COMMENTARIES ON BAUCHER. BAUCHER was certainly the greatest and most clever high- school rider we have ever had.* There is no doubt that he did not invent the high school, which is the result of the work of many generations of horsemen, but he invented and co- ordinated a new and astonishing method. Prior to his time, no horseman had obtained such marvellous results. \| He con- quered many difficulties, and removed a great number of obstacles which a man who wishes to break-in a school horse always meets. He invented new exercises, which he did with remarkable The names of the masters to whom the horsemen of to-day are beholden would form a long list. France can boast of an admirable host of great horsemen. If Italy can name Pignatelli; England, Newcastle; and Germany, Count von Schweppe, France can put forward hundreds of illustrious names, at the head of which shine—to speak only of bygone celebrities—such horsemen as Dupaty de Clam, La Guériniere, the Chevalier d’Abzac, the Marquis of Bigne, and others. The school. of Hanover, which is the most celebrated of foreign schools, is the direct offspring of the great school of Versailles. Beyond all dispute, France is the classic country of equitation. Digitized by Microsoft® Digitized by Microsoft® COMMENTARIES ON BAUCHER. 333 precision. His method is specially admirable from the fact that it keeps a horse sound by proper distribution of weight, rational gymnastics, suppling, and a correct development of his powers.* Also it has the great advantage over the old methods of quickly giving results. Thus, with Baucher’s method, we can break a hack in two months, and a school horse in eight or ten months. Formerly, the latter result took two or three years to obtain; and, besides, the old time horsemen never attempted the difficulties which Baucher conquered.t I go further, and say that they did not even know that such difficulties existed. We should therefore bow with gratitude before this master of the equestrian art. Is this equivalent to saying that Baucher should be exempt from criticism? Certainly not; and for my part, I am far from agreeing with all his ideas. 1 even think that it is my duty to oppose those proceedings of his which I have found to be wrong. In this book I have had occasion several times to show up certain mistakes of the great horseman. * T have adopted in my work what I call the three golden keys of Baucher’s method ; namely, his complete suppling exercises, with greater elevation of the head and neck; his attacks with the spurs, to inclose the horse; and his rassembler, which I have perfected. Apart from this, I have worked more on the lines of the old school of Versailles than on those of Baucher, in that I always advocate fast paces, and that the horse should be allowed to extend himself. t Up to the present time I have broken thirty-five school horses, which is more than anyone else has done. Baucher, who died when he was seventy-four, broke twenty-six. Being only fifty-six, I hope to double that number when I amas old as the great rider. I have broken hundreds of hacks. Ido not pretend to be cleverer than my illustrious predecessors. I know that before my time others have done as well, and perhaps better. I mention these facts only to show the results of my method. Iam certain that every horseman who wishes to follow it will be able to obtain similar results, provided that he is properly endowed by Nature, and that he loves horses. Few authors have described a correct method of breaking in the proper sense of the word. I have read all the books on equitation, and find that none of them are perfect. Digitized by Microsoft® t Up to the present time I have broken thirty-five school horses, which is more than anyone else has done. Baucher, who died when he was seventy-four, broke twenty-six. Being only fifty-six, I hope to double that number when I amas old as the great rider. I have broken hundreds of hacks. Ido not pretend to be cleverer than my illustrious predecessors. I know that before my time others have done as well, and perhaps better. I mention these facts * T have adopted in my work what I call the three golden keys of Baucher’s method ; namely, his complete suppling exercises, with greater elevation of the head and neck; his attacks with the spurs, to inclose the horse; and his rassembler, which I have perfected. Apart from this, I have worked more on the lines of the old school of Versailles than on those of Baucher, in that I always advocate fast paces, and that the horse should be allowed to extend himself. COMMENTARIES ON BAUCHER. I shall now criticise some of his opinions. Ido not pretend to be cleverer than my illustrious predecessors. I know that before my time others have done as well, and perhaps better. I mention these facts only to show the results of my method. Iam certain that every horseman who wishes to follow it will be able to obtain similar results, provided that he is properly endowed by Nature, and that he loves horses. Few authors have described a correct method of breaking in the proper sense of the word. I have read all the books on equitation, and find that none of them are perfect. Digitized by Microsoft® COMMENTARIES COMMENTARIES ON BAUCHER. 334 Thus, in the first place, I say that Baucher’s method, which consists in keeping the horse completely and constantly closed between the hands and legs of the rider, is dangerous for people who are indifferent riders, and also for those who work or break their first horses without being under the eye of a master.* It is, therefore, not within the compass of everyone, and perhaps it cannot be practised without trouble, except by those who have seriously studied it. Baucher says that the mouths and sides of all horses are the same, which is an entirely erroneous idea. I maintain, on the contrary, that it is impossible to find two horses whose mouths and sides are equally sensitive. Without doubt, the differ- ence, like that between the leaves of a tree, may not be very apparent in all cases, but it exists, and cannot be denied. It is possible that we can succeed in making all horses light in hand and sensitive to the spurs, but I deny that we can give them all the same lightness and sensitiveness. Baucher adds that in the case of a puller, his mouth is not at fault, and that it is sufficient to change his equilibrium. Let us test his statement by taking, for instance, racehorses in training, and leaving them to make their own distribution of weight, with their heads low, necks stretched out, and croups high. What will happen? Some will not go up to the bridle and will run badly ; others will pull just enough to run well; and a third variety will pull too hard, and will run away, notwithstanding the strength of their riders’ arms. * This is probably the reason why many true followers of Baucher have made their horses restive. Digitized by Microsoft® COMMENTARIES ON BAUCHER. What does this prove, except that they all have not the same sensitiveness of mouth, as they are all in the same position? We should note that all horses in training are bitted in the same way, namely, with a plain snaffle. We therefore cannot blame the curb for their different ways of pulling. The experiments I have made have thoroughly cleared up Digitized by Microsoft® COMMENTARIES ON BAUCHER. 335 this point, and I have no hesitation in laying down the principle that the mouth of each horse has its own peculiar degree of sensibility, no matter what is the pace, work, or distribution of weight. this point, and I have no hesitation in laying down the principle that the mouth of each horse has its own peculiar degree of sensibility, no matter what is the pace, work, or distribution of weight. Thus, I owned and broke to high school work the thorough- breds Redoubt by Parmesan, and Gant by Gantelet, out of Mlle. de la Romanerie, both of which had won races. I did not get them until they were five years old, and I made them remarkably good school horses. Redoubt had a very light mouth, and could do the perfect rassembler almost without my having to touch the reins. Gant had a harder mouth, and required to have the reins drawn tightly, in order to do the rasseméler to the same extent. Riding them alternately, I went to the training ground be- hind Bagatelle, at a walk, trot, and canter, and observed, during these paces, the same difference of mouth which I had noticed in the manége, when doing the finest high school work. Having brought them on the race-course, with the curb chain removed and the curb reins knotted on the neck, I sent them a short preliminary canter. Both of them quickly got into their racing position and speed. After that I sent them a full speed gallop of about five furlongs, and they performed in a manner which would have given no one the impression that they had not galloped for eighteen months ; but each of them showed in the full gallop the same difference of mouth which they had done in the riding school and when hacking. Digitized by Microsoft® COMMENTARIES ON BAUCHER. During the gallop on the race-course, Redoubt pulled just enough to run well; but Gant took a firm hold of the snaffle, pulled hard, and tried to run away. I easily stopped the former in a few strides, but I succeeded in pulling up the latter only at the end of a hundred and fifty or two hundred yards, and more by the voice than by the hands. After the gallop, I walked them back to the stable to let them catch their wind, and as soon as \| arrived, I made them COMMENTARIES ON BAUCHER. 330 do their high school work without taking off their bridles. Here, also, I found their respective mouths to be exactly the same as before. While hacking, these horses were light in hand, without being on their haunches.* On the race-course they pulled just as much as if they had never been taught high school work; and, having returned to the school with their hind legs well under them, they were as light in hand as if they had never been galloped on a race-course. We should note that in the experiments which I have just described, the respective weights, with these horses, were dis- tributed in three absolutely different ways. 1. When going to the course it was distributed in the proper manner for hacking, and was the result of an ordinary feeling on the reins. 2. During the gallop, the preponderance of weight was on the forehand. 2. During the gallop, the preponderance of weight was on the forehand. 3. In the manege, on the resumption of high school work, the preponderance was on the hind quarters. Consequently, there were three modifications in the distribution of weight. The application of the “aids” was also entirely different, according to the nature of the work. But the essential point on which I differ from Baucher is, that a horse’s mouth remains the same, no matter what is the work, use of the “aids,” or distribution of weight. In the three distributions of weight I found the same difference of mouth. I repeated this experiment with twenty thorough-breds which had been broken for high school work, and they all brought me to the same conclusion: that the nature of the mouth does not vary according to the distribution of weight. * Horizontal equilibrium is the equilibrium for hacking. Digitized by Microsoft® * We might as well believe that all men are equally ticklish. t Besides, I have already said that we should not use the simple ramener except with a high-spirited horse which goes freely up to his bridle. Digitized by Microsoft® COMMENTARIES ON BAUCHER. It is sometimes evident that a horse which holds his head low makes the rider carry his head and neck ; but if the rider Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® COMMENTARIES ON BAUCHER. 337 changes the distribution of weight by raising the head and neck, they will not bear so heavily on his hands. Baucher confused the sensitiveness of the mouth with the lightness which results from a change in the position of the head and neck. Let us now consider the sides of a horse. Who will believe that the sides of a common, heavy, lymphatic horse are as sensitive as those of a strong, wiry and free-going animal? * It is possible to make the former do almost the same exercises as the latter; but at what cost? We would have to dig the spurs into him to make him move; but with the free-goer, the mere approach of the heels would be enough. A ticklish, impatient horse will never bear the spurs with the same docility as a good-tempered, placid animal. In no case can we succeed in completely changing the work of nature. Further, a horse which is naturally ticklish, becomes still more so from the continual contact of the spurs. It may even happen that a horse which is not naturally ticklish, becomes ticklish from the use of the spurs during breaking. Also, a horse which has bad loins, weak hocks, or any other defect, will not be cured by Baucher’s method. Often his imperfections will only be increased by the sufferings which he will endure, on account of being made to take up certain positions, as in ordinary reining in, without the pressure of the legs,f or in the complete vassembler. Baucher, therefore, was far from being right when he asserted that his method cured all ills. As a general rule, the weaker and more unsound a horse is, the less should be expected from him, under pain of making him incapable of any kind of work. With such an animal, 22 22 Digitized by Microsoft® COMMENTARIES ON BAUCHER. 338 there is no question of fine equitation, for he cannot bear the vassemler. ‘Ne should simply require him to go forward, and we should feel happy if we make him a passable hack. * The legs are ‘“‘aids’’ of impulsion ; the hands, ‘‘aids” of retention and direction. Digitized by Microsoft® COMMENTARIES ON BAUCHER. Baucher appears to me to be absolutely wrong in stating that we should destroy the “ instinctive forces” of the horse, and act only on the “transmitted forces.” This theory is so strdnge and so contrary to the nature of things, that I cannot help thinking that the expression used by Baucher incorrectly translated his thoughts. The “instinctive forces” are apparently the natural forces, or muscular power. If it is destroyed, what remains? What are the “transmitted forces”’ of which Baucher talks? Whence do they come? We can transmit an electric current or a moral effect, but to transmit a force we must, first of all, produce it, and we know that the rider should not display force, because he ought always to remain supple; and even if he displayed force, it would be insignificant as compared to that of the horse. What means can the rider employ? He has only his legs and hands.* Without doubt the legs awake the vigour of the horse when they are well closed, but they do not add any force to it. They simply stimulate him to put forth the energy he possesses. The hands ought not to display force. If, in an excep- fional case, they make an effort, its object will only be to thwart that of the horse. ‘They do not transmit force to him, but more or less check his force. Supposing that two jockeys are finishing almost together, and that their horses are equally exhausted. If one of them is very strong, he can, by displaying great energy of hands, legs and whip, appear to communicate the force of propulsion to Digitized by Microsoft® Digitized by Microsoft® COMMENTARIES ON BAUCHER. 339 his horse, and thus obtain two or three powerful strides, which will enable him to win. Such a force has the appearance of being transmitted, but in reality the jockey simply stimulates by his vigorous action, all the remaining energy of the horse, and makes him go at his maximum speed for two or three seconds. Let us take another case of horses being exhausted when returning from a long hunt, and when going down a steep incline, at the bottom of which there is a ditch, at which some of them, not being well supported by their rider’s legs, stumble or fall. COMMENTARIES ON BAUCHER. One of the riders, on the contrary, spurs his animal sharply and thus prevents him making a mistake, by doing which he appears to transmit force to him at that moment. in reality, he only stimulates the energy of his horse, whose natural force saves them both. Let us take a high school horse, when he is at the end of his work and has lost his “go,” as sometimes happens. _ If I wish him to do some energetic movements, I am obliged to have recourse to the spurs, and I use a certain amount of vigour to make him answer to their attacks; but instead of transmitting force to him, I merely awaken his energy, which he puts into action. To sum up: the rider does not transmit force to his horse, whose natural forces he directs, moderates, or excites at his pleasure. I am inclined to think that Baucher simply wished to say that whenever a horse tries to take the initiative in using his ewn natural forces, the rider should stop or direct them, especially if the animal wants to “ play up,” as, for instance, by plunging, rearing, or spinning round. But when a horse puts forth all his energy to do a good walk, a grand trot, or a well- marked canter, I don’t think that we ought to try to destroy it. Baucher neither destroyed nor transmitted forces ; he directed them. He obtained control of the horse’s powers by suppling 22* 22* 22* Digitized by Microsoft® COMMENTARIES ON BAUCHER. 340 exercises, and by rational training, and consequently he ruled his horse by preventing him using these forces in his own way. Finally,a horse can always escape from strength by strength, in which case the rider will soon be exhausted. Therefore the rider ought always try to prevent the horse knowing his own strength, so that he may hinder him from using it against himself. He can do this by feeling and divining in advance the intentions of the animal. A rider who has tact foresees the defences of his mount, feels them coming, and wards them off before the animal can make them. Baucher, after having passed his life in breaking horses such as Partisan, Buridan, Capitaine, Stades, and others, complains that his horses were not always light in hand during changes of direction. Digitized by Microsoft® COMMENTARIES ON BAUCHER. The reason which he gave was that, with his way of working he obtained only eguilibrium of the second kind, which I call “ taeperfect equilibrium” or “ incomplete rassem- bler.”” According to him, “eguzlilrium of the first kind,” which I call “ perfect equilibrium” or “ complete rassembler,” is obtained by using the hands without the legs, or the legs without the hands. But in this, Baucher seriously deceived himself, because the fact of his finding “equilibrium of the first kind” towards the end of his career was due to the eleva- tion of the head and neck, which he finally adopted at a time when he was no longer able to ride. He was not able to obtain the complete rassembler by placing the head and neck of his horses in the way he used to do. Vhe head was too low, and the neck was bent in the middle. Therefore it fre- quently happened that his horses were badly balanced. It is simply absurd to say that the “complete rassembler” can be obtained by the hands without the legs, or by the legs without the hands. We read on page 82 of the fourteenth and last edition of Baucher’s book that “my method places the horse so much Digitized by Microsoft® COMMENTARIES ON BAUCHER. 341 under the control of his rider that, by the combination of the effects of the legs and hands, our slightest movements suffice to direct the energy of this powerful animal, according to our will.” Nothing could be truer. But why does he maintain on page 178 of the same edition, that the only true kind of riding should be done by “legs without hands, and hands without legs”? How did he not see that these two assertions are contradictory? The office of the legs of the rider is to make the horse energetic in his hind quarters, and that of the hands, to make him “amiable in his mouth.” Without the simultaneous co-operation of these two “aids,” we may do wonderful tricks, but not good horsemanship. under the control of his rider that, by the combination of the effects of the legs and hands, our slightest movements suffice to direct the energy of this powerful animal, according to our will.” Nothing could be truer. Digitized by Microsoft® COMMENTARIES ON BAUCHER. But why does he maintain on page 178 of the same edition, that the only true kind of riding should be done by “legs without hands, and hands without legs”? How did he not see that these two assertions are contradictory? The office of the legs of the rider is to make the horse energetic in his hind quarters, and that of the hands, to make him “amiable in his mouth.” Without the simultaneous co-operation of these two “aids,” we may do wonderful tricks, but not good horsemanship. The fact that Baucher broke his horses with the two “ aids,” and afterwards used only one of them, proves that he deceived himself by thinking that he had obtained perfect equilibrium with “legs without hands and hands without legs.” But as the legs are necessary for impulsion and the hands for direc- tion, he hastened to go back to the two “ aids.” Under these conditions, why did he abandon an “aid” which he was obliged to use every moment? Is the proof required? When working on “two tracks” without the legs, there will be nothing to indicate to the horse that his haunches should move, especially if we want him to do the “ two tracks ” at the canter or fassage. If we demand the Spanish trot without legs, the horse will raise his fore legs a little without gaining ground, but nothing will give him the impulsion necessary for the trot. Change the “aids,” and demand the same work without using the hands. The action of the legs to obtain the elevation and extension of the fore legs will have the result of sending the horse abruptly forward, because there is nothing to restrain him and to raise the fore hand. It will be the same in all other movements. I admit, however, that when the education of the school horse has been carried to its highest degree of perfection, he Digitized by Microsoft® COMMENTARIES ON BAUCHER. 342 will do, so to speak, all the movements without the help of the “aids,” it being sufficient to indicate to him what he has got to do, by the slightest approach of the leg, with the reins slack on his neck. In fact, this is a question of zwances (minute dif- ferences): but all art is made up of zuances. Digitized by Microsoft® COMMENTARIES ON BAUCHER. Be that as it may; but since Baucher acknowledges that he could get only the “ equilibrium of the second kind ” (incomplete rassembler), 1 am justified in thinking that my school horses are superior to his ; because, for the last ten years, I have ob- tained “ equilibrium of the first kind” (complete rassemzbler). I hasten to add that, thanks to Baucher, I succeeded in finding this rassembler, which is possible only when the head and neck are kept very high. Also, when I say that my school horses are superior to those of the illustrious master, I do not pretend that they were more precise in their movements than his, which were perfectly correct. I wish simply to say that I obtained the same school movements in as good form as my learned pre- decessor, but with greater elevation of head, neck, and limbs ; that is to say, with more complete equilibrium, which conse- quently required less effort, and above all things with more impulsion. In his books on equitation, Baucher said little about riding in the open. This was an evident omission. The fact is that Baucher never rode outside. Without being his pupil, I followed and studied him during his journeys to Austria, Italy, Switzerland, etc., from 1847 to 1850. But during these three years I never saw him go out on horseback. It has been very incorrectly stated that his seat in the saddle was weak, and for this reason he was afraid to ride hacking or hunting. I admit that he was not sucha fine rough rider, and had not such a strong seat as the Count d’Aure, but that does not prove he was afraid to ride outside. The fact that he broke many horses proves that he had a strong seat; because there are always more or less violent struggles during break- Digitized by Microsoft® Digitized by Microsoft® COMMENTARIES ON BAUCHER. 343 ing. We must therefore seek elsewhere for the reason which prevented Baucher from riding in the open. ing. We must therefore seek elsewhere for the reason which prevented Baucher from riding in the open. Baucher being a reformer and consequently a secker, had no pleasure in leaving a horse to himself, as is done when hacking. Digitized by Microsoft® COMMENTARIES ON BAUCHER. He devoted all his life to his work in order to show us the way, which was the only thing that interested him. Riding without working was only a weariness tohim. There- fore he never studied the character or manner of riding a hack or hunter; or the enormous difference between a “closed-in” school horse and an ordinary saddle horse, which is left a good deal to himself. As he did not ride outside, he never rode his horses at fast paces, which was wrong; because a horse passes very easily from an extended position to the rassembler. Full speed, provided that it is not too prolonged, has the advan- tage of allowing the animal to extend himself, and conse- quently to obtain rest, by changing his equilibrium, while at the same time it develops and strengthens his lungs. The chief fault of Baucher was that of keeping his horse constantly “enclosed.” I think we ought to observe the principle of letting the horse extend himself after each con- cession, during breaking. Finally, 1 maintain that as a general rule during breaking, we should accustom our mount to extend himself from time to time at the walk, trot and canter. Every form of equilibrium and position ends by fatiguing the horse. If we change one of them, the horse will return to it with pleasure. As a last observation I may remark that on page 103 of the fourteenth edition of his book, Baucher repeats a conversation which he had at Berlin with some German officers who were supposed to know something about horses. They said: “ We like to have our horses zz front of the hand.” Baucher replied: “TI like mine to be Jehzud the hand, and in front of the legs.” Digitized by Microsoft® COMMENTARIES COMMENTARIES ON BAUCHER. 344 Personally, I share neither the opinion of the German officers nor that of Baucher. The horse ought to be in front of the legs and lightly oz the hand. It is always a question of nuances; at least, when army horses have not been con- sidered, because with them there is something quite different from a zuance, and in this case I differ entirely from Baucher. I even dare to say that the single fact of his stating that horses should be behind the hand, ought to be sufficient to exclude his method from the army. COMMENTARIES ON BAUCHER. Such an opinion put into practice would only make the horse hesitating ; because the hand directs him. The horse always hesitates when he does not feel the hand. But if he is dehind the hand he does not feel the reins. The army horse ought always to go freely up to his bridle (on the hand). These are the principal points of Baucher’s method with which I disagree. Nevertheless, I have a sincere admiration for him. Baucher was a creator, and every one who rides ought to respect him as a master. He had the great merit of not describing anything which he could not do. Many who have come after him have written at great length on riding, and often with the object of describing magnificent movements which they have never done. Baucher proved the superiority of his theory by putting it into practice. Digitized by Microsoft® 345 Digitized by Microsoft® TESTS OF HORSEMANSHIP. To be an accomplished rider, or at least to be a fair per- former in the saddle, a man should be able to pass the follow- ing tests :— 1. To ride a difficult horse. 2. To ride in a steeplechase. 2. To ride in a steeplechase. 3. To ride a trotting match, 3. To ride a trotting match, 4. To ride a flat race. 4. To ride a flat race. 5. To be able to break and ride a school horse. Riding a difficult horse, when it is only a qu 5. To be able to break and ride a school horse. Riding a difficult horse, when it is only a qu Riding a difficult horse, when it is only a question of remaining on his back, is simply an affair of strength of seat and pluck. It is sufficient to be a good rider and a bit of a dare-devil. But we require a knowledge of reasoned-out equitation to anticipate and thwart his defences. I place steeplechase riding in the second rank, although it requires great strength of seat and pluck. A cross-country jockey has to be a good rider, rather than a good horseman ; but steeplechase riding requires less delicacy of handling and tact than the following tests. I think match trotting comes in the third rank as regards judgment and knowledge. It is evident that if the rider of a trotter cannot accurately tell when his horse is at the top of his speed, he will continue to push him, and will make him break into the gallop. But judgment of pace is much more Digitized by Microsoft® TESTS OF HORSEMANSHIP. 346 easy in trotting than in flat-race riding, to succeed in which a jockey should know what is the highest speed of his horse, should always keep near it, and should demand it only at the critical moment. I put flat-race riding in the fourth rank, considering that it is truly an art which only such men as F. Archer, Watts, Cannon, Webb and others can acquire. A flat-race jockey has to be an exceedingly good judge of pace, and if he does not know what speed his animai can maintain without becom- ing exhausted, he will never make a name for himself. Digitized by Microsoft® TESTS OF HORSEMANSHIP. When we think that the highest speed of a racehorse is about five furlongs in a minute, we will understand how difficult it is to judge pace within a second or two. If it is simply a matter of one following the other, the first comer can do it. The difficulty is to set and maintain the pace which suits the horse best, and if the jockey cannot place him as he likes, without fighting with him, he will do no good. If the jockey takes too strong a bearing on the reins, the horse will exhaust himself by the efforts he makes against the hands, in which case his mouth suffers much less than his loins and hocks, the result being that the animal will not be able to finish in good style. I, on the contrary, the jockey does not keep a proper hold of his horse’s head, the animal will go too fast, and will be unable to struggle at the critical moment. The jockey should therefore have sufficient sense not to fall into either of these extremes. Breaking a school horse comes under the last term. To succeed at it, a man should possess knowledge, delicacy of touch and tact to a supreme degree, and should have an exact acquaintance with the capabilities of a horse, so that he may break him without making him unsound. In the breaking of a school horse, we require not only a perfect knowledge of the effects of the hands and legs, but we Digitized by Microsoft® TESTS OF HORSEMANSHIP. 347 should also, by our seat, be able to feel the slightest move- ments of the hind quarters, for we learn by our seat what passes under us. Consequently, we can check the slightest fault, and immediately reward the faintest sign of good will. This is the entire secret of breaking. Further, the riding master who breaks a school horse, acts alone, and depends only on himself. Every fault committed and every good movement done are his work. This is true, only in the school. The racehorse, to mention his case only, passes through many hands, such as those of trainers, riding lads, and jockeys, and if the animal commits a fault with any of them, the man can put it down to his next-door neighbour. Only the school horse is the exclusive work of the person who broke him. TESTS OF HORSEMANSHIP. Count d’Aure replied one day to a criticism of Baucher that, “T am not a horse breaker.” Then, what did he break? Did the word breaker jar on his ears? For my part, I know no other. Of course we should not regard in the same light the horseman who breaks a horse in good style, as the groom who takes the rough edge off him, and \| venture to say that no one can be a real horseman, if he cannot break-in a horse. Breaking is the horseman’s touchstone. The broken horse is the proof of the dreaker. Digitized by Microsoft® 348 Digitized by Microsoft® PROGRAMME OF HORSE-BREAKING, done under the direction of Mr. JAMES FILLIs. done under the direction of Mr. JAMES FILLIs. IST LESSON. Lunging the horse for some days, to the right and to the left, to make him obedient. 1. Putting him in hand. Flexion of the jaw, standing still, and then in movement. (The right hand holding both reins of the bridle at two inches from the bit, and high, in order to raise the neck. The left snaffle rein in the left hand, which is carried forward in opposition to the right hand.) Pat the horse on the neck and begin again as soon as he yields. 2. Make the horse yield to the right leg and to the left leg. Digitized by Microsoft® WITH BELGIAN OFFICERS. [ was giving performances at Brussels, when Count d’Oultremont asked me to give an exhibition with my horses at the Royal Circle of which he was president. He expressed the desire that this performance would be accompanied by explanatory remarks, “Your ‘aids’ are so delicate,” he said to me, “that we do not always see the precise means you arrive at a result which we witness. We wish to know how you obtain so much with such a small effort. Give us explanations.” The exhibition took place one afternoon in the month of December, 1890. All the superior officers who were members of the Circle were present. I worked my horses, but not as in a public show. Sometimes placing my mount in a good position to obtain the desired result under correct conditions ; sometimes placing him in a bad position, in order to show its fallacy by making a mistake ; I accompanied each part of the work by explanations, which were practically confirmed on the spot. After the performance, the officers of the second regiment of Guides asked me, through Commandant Fivé, to be good enough to make out a course of instruction for their use. I accepted with great pleasure, and the riding school of the 2nd Guides was placed at my disposal every day from ten Digitized by Microsoft® Digitized by Microsoft® WITH BELGIAN OFFICERS. 340 o'clock to half-past twelve. The course consisted of thirty lessons.* The following was the programme which I laid down, and which was carried out consecutively :— 2ND REGIMENT OF GUIDES of H.M. the King of the Belgians. PROGRAMME OF HORSE-BREAKING, PROGRAMME OF HORSE-BREAKING, done under the direction of Mr. JAMES FILLIs. 4TH LESSON, . Repetition of the 3rd lesson. Short trot, and change of hand Short trot, and change of hand on the diagonal. Canter to the right and to the left. Canter to the right and to the left. . Getting the horse into hand. Be Ns . Getting the horse into hand. 3RD LESSON. . Repetition of the 2nd lesson. . Repetition of the 2nd lesson. . Change of the diagonal hand, to two-thirds of the change of hand; making the horse yield to the leg on two tracks (to both hands). Shoulder-in: Repeat this movement to both hands. WITH BELGIAN OFFICERS. WITH BELGIAN OFFICERS. 350 5TH LESSON. . Repetition of the 4th lesson. . Canter on the diagonal. . Circling. . Change of hand by passing into the trot. mp wind . Collecting the horse. 6TH LESSON. . Lateral flexion of the head on the neck (the reins held as in the flexion of the jaw). . Canter on the diagonal. Circling and changing the hand. . Shoulder-in. Canter on a straight line and change of hand. . Collecting the horse. Aw kh wY nN . Collecting the horse. 2ND LESSON. 1. Flexions of the jaw, the man being on foot; same flexions when mounted. 2. Short trot: (a) On the left diagonal biped. ' (6) On the right diagonal biped. ' (6) On the right diagonal biped. Rising in the stirrups at the trot. 3. Shoulder-in: To the right hand, and to the left hand. 4. Reversed volte. 4. Reversed volte. 4. Reversed volte. * Tt was the same with the 1st Regiment of Guides, who placed their manége at my disposal every day from nine till half-past ten. * Tt was the same with the 1st Regiment of Guides, who placed their manége at my disposal every day from nine till half-past ten. 7TH & 8TH LESSONS. . Direct and lateral flexions on foot. Digitized by Microsoft® 12TH, 13TH & I4TH LESSONS. . Repetition of the 11th lesson. IOTH LESSON. 1. The same order of work. 2. Rassembléd trot, and movements on two tracks. 3. Half a turn on the haunches. QTH LESSON. 1. Repetition of the 7th lesson. 2. Cadence of the trot, and continued vassemdler. 3. Increase the collection. Digitized by Microsoft® WITH BELGIAN OFFICERS. 351 CO ONG ce Repeat the flexions while moving. Short trot. Collecting the horse. Canter on the inward and outward leg. Shoulder-in. . Shoulder to the wall. . Rassembler while moving. Repeat the flexions while moving. Canter on the inward and outward leg. Shoulder-in. . Shoulder to the wall. CO ONG ce . Rassembler while moving. IITH LESSON. . Repetition of the preceding lesson. . Repetition of the preceding lesson. Canter on the inward and outward leg in a straight line I5TH LESSON, Repetition of the preceding work. Repetition of the preceding work. Increase of the vassembler and of collection. Shoulder-in on the circle. Shoulder-out on the circle. Sh Qo ly Starting into the canter from the halt. I6TH LESSON. Short trot. Collecting the horse. Short trot. Collecting the horse. Flexion with the curb, on foot. Digitized by Microsoft® WITH BELGIAN OFFICERS. 352 3. School walk: Shoulder-in. fe » Shoulder-out. Change of hand on two tracks. The same movements on the circle. 3. School walk: Shoulder-in. fe » Shoulder-out. Change of hand on two tracks. The same movements on the circle. Canter on the inward and outward leg. Changes of hand by passing to the trot. A higher degree of collection. WoO I8TH LESSON. Repetition of the preceding lesson. Repetition of the preceding lesson. 17TH LESSON. Same succession of work. Increased collection. eNom Half turn on the haunches. Digitized by Microsoft® 3. School walk: shoulder-in. 3. School walk: shoulder-in. BS » shoulder-out. Change of hand on two tracks. . Same movements on the circle. wR Canter on the inward and outward leg. Change of hand by passing to the trot. Getting the horse completely in hand. IQ9TH LESSON. Collecting the horse at the trot. Collecting the horse at the trot. Flexions with the curb on foot. Flexions with the curb on foot. Shoulder-in and shoulder-out at the school trot. Shoulder-in and shoulder-out at the school trot. Half turn on the shoulders. Canter on the inward and outward leg. Canter on the inward and outward leg. An wn Change of hand at the canter, by arriving at the opposite track, at the walk, and starting at the canter by the position of the head (to each hand). 7. Ona straight line: canter to the right: a few strides— at the walk—canter to the left—repeat several times the same movement. 8. On the circle: Shoulder-in—shoulder-out. g. At the canter: Shoulder-in. (Never shoulder-out at the canter, so as not to accustom the horse to go sideways.) o. At the walk: getting the horse completely in hand. to. At the walk: getting the horse completely in hand. Digitized by Microsoft® Digitized by Microsoft® WITH BELGIAN OFFICERS. 21ST LESSON. . Flexions with the curb (by going with the head high). . Flexions with the curb (by going with the head high). Rassembler—school trot—shoulder-in—shoulder-out. 3. Halting—Reining back—Forward. (Repeat the same 3. Halting—Reining back—Forward. (Repeat the same movement several times.) 4. School walk: demi-volte and shoulder-in by finishing the demi-volte (to both hands). 5. Canter on a straight line with both fore legs alternately leading. Change of hand at the canter by passing into the walk and changing the leg. 6. Ve Demi-volte at the canter ; change of leg at the track. Shoulder-in, a canter, to both hands. Ve Shoulder-in, a canter, to both hands. Circling at the canter on the inward a 8. Circling at the canter on the inward and outward leg. Collecting the horse completely at the walk. OQ. Collecting the horse completely at the walk. WITH BELGIAN OFFICERS. 353 1. Repetition of the preceding lesson. 1. Repetition of the preceding lesson. Canter down the centre. 1. Repetition of the preceding lesson. Canter down the centre. 2. Canter down the centre. Reversed volte, straightenin 2. Canter down the centre. Reversed volte, straightenin Digitized by Microsoft® 20TH LESSON. 1. Trot with the horse in hand. Flexion with the curb on foot. 2. Canter down the centre. Reversed volte, straightenin Reversed volte, straightening the horse on the same leg, change of leg. Reversed volte, straightening the horse on the same leg, change of leg. to ww 23RD LESSON. 1. Repetition of the preceding lesson. Getting the horse more completely in 1. Repetition of the preceding lesson. Getting the horse more completely i Getting the horse more completely in hand than before, to finish. . Preparation for the passage. . Preparation for the passage. . Preparation for the passage. Digitized by Microsoft® WITH BELGIAN OFFICERS. 354 Ordinary volte with shoulder in, same movement to the other hand. Ordinary volte with shoulder in, same movement to the other hand. . Walk. Canter to the right, canter to the left, three strides on each leg. Canter to the right, canter to the left, three strides on each leg. Collecting the horse. Preparation for the pa Collecting the horse. Preparation for the pa Preparation for the passage. Preparation for the passage. 24TH LESSON. . Repetition of the 22nd lesson. . Repetition of the 22nd lesson. to Each day more complete vasseméZer than before, so as to obtain the passage. 25TH LESSON. 1. Repetition of the 22nd lesson. Getting the horse more complet 1. Repetition of the 22nd lesson. Getting the horse more complete Getting the horse more completely in hand than before, to finish. 26TH, 27TH, 28TH, 29TH, AND 30TH LESSONS. Digitized by Microsoft® 26TH, 27TH, 28TH, 29TH, AND 30TH LESSONS. . Repetition of the 22nd lesson. Getting the horse in hand more and more completely. . A few strides of the passage, pat the horse on the neck, begin again. F. DE Hase, Commandant Adjutant-Major. Brussels, 5th January, 1891. . A few strides of the passage, pat the horse on the neck, begin again. The results were excellent, as the following letter shows, and I most cordially thank the gentlemen who signed it :— Digitized by Microsoft® Digitized by Microsoft® WITH BELGIAN OFFICERS. 355 Digitized by Microsoft® “DEAR MR. FILLIS,— “DEAR MR. FILLIS,— “ At the moment when you are going to bring out a new edition of your ‘Breaking and Riding,’ we are anxious to express our admiration of the excellent principles you have taught us. “You have shown us, in breaking, how to combine reasoned- out energy with victorious patience. “No more excitement! No intemperate punishment, which is prejudicial to success. “Also, thanks to your method and your instruction, we have succeeded in thirty lessons to break horses of entirely different tempers without injuring them, and we fully recog- nise that the principles we previously employed did not give us such rapid and satisfactory results. “We are anxious, dear Mr. Fillis, to add our tribute to the numerous testimonies of admiration which you have received everywhere, and we beg you to accept the expression of our distinguished regards. “FF. DE HasE, Commandant Adjutant-Mayjor. “ LAMBERT, Captain Commandant. “ LAMBERT, Captain Commandant. “Frye, Commandant. “Crc. HERG, Lieutenant. “DE FORMANOI, Captain. “P. BIOURGE, Ist Lieutenant. “LE GRAND, Lieutenant. ‘“G, JEIDELO, Lieutenant. “M. LECLERQ, Captain. “FE. Doca, Lieutenant. “COMTE JEAN DE MEROD, Ist Lieutenant. “M. D’'HESPEL, 1st Lieutenant. “R. PYCKE, Ist Lieutenant. “VANLOQUERE, Ist Lieutenant. “ Bra, Captain.” Digitized by Microsoft® WITH BELGIAN OFFICERS. 356 I ought to mention that in Belgium no one ever thought of blaming me for riding in a circus, which is considered in that country to be the only place for equestrian work, and that it is as natural for a horseman to ride in a circus, as for a lyric artist to show himself at the opera, or for an Academician to dress himself in green, in order to croak out an address under the cupola of the Institute. J ought also to mention that at Brussels I did not meet any military distrust of civilians. The people only required a_ riding master to thoroughly know what he taught. Cavalry officers are obliged to learn not only riding, but also an infinite number of other things which make it very difficult for them to afford time to study equestrian science to its utmost limits. Hence the necessity for specialising, as well for the instruction of the military rider as for others. I believe I faithfully convey the opinions, on these matters, of the Belgian cavalry officers whom I had the honour of meeting. Digitized by Microsoft® IN DEX, ee ee * AIns,’’ 19. America, 25. Amour, 251. Andrews, H., 203. Arab horses, 158. Archer, Fred, 208, 346. Archiduc, 206. Army horse, the, 213-235. BARON FINOT, 202. >» de Vaux, 171. Baucher, 43, 44, 56, 58, 62, 63, 64, 65, 82, 105, 106, 110, 126, 156, 171, 261, 306, 319, 332-344- Baudres, 204. Beginners, 23. Belgian officers, 348-356. Bit, getting behind the, 117. Bitting, 10. Bucking, 78. Buridan, 340. CANNON, 346. Canter on three legs, 302-305. », 5 the, 153-163, 180. », tothe rear, 314, 325. “5 when rassemblé’d, 261. » Without gaining ground, 314, 325. Cantering, ‘‘ false,’ 168. Capitaine, 340. Caron, Francois, 332. Carrots, 6, 70. Castillon, 178. Changes of direction, 87, 166. 3 », leg, 166-172, 270-275. Chevalier d’Abzac, 332. Clocher, 178. Close work, 40. Collecting a horse, 44, 123. Count d’Aure, 332, 342, 347- +» von Schweppe, 332. Counter-demi-volte, 92. “* Cracking Nuts,’’ 126. Crimea, the, 214. Curb, 10, 150, 198. >, chain, 13. ¢ DAISY CUTTING,” 52, 173, 179. Defences, 82, 281. Demi-voltes, 91, 163. Diagonal effects, 109. Direction, changes of, 87, 166. Doubler, 90. Dozulé, 151. ENGLAND, 25. English stiffness, 22. Equestrian tact, 129, 130. Equilibrium, 336, 340, 342. FACE, EXPRESSION OF, 6. Feeding, 4, 226, 227. Fishing rod, 53. Flat racing, 206-209. Flexions, direct, 44. » » lateral, 96. Fra Diavolo, 206. Franconi, Victor, 332. GALLOP, 180. Gant, 319, 320, 335. AIns,’’ 19. America, 25. Amour, 251. Andrews, H., 203. Arab horses, 158. Archer, Fred, 208, 346. Archiduc, 206. Army horse, the, 213-235. Arab horses, 158. Archer, Fred, 208, 346. Army horse, the, 213-235. Army horse, the, 213-235. BARON FINOT, 202. >» de Vaux, 171. Baucher, 43, 44, 56, 58, 62, 63, 64, 65, 82, 105, 106, 110, 126, 156, 171, 261, 306, 319, 332-344- Baudres, 204. Beginners, 23. Belgian officers, 348-356. Bit, getting behind the, 117. Bitting, 10. Bucking, 78. Buridan, 340. CANNON, 346. Canter on three legs, 302-305. », 5 the, 153-163, 180. », tothe rear, 314, 325. “5 when rassemblé’d, 261. » Without gaining ground, 314, 325. Cantering, ‘‘ false,’ 168. Capitaine, 340. Caron, Francois, 332. Carrots, 6, 70. Castillon, 178. Changes of direction, 87, 166. 3 », leg, 166-172, 270-275. >, chain, 13. Equilibrium, 336, 340, 342. FACE, EXPRESSION OF, 6. Feeding, 4, 226, 227. Fishing rod, 53. Flat racing, 206-209. Flexions, direct, 44. » » lateral, 96. Fra Diavolo, 206. Franconi, Victor, 332. GALLOP, 180. Digitized by Microsoft® IN DEX, Gant, 319, 320, 335. Cantering, ‘‘ false,’ 168. Capitaine, 340. Caron, Francois, Fra Diavolo, 206. Franconi, Victor, 332. Changes of direction, 87, 166. 3 », leg, 166-172, 270-275. Digitized by Microsoft® Digitized by Microsoft® INDEX. 358 EX. La Guérinibre, 288, 332. Ladies, 3, 163. Lady, putting up a, 29. Lady's dress, 27. ,, school horse, 326. Lancade, 79. Lateral effects, 108. Lenoble du Theil, 177. Little Duck, 207. Louis d'Or, 207. ‘ Lunging a horse, 32. “MAKING MUCH oF A Horsg,”’ 8. Man, I. Mares, 3. Markir, 177, 252, 276, 282, 287. Marquis de Bigne, 332. Martingale, 13. Memory, horses’, 114. Mount, making quiet to, 66. Mounting, 66. Mouth, 49, 50, 334-336. », » hard and soft side of, 78. NEGRO, 296. Nervous horses, 184. New school movements, 322. » 99» the, 300, 30r. Newcastle, 332. Normandy horses, 217, 220. “Nuts, cracking,” 126. PARADOX, 208. Parmesan, 178. Partisan, 340. Pascal, 151. Passage, 275-301. Passage to the rear, 305. Pats on the neck, 8. Piaffer, 275-301. oy - see-saw, 306. Pignatelli, 332. Pirouettes, 117, 246, 262. ue with the feet crossed, 246. ss »veversed, III, 117, 245, 240. Gantelet, 178. La Guérinibre, 288, 332. Gaulois, 81. Ladies, 3, 163. German stiffness, 22. Lady, putting up a, 29. Germans, 22, 235. Lady's dress, 27. Germinal, 252, 261, 276, 305, 320, ,, school horse, 326. 321, 326. Lancade, 79. Girthing up, 69. Lateral effects, 108. “* Give and take,’”’ 125. Lenoble du Theil, 177. Little Duck, 207. Louis d'Or, 207. HACK, THE, -183. ‘ igus ai alata Lunging a horse, 32. Half-rear, 79. Halt, 85. Hand, behind the, 343. “MAKING MUCH oF A Horsg,”’ 8. » » getting horse in, 125. Man, I. »» » 1m front of the, 343. Mares, 3. 4, , on the, 126. Markir, 177, 252, 276, 282, 287. ‘IIands,’’ 48. Marquis de Bigne, 332. Hatchet, 203. Martingale, 13. Head about, throwing the, 188. Memory, horses’, 114. Heavy in front, 53. Mount, making quiet to, 66. Heels, 2. Mounting, 66. High-school riding, 236-331. Mouth, 49, 50, 334-336. Horse, cold, 176. », » hard and soft side of, 78. sg- -g Ob, 76; TST »> » intelligence of the, 5. NEGRO, 296. » » sluggish, 175. Nervous horses, 184. », 4 the, 1. New school movements, 322. » » the Army, 213-235. » 99» the, 300, 30r. Horsemanship, tests of, 345. Newcastle, 332. aa , high-school, 221. JOCKEYS, 202, 207-209, 338. Jumping, 195-205. Immobtlité, 81. IN DEX, Normandy horses, 217, 220. Horses, impetuous, 8, 175. “Nuts, cracking,” 126. >» 5 nervous, 184. », with vices, 184-194. PARADOX, 208. +, sides, 337. Parmesan, 178. Humming a tune, 298. Partisan, 340. Hunter, English, 211. Pascal, 151. Passage, 275-301. Passage to the rear, 305. Pats on the neck, 8. Piaffer, 275-301. oy - see-saw, 306. Pignatelli, 332. Pirouettes, 117, 246, 262. ue with the feet crossed, 246. JOCKEYS, 202, 207-209, 338. ss »veversed, III, 117, 245, Jumping, 195-205. 240. » 4 the, 2ro-or, Hurdle racing, 202. Immobtlité, 81. Impetuous horses, 8, 175. Intelligence of the horse, 5. Gantelet, 178. Gaulois, 81. German stiffness, 22. Germans, 22, 235. Germinal, 252, 261, 276, 305, 320, 321, 326. Girthing up, 69. “* Give and take,’”’ 125. Gantelet, 178. Gaulois, 81. German stiffness, 22. Germans, 22, 235. Germinal, 252, 261, 276, 305, 320, 321, 326. Girthing up, 69. “* Give and take,’”’ 125. HACK, THE, -183. igus ai alata Half-rear, 79. Halt, 85. Hand, behind the, 343. » » getting horse in, 125. »» » 1m front of the, 343. 4, , on the, 126. ‘IIands,’’ 48. Hatchet, 203. Head about, throwing the, 188. Heavy in front, 53. Heels, 2. High-school riding, 236-331. Horse, cold, 176. sg- -g Ob, 76; TST »> » intelligence of the, 5. » » sluggish, 175. », 4 the, 1. » » the Army, 213-235. Horsemanship, tests of, 345. aa , high-school, 221. Horses, impetuous, 8, 175. >» 5 nervous, 184. », with vices, 184-194. +, sides, 337. Humming a tune, 298. Hunter, English, 211. JOCKEYS, 202, 207-209, 338. Jumping, 195-205. » 4 the, 2ro-or, Hurdle racing, 202. Immobtlité, 81. Impetuous horses, 8, 175. Intelligence of the horse, 5. ‘ Lunging a horse, 32. »» » 1m front of the, 343. 4, , on the, 126. Markir, 177, 252, 276, 282, 287. Marquis de Bigne, 332. Head about, throwing the, 188. Heavy in front, 53. » 4 the, 2ro-or, Hurdle racing, 202. Digitized by Microsoft® INDEX. 359 Pirouettes, reversed, on three legs, 245. \| Shoulder-in, 135-140, 260. Pivot, making horse, 110. Shying, 187. “« Playing up,’’ 82, 281. Side-steps, 133. Plunging, 42. Snaffle, 10, 198. Pointe, 79. Spanish trot, 251-260, 301, 325. Pudeldressirung, 298. 55 walk, 237. Pulling, 334-336. 4 o> 9 NeW, 322. Punishment, 9, 112. Spurs, 18, 113, 115, 288, 206. Stades, 306, 340. Standing still, 85. Steeplechasing, 202. Stirrups, 15. », » length of, 21. Printed at The Chapel River Press, Kingston, Surrey. SADDLES, 15, 28. School riding, high, 236-331. >» walk, 118. Seat, lady's, 24. 5» » man’s, 18. See-saw piaffer, 306. Serpentine, 302, 325. IN DEX, Swimming horses, 230. RACING, FLAT, 206-209. Rameiner, 124, 125. Rassembler, 124, 127, 129, 130-132. Rearing, 42, 79, 80. Redoubt, 335. Rein back, 119, 247. Reining back without reins, 247. ‘TAKE AND GIVE,” 125. Reins, how to hold the, 72. Thorough-breds, 1, 2, 177-179, 183, Remounts, 215-233. 217-219, 222. Reward, 9, 112. Tongue over bit, 49. Riding, high school, 236-331. Trot, the, 140-153. »» ahorse first time, 78. Trotters, 150, 217. »> , ordinary riding, 32-183. ‘Two tracks,” 133-140. Rocking the forehand, 248. »» haunches, 249. VERMOUTH, 178. Rotation of croup and shoulders, 108. Vices, horses with, 184-194. Routined,’’ 251. Voltes, 90, 118, 163. Run-aways, 189-194. >> » reversed, 118. Running back, 43. WALK, 85. SADDLES, 15, 28. », 5 school, 118. School riding, high, 236-331. Watts, 207, 346. >» walk, 118. Webb, 346. Seat, lady's, 24. Whip, 18, 76, 113, 298, 300. 5» » man’s, 18. », » teaching by the, 76. See-saw piaffer, 306. Serpentine, 302, 325. Zur, 178. \| Shoulder-in, 135-140, 260. Shying, 187. Side-steps, 133. Snaffle, 10, 198. Spanish trot, 251-260, 301, 325. 55 walk, 237. 4 o> 9 NeW, 322. Spurs, 18, 113, 115, 288, 206. Stades, 306, 340. Standing still, 85. Steeplechasing, 202. Stirrups, 15. », » length of, 21. Swimming horses, 230. ‘TAKE AND GIVE,” 125. Thorough-breds, 1, 2, 177-179, 183, 217-219, 222. Tongue over bit, 49. Trot, the, 140-153. Trotters, 150, 217. ‘Two tracks,” 133-140. VERMOUTH, 178. Vices, horses with, 184-194. Voltes, 90, 118, 163. >> » reversed, 118. WALK, 85. », 5 school, 118. Watts, 207, 346. Webb, 346. Whip, 18, 76, 113, 298, 300. », » teaching by the, 76. Zur, 178. Pirouettes, reversed, on three legs, 245. Pivot, making horse, 110. “« Playing up,’’ 82, 281. “« Playing up,’’ 82, 281. Plunging, 42. Plunging, 42. Pudeldressirung, 298. Pulling, 334-336. Punishment, 9, 112. Spurs, 18, 113, 115, 288, 206. RACING, FLAT, 206-209. RACING, FLAT, 206-209. Rameiner, 124, 125. Rassembler, 124, 127, 129, 130-132. Rearing, 42, 79, 80. Redoubt, 335. Rein back, 119, 247. Reining back without reins, 247. Reins, how to hold the, 72. Remounts, 215-233. Reward, 9, 112. Riding, high school, 236-331. »» ahorse first time, 78. »> , ordinary riding, 32-183. Rocking the forehand, 248. »» haunches, 249. Rotation of croup and shoulders, 108. Routined,’’ 251. Run-aways, 189-194. Running back, 43. SADDLES, 15, 28. School riding, high, 236-331. >» walk, 118. Seat, lady's, 24. Digitized by Microsoft® IN DEX, 5» » man’s, 18. See-saw piaffer, 306. Serpentine, 302, 325. Rameiner, 124, 125. Rassembler, 124, 127, 129, 130-132. Rearing, 42, 79, 80. Redoubt, 335. Rein back, 119, 247. Reining back without reins, 247. Reins, how to hold the, 72. Remounts, 215-233. Reward, 9, 112. Rid Reward, 9, 112. Riding, high school, 236-331. »» ahorse first time, 78. »> , ordinary riding, 32-183. Rocking the forehand, 248. »» haunches, 249. Rotation of croup and shoulders, 108. ** Routined,’’ 251. Run-aways, 189-194. Running back, 43. SADDLES, 15, 28. School riding, high, 236-331. >» walk, 118. Seat, lady's, 24. 5» » man’s, 18. See-saw piaffer, 306. Serpentine, 302, 325. Zur, 178. Zur, 178. Printed at The Chapel River Press, Kingston, Surrey. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft®
https://openalex.org/W4382046964	https://www.frontiersin.org/articles/10.3389/fpubh.2023.1090436/pdf	English	null	The spatial linkage mechanism: medical level, public health security, and economic climate from 19 OECD EU countries	Frontiers in public health	2,023	cc-by	11,939	OPEN ACCESS OPEN ACCESS EDITED BY Dilaver Tengilimoğlu, Atılım University, Türkiye REVIEWED BY Muhammad Farhan Bashir, Shenzhen University, China Çağdaş Erkan Akyürek, Ankara University, Türkiye CORRESPONDENCE Tao Liu leo.liutao@163.com RECEIVED 05 November 2022 ACCEPTED 08 June 2023 PUBLISHED 26 June 2023 CITATION Fu R, Zheng B, Liu T and Xie L (2023) The spatial linkage mechanism: medical level public health security, and economic clim from 19 OECD EU countries. Front. Public Health 11:1090436. doi: 10.3389/fpubh.2023.1090436 Rong Fu 1, Binbin Zheng 1, Tao Liu 2 and Luze Xie 1 1 College of Economics, Hangzhou Dianzi University, Hangzhou, China, 2 Department of Sociology, Hangzhou Dianzi University, Hangzhou, China Introduction: The global spread of the COVID-19 has brought about global changes, especially in terms of economic growth. Therefore, it has become a global issue to explore the impact of public health security on the economy. Methods: Employing a dynamic spatial Durbin model, this study analyzes the spatial linkage mechanism of medical level, public health security, and economic climate in 19 countries as well as investigates the relationship between economic climate and COVID-19 by the panel data of 19 OECD European Union countries from March 2020 to September 2022. Results: Results show that an improvement in the medical level can reduce the negative impact of public health security on the economy. Specifically, there is a significant spatial spillover effect. The degree of economic prosperity hurts the reproduction rate of COVID-19. © 2023 Fu, Zheng, Liu and Xie. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. © 2023 Fu, Zheng, Liu and Xie. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS Discussion: Policymakers should consider both the severity of the public health security issues and the economic level when developing prevention and control policies. Given this, corresponding suggestions provide theoretical support for formulating policies to reduce the economic impact of public health security issues. COVID-19, public health, economic climate, economic growth, dynamic spatial Durbin model, OECD TYPE Original Research PUBLISHED 26 June 2023 DOI 10.3389/fpubh.2023.1090436 TYPE Original Research PUBLISHED 26 June 2023 DOI 10.3389/fpubh.2023.1090436 frontiersin.org 1.2. Strategic measures under COVID-19 The relationship between economic growth and the COVID-19 pandemic is complex. In the long run, the harm caused by the pandemic to the country and society is continuous, but the positive environmental impact is only temporary. The outbreak of COVID-19 has slowed or even stalled the global economy, reduced carbon emissions, and improved air quality in many cities around the world. However, when the pandemic subsides, carbon and pollutant emissions will return to the same levels as before, and the positive environmental impact of the pandemic will be lost (26). The mutation and invasion of COVID-19 strains require government departments to develop more powerful strategies to overcome the threat caused by COVID-19 (27). In this regard, Akighir et al. (28) estimated the macroeconomic development level of Nigeria after adopting the economically sustainable development plan, indicating that the sustainable economic development policy of the government has a positive impact on national economic growth, employment, inflation, and so forth. Dorn et al. (29) studied a balanced strategy that can meet the co-benefits of health protection and the economy, and also reduce economic losses without compromising medical goals. Contrary to the economic pain caused by the COVID-19 pandemic to the general public and the non-investment class, the market has brought immeasurable rewards to those at the top (30). For example, the pharmaceutical industry benefitted greatly from the shortage of drugs in the early stage of the pandemic (31). The development of health tourism has also helped the economic growth of countries such as Turkey. The national government can alleviate the economic losses caused by COVID-19 by supporting health tourism (32). In addition to social isolation, the national government should introduce policies to control the spread of COVID-19. Under the COVID-19 transmission model, every country will be affected by economic conditions and government intervention measures. Good economic conditions will exacerbate the spread of COVID-19, while appropriate government intervention measures will greatly reduce its spread (14). Specifically, the COVID-19 pandemic has reduced the activities of the service and manufacturing industries, resulting in an increase in the number of unemployed people. To reduce the negative impact of the pandemic and intervention measures on the economy, the government should take financial, monetary, and other economic measures to expedite economic recovery (15, 16). 1. Introduction At the beginning of 2020, the COVID-19 pandemic affected national economic growth and individuals’ basic life necessities. It has been the largest global public health crisis since the influenza pandemic in 1918 (1) and is still prevalent worldwide. Under the COVID-19 pandemic, global change is inevitable. Thus, it has become urgent to analyze and interpret the impact of the COVID-19 pandemic as well as what measures should be implemented to handle similar crises. Similar to the COVID-19 pandemic, the SARS outbreak in 2003 had a profound impact on the Asian tourism industry and affected the economic growth of Asia and the world. At the same time, it showed that the ban and measures adopted can effectively prevent the spread of the virus, but they may exacerbate panic (2). From the data available now, the impact of SARS is much smaller than our estimate of its current occurrence (3). The features that differentiate COVID-19 from recent encounters are its wide geographical spread in terms of contagion and its high mortality rate (4). Compared to the impact of the SARS outbreak, the economic impact and spillover effects on the European Union (EU) increased significantly when the COVID-19 pandemic occurred. China’s economic growth was affected at the initial stage of COVID-19, but it soon recovered. Thanks to the increase in China’s influence, the country was less affected by the negative spillover Frontiers in Public Health 01 frontiersin.org Fu et al. Fu et al. 10.3389/fpubh.2023.1090436 effects of other countries during the pandemic (5). Accordingly, we can maintain a wait-and-see attitude for the current prediction of losses caused by the COVID-19 pandemic. can affect GDP to some extent, and its impact is not entirely linear; increased health spending in a country increases human capital, either directly or indirectly, leading to higher productivity and an increase in GDP (20, 21). Healthcare levels and economic development go hand in hand. For example, Bangladesh’s economic growth is hindered by underdeveloped medical care (22). Therefore, the government should increase medical funding to mitigate the impact of the COVID-19 pandemic on residents’ lives and economic growth (23). COVID-19 affects not only current economic growth but also the future expectations of investors and consumers. Taking the United States as an example, the study finds that the health crisis and economic downturn will have a negative impact on investors. 1. Introduction At the same time, the health crisis in other countries will also have a negative spillover effect on investor expectations (24). The economic recession brought about by the COVID-19 pandemic will inevitably affect the unemployment rate, which will in turn reduce national tax revenue and increase government spending. Therefore, the federal government must avoid major deficits and harmful cuts, and improve the healthcare safety net by increasing Medicaid (25). 1.2. Strategic measures under COVID-19 At a time when rapid antigen diagnostic tests (RTDs) are widely used in COVID-19 detection, the government can turn the COVID-19 pandemic into a controllable infection through rapid testing (17). Standing at the crossroads of this choice, governments should learn a lesson. It is important to revive the economy, but once everyone is dead, then no one contributes to the economy (18). In emergency response, it is usually better for the government to overreact and then scale down when necessary, rather than to react too late (19). Therefore, the government must grasp the intensity of intervention, both development and pandemic prevention and control. Health spending 1.1. COVID-19 pandemic and economic growth: a historical overview The most obvious harm of global change caused by the COVID-19 pandemic is the increased medical burden. In Italy, for example, the number of deaths due to COVID-19 in the first quarter of 2020 reached 18,000, resulting in a loss of economy and productivity (6). The levels of anxiety, depression, and stress of healthcare employees were also significantly affected (7, 8). In the São Paulo intensive care ward, Brazil, the rate of hand hygiene during the COVID-19 pandemic decreased significantly compared to that before the pandemic. This was because the increased medical burden caused by the pandemic and other diseases had not resulted in increased hand hygiene compliance (9). Moreover, the high cost of COVID-19 treatment has exacerbated the existing burden on developing countries. At the same time, economic growth also affects the regional virus transmission rate. Especially in areas with unfavorable economic development, the transmission rate of COVID-19 cannot be well controlled. This situation has formed a vicious circle of the poorer the more serious, the more serious the poorer (10). Developed countries have more medical experience and higher medical standards, which has enabled them to the second and third waves of the handle pandemic better than developing countries (11). On this basis, the national government of developing countries can solve the problems through reasonable measures to reduce the cost of treatment and implement tiered charges for both rich and poor areas (12). The indirect impact of the COVID-19 pandemic is also huge, especially its negative impact on economic growth. Under the conditions of the COVID-19 outbreak, the medical capacities of hospitals were lacking, which inevitably led to economic losses. The cost of adopting different means of prevention and control of COVID-19 varies, and isolation may be the best way to deal with it (13). Frontiers in Public Health 2.2. Public health safety calculation Most infectious diseases that broke out in recent years have the characteristics of widespread contagion and profound impact. Many human infectious diseases have evolutionary patterns, such as AIDS, malaria, and hepatitis B. Their initial appearance led to a pandemic, with periodic outbreaks experienced in the process of human society, eventually forming endemic diseases and likely to erupt in the future (37). Especially in the process of globalization, geographical restrictions have weakened, and infectious diseases such as tuberculosis have moved from high-prevalence areas to low-prevalence areas with the deepening of international exchanges, thus affecting global public health security (38). COVID-19 has also caused a severe blow to tourism, hotel, education, and other industries (39–41). In addition, the losses caused by the pandemic do not only exist in the present. For instance, many patients have developed long-term physical and even psychological problems due to the disease. Indeed, the impact of the disease is prolonged. For a long time, the incidence of infectious diseases alone could not accurately measure the regional PHS indicators. Compared to disease incidence and mortality, disease burden more comprehensively reflects the regional disease severity and regional public health security level. Disease burden refers to the economic, life, and quality of life loss of patients after the occurrence of the disease (42). Moreover, when the Harvard T.H. Chan School of Public Health, the World Bank, and the World Health Organization (WHO) cooperated to assess the global disease burden in 1993, a new indicator of DALY was introduced in assessing the global disease burden (43). Since then, the DALY measurement has become a common disease burden measurement method. In view of this, the present research uses DALY to measure disease burden and constitute a PHS index. The construction process is as follows (44): Hypothesis 1: There is a spatial spillover effect of the medical level on public health safety issues and economic climate. Hypothesis 2: Increased public health safety issues can inhibit current economic growth; however, current economic growth cannot alleviate public health security issues. This paper only considers the extent to which the medical level and the strictness of policy response affect the public health safety issues and the economy. Other variables do not have a significant impact on the model. Therefore, the study assumes that other variables such as demographic structure, industrial structure, and psychological factors are not significantly different across countries. 2. Materials and methods YLD I DW L e r rLD = × × 1− − ( ) (2) YLL N r e rL = 1− − ( ) (3) YLD I DW L e r rLD = × × 1− − ( ) (2) (2) 1.3. Aims and contributions of the study As the most influential public health security issue at present, COVID-19 has had an impact on all aspects of the world. Especially in the economic aspect, the pandemic has brought a huge blow to the economy. With globalization enhancing international exchanges, the 02 frontiersin.org Fu et al. Fu et al. 10.3389/fpubh.2023.1090436 monthly average government response strictness index; and reproduction rate (RR) is the country’s monthly virus reproduction rate. spillover effect of the pandemic cannot be ignored. Many studies have used different models to study the relationship between COVID-19 and the economy (33–35), especially the direct impact of the pandemic on the economy. Different medical levels between countries lead to differences in the way and effectiveness of pandemic prevention in each country, and the medical level becomes an influential factor during a pandemic. The present study combines the economic level matrix and the geographical distance matrix to build a dynamic spatial Durbin model (36). We are more concerned with the indirect impact of the pandemic on neighboring countries. The dynamic spatial Durbin model can well integrate geographical distance into the model, expand the impact of the COVID-19 pandemic from one country to neighboring countries, and more comprehensively describe the spread and harm of the pandemic in the context of globalization. Expanding from COVID-19 to general public health security issues informs future public health security issues. Based on this, the study proposed two main hypotheses. Frontiers in Public Health 2.2. Public health safety calculation Moreover, differences in topography and landscape across countries do not affect the construction of the geographical distance matrix. Compared with the existing literature, this paper possibly makes the following contributions: first, adopting the COVID-19 disability- adjusted life year (DALY) as a public health safety (PHS) indicator and using it to analyze the impact of public health safety issues on the economic climate; and second, adding control variables to the Durbin model (strictness of policy response), which reduces the impact of different national pandemic prevention policies on the results. DALY YLD YLL = + (1) (1) In Formula (1), YLD represents the number of years of health lost due to disability, and YLL represents the number of years of life lost due to death. frontiersin.org 2.3. Spatial correlation test The basic measure of spatial autocorrelation analysis is Moran’s I, which is derived from the Pearson correlation coefficient in statistics and can reveal the laws of geographic space (46). In this research, Moran’s I is used to test the spatial correlation of the indicator of the economic climate. The statistical calculation process of the global Moran’s I to measure the spatial correlation is as follows: I n W Y Y Y Y S Y Y i n j n i j i J i n i = − ( ) − ( ) − ( ) = = = ∑∑ ∑ 1 1 0 1 2 , (4) (4) In Formula (4), Yi is the OECD CLI of each country, Wi j, is the economic geospatial weight between countries i and j, n is the total number of countries, and S0 is the aggregation of spatial weights: 0 1 1 n n i j i j S W ∑∑ , = = = (5) (5) growth of neighboring countries, and the spatial clustering effect really exists. This paper then draw a LISA scatterplot of the Anselin Local Moran’s I (Figure 1). The cross-sectional data in February 2021 and November 2021 are analyzed, and it is found that there were high-high aggregation and low-low aggregation between countries, with a strong spatial clustering effect. Since countries within the EU are not only connected by distance but also by mutual economic activities, an economic geospatial nested matrix is constructed (47). The construction process is as follows: w if i j d if i j i j i j , , , , 1 2 0 1 = = ≠    (6) w if i j d if i j i j i j , , , , 1 2 0 1 = = ≠    w if i j d if i j i j i j , , , , 1 2 0 1 = = ≠    (6) w w diag y y y i j i j n , , = ( ) 1 1, , , 2  (7) (6) 2.1. Variable selection and data interpretation YLL N r e rL = 1− − ( ) (3) This study selects data from the official statistics of the OECD and the data provided by Martin College of Oxford University and the Global Clinical Development Lead. In particular, it uses the Comprehensive Leading Indicator (CLI) to measure the degree of future economic prosperity; the quadratic interpolation to convert quarterly GDP into monthly GDP, which is then taken as the economic climate level of each country; and the COVID-19 DALY as a public health safety (PHS) indicator. Three control variables are selected: medical level (ML), which is the number of hospital beds per 1,000 people in each country, is selected as a measure of the medical level of each country during the COVID-19 pandemic; policy response strictness (PRS) is the (3) In Formula (2), I represent the number of patients infected by COVID-19 (this study adopts the number of cases per 100,000 people), and DW represents the weight of the disease. According to the research on the weight of the disease proposed by Saloman et al. (45), the DW of the COVID-19 pandemic is equal to 0.133; LD is the disease duration, usually two weeks, or 0.0038 years; and r is the discount rate, usually 0.03. In Formula (3), N represents the number of deaths due to COVID-19 (this study adopts the number of deaths 03 frontiersin.org Fu et al. 10.3389/fpubh.2023.1090436 TABLE 1 Global Moran’s I from March 2020 to September 2022. TABLE 1 Global Moran’s I from March 2020 to September 2022. per 100,000 people), and L represents the life expectancy. The final estimated DALY included panel data of 31 months for 19 countries. TABLE 1 Global Moran s I from March 2020 to September 2022. Month Moran’s I Month Moran’s I 2020/03 −0.024 2021/07 0.658* 2020/04 −1.214** 2021/08 0.675* 2020/05 −0.996** 2021/09 0.735* 2020/06 −0.812* 2021/10 0.746* 2020/07 −0.034 2021/11 0.675* 2020/08 0.111 2021/12 0.525 2020/09 0.283 2022/01 0.306 2020/10 0.555 2022/02 0.043 2020/11 0.767* 2022/03 −0.200 2020/12 0.941 2022/04 −0.368 2021/01 1.156* 2022/05 −0.459 2021/02 1.211* 2022/06 −0.476 2021/03 0.998 2022/07 −0.407 2021/04 0.905 2022/08 −0.328 2021/05 0.788 2022/09 −0.252 2021/06 0.670* 10% significance level, 5% significance level, *1% significance level. Frontiers in Public Health 10% significance level, 5% significance level, 1% significance level. 2.5. Panel model of interaction mechanism TABLE 2 Test results of traditional mixed panel model. Test CLI GDP LM-lag 50.950 567.365* LM-error 59.799* 536.225 Hausman 37.07* 34.61* 5% significance level, 1% significance level. The impact of the COVID-19 pandemic on national economic growth is described above, but the impact between the two is mutual. Health infrastructure, pandemic prevention, and control policies, urban density, urban environment (51), and economic growth will all have an impact on the severity of the COVID-19 pandemic in a region. Structural changes have an important impact on the discovery of causality, for example, Xu et al. (52) found that economic activities mainly caused environmental pollution through the shock of the COVID-19 pandemic. This paper refers to Guven et al. (53) fixed- effect panel model to reset the model and further analyze the impact of economic growth level on the severity of the pandemic. After the Hausman test, it can be seen that the fixed effect model is more suitable, and so the following fixed effect model is set: 0 1 , 1 1 2 2 1 1 3 3 4 4 1 1 5 5 5 5 1 1 n n i,t i t i, j j,t i,t i, j j,t j j n n i,t i, j j,t i,t i,t i, j j,t j,t j j n n i,t i, j j,t i,t i, j j,t i i,t j j Y Y W Y PHS W PHS ML W ML PHS ML W PHS ML PRS W PRS RR W RR β β δ β δ β δ β δ β δ β δ µ ε = = = = = = = + + + + + + + × + × + + + + + + ∑ ∑ ∑ ∑ ∑ ∑ − (10) (10) RR Y Z i,t i t i,t i i,t = + + + + β β β µ ε 0 1 2 , (11) (11) In Formula (10), Y is the explained variable future economic climate (CLI) and economic climate (GDP). The degree of PHS is the explanatory variable, ML is the moderating variable, and PRS and virus RR are the control variables.h The dependent variable is the regional COVID-19 pandemic severity (RR). Zit represents the control variables PHS and PRS, and Yi t, represents the future economic climate (CLI) and economic climate (GDP). 2.4. Constructing the dynamic spatial Durbin model based on the moderating effect theory 0 1 , 1 1 2 2 1 1 3 3 4 4 1 1 5 5 5 5 1 1 n n i,t i t i, j j,t i,t i, j j,t j j n n i,t i, j j,t i,t i,t i, j j,t j,t j j n n i,t i, j j,t i,t i, j j,t i i,t j j Y Y W Y PHS W PHS ML W ML PHS ML W PHS ML PRS W PRS RR W RR β β δ β δ β δ β δ β δ β δ µ ε = = = = = = = + + + + + + + × + × + + + + + + ∑ ∑ ∑ ∑ ∑ ∑ − (10) TABLE 2 Test results of traditional mixed panel model. Test CLI GDP LM-lag 50.950 567.365* LM-error 59.799* 536.225 Hausman 37.07* 34.61* 5% significance level, 1% significance level. TABLE 2 Test results of traditional mixed panel model. Frontiers in Public Health 2.5. Panel model of interaction mechanism After testing, it is known that the inflation factor of each explanatory variable is less than 10, so there is no multicollinearity. The following model tests are based on a mixed panel data model with the interaction term removed from the model set up above. On this basis, the LM test is carried out to show the rationality of choosing the spatial Durbin model. As shown in Table 2, both the spatial lag model test and spatial error model test are significant, indicating that both models are supported and the mixed OLS model is rejected. Thus, the rationality of using the spatial Durbin model is confirmed. 2.4. Constructing the dynamic spatial Durbin model based on the moderating effect theory (7) If the relationship between variables X and Y is represented by a function with variab+le M, then M is the moderating variable (48). Compared to the interaction effect, the independent variable and moderating variable in the moderating effect are asymmetric and cannot be interchanged. The current study adopts the most commonly used adjustment model proposed by Wen et al. (49) to analyze the adjustment effect of medical level on public health security issues. The specific test equation is as follows: where yi refers to the GDP per capita of each country from 2020 to 2021 (calculated by purchasing power parity), di j, refers to the distance between national capitals (the unit is kilometers), and wi j, is standardized on the basis of Formula (7): 1 i,j i,j n i,j j w W w ∑= = (8) (8) Y aX bM cXM e = + + + (9) (9) By Formula (8), the wi j, matrix is made dimensionless to make it reflect the spatial correlation structure more clearly, which is convenient for the subsequent drawing of the local Moran exponent map and the establishment of the spatial Durbin model. Since there is a time lag in the degree of economic prosperity, the time lag term of the degree of economic prosperity is introduced into the standard static spatial Durbin model. The exogenous variable PRS is introduced as a control variable, and a dynamic spatial Durbin model is established based on the adjustment effect (50): From August 2020 to February 2022, Moran’s I is positive and significant (Table 1), indicating that the CLI of European countries has a significant positive correlation in the spatial distribution, that is, there is a clustering trend in the economic 04 frontiersin.org Fu et al. Fu et al. 10.3389/fpubh.2023.1090436 FIGURE 1 Lisa scatterplot of the AnselinLocal Moran’s I. (A) Lisa scatterplot in February 2021; (B) Lisa scatterplot in November 2021. FIGURE 1 Lisa scatterplot of the AnselinLocal Moran’s I. (A) Lisa scatterplot in February 2021; (B) Lisa scatterplot in November 2021. 3. Results 3.1. Comparative analysis of Spanish flu and COVID-19 3. Results 3.1. Comparative analysis of Spanish flu and COVID-19 Then, the Hausman test is carried out on whether the model adopts the fixed-effect model or the random-effect model. The p value of the Hausman test is less than 0.05, which proved that the fixed- effect model should be selected. The LR test and Wald test are then carried out on the model to analyze whether the spatial Durbin model will degenerate into a spatial auto-regression model and a spatial error model, which shows that the choice of the spatial Durbin model is very reasonable. The results are shown in Table 3. As the most lethal infectious disease in human history, the Spanish flu changed human life. Like COVID-19, which is currently prevalent, hatred makes people regard a country as the culprit. Most infected people have low immunity due to illness and die of other diseases (54). Both pandemics have a severe blow to the global medical and healthcare system. In the early days of pandemics, when confronted with these two pandemics, people underestimated the infection and 05 frontiersin.org Fu et al. 10.3389/fpubh.2023.1090436 Fu et al. TABLE 3 Test results of the fixed effect model. TABLE 3 Test results of the fixed effect model. location and primarily concentrated in central Europe, while the first type of countries is primarily concentrated in western Europe and coastal areas. This classification shows that the severity of public health security issues between countries with relatively close geographical distances is correlated, and it preliminarily confirms that there is a spatial spillover effect on public health security issues. Test CLI GDP LR-test SDM-SAR SDM-SEM SDM-SAR SDM-SEM 18.52** 7.97* 49.05* 107.32* Wald- test Unconstrained Constraint Unconstrained Constraint 18.99*** 8.12* 49.01* 65.23* 10% significance level, 1% significance level. According to the clustering results, this study draws a line chart of the PHS of the COVID-19 pandemic in these two types of countries (Figure 3). The changes in PHS during the COVID-19 pandemic vary in different countries, but most of its peaks are concentrated from November 2020 to April 2021. Although the cold weather can inhibit the activity of the virus to a certain extent, the outbreak in winter has brought huge disasters to the residents of all countries due to the unfavorable supervision of the pandemic in European countries. 3.1. Comparative analysis of Spanish flu and COVID-19 In November 2021, the COVID-19 pandemic severity increased sharply, indicating that the pandemic is threatening and recurrent, and therefore countries should control it scientifically and rationally. After the lockdown was lifted in February 2022, individual countries stopped implementing mandatory closure measures and replaced them with home quarantine measures for sick people. Preventive and control measures are still in place, but they are much more relaxed than they were during the initial period of the pandemic. The economy is also gradually recovering, and the pandemic is steadily declining in both groups of countries. Scientific prevention and control measures can more effectively control the pandemic and promote economic recovery.t TABLE 4 Comparison between COVID-19 and Spanish flu. TABLE 4 Comparison between COVID-19 and Spanish flu. p pl COVID-19 Spanish Flu Influenza duration December 2019 - 25 months Scope of influence Global Less than half of the countries Main age group of dead patients Age more than 65 years 25–40 years old Proportion of deaths to total population (Italy) 0.2% 5% Economic loss (Mexico) 180 billion dollars 9 billion dollars The number of Spanish flu deaths in Italy is based on the study of Burdekin (57). COVID-19 Spanish Flu Influenza duration December 2019 - 25 months Scope of influence Global Less than half of the countries Main age group of dead patients Age more than 65 years 25–40 years old Proportion of deaths to total population (Italy) 0.2% 5% Economic loss (Mexico) 180 billion dollars 9 billion dollars The number of Spanish flu deaths in Italy is based on the study of Burdekin (57). mortality rates of the viruses associated with them (55). Therefore, this study compares the two pandemics (56).l As shown in Table 4, the death toll of the Spanish flu was higher than that of COVID-19, due to war and low medical levels at that time. The current medical level is far higher than that in 1918, but the proportion of deaths in the total is still not low. We can see the changes in the scope of influence: the world links closely in the context of globalization. The scope of influence of COVID-19 is much larger than that of the Spanish flu. The economic losses from COVID-19 were much higher than those from the Spanish flu due to the globalization of the economy, and the spillover from the COVID-19 pandemic has dealt a severe blow to the global economy. 3.1. Comparative analysis of Spanish flu and COVID-19 Therefore, the spillover effect between regions needs to be considered in public security research to prepare for the next attack on public health security. Frontiers in Public Health frontiersin.org 3.1. Comparative analysis of Spanish flu and COVID-19 The aggravation of the pandemic situation in European countries in winter also shows that the pandemic is no longer a disaster for one country but a disaster for the whole world. The impact of the spillover effect of the pandemic on neighboring countries is inestimable, and the pandemic should be jointly managed and controlled to prevent its spread. The first category of countries increased significantly in the early stage of the pandemic. It is very likely that this category includes mostly Western European countries with developed tourism, which led to the outbreak of the pandemic due to the flow of tourists in the early stage. After the inflection point in May 2020, it surged after November 2020, but the severity was weaker than that of the second category of countries. It is speculated that the first category of countries had already dealt with the outbreak in the early stage of the pandemic, and so the pandemic control during the second outbreak was more in place than the second category of countries. The second category of countries had relatively mild pandemics before September 2020 and concentrated outbreaks from October 2020 to May 2021, with a sharp increase after October 2021. These countries paid more attention to pandemic supervision in the early stage of the pandemic, such that there was no large-scale outbreak of the pandemic. However, the pandemic was repeated. Owing to the mitigation of pandemic control by the first type of countries, the pandemic broke out in October 2020. At the same time, the countries quickly implemented pandemic supervision and prevention policies such that the COVID-19 pandemic severity gradually decreases after peaking in March 2021. In November 2021, the COVID-19 pandemic severity increased sharply, indicating that the pandemic is threatening and recurrent, and therefore countries should control it scientifically and rationally. After the lockdown was lifted in February 2022, individual countries stopped implementing mandatory closure measures and replaced them with home quarantine measures for sick people. Preventive and control measures are still in place, but they are much more relaxed than they were during the initial period of the pandemic. The economy is also gradually recovering, and the pandemic is steadily declining in both groups of countries. Scientific prevention and control measures can more effectively control the pandemic and promote economic recovery. 3.1. Comparative analysis of Spanish flu and COVID-19 Since the EU proposed to completely lift the lockdown in According to the clustering results, this study draws a line chart of the PHS of the COVID-19 pandemic in these two types of countries (Figure 3). The changes in PHS during the COVID-19 pandemic vary in different countries, but most of its peaks are concentrated from November 2020 to April 2021. Although the cold weather can inhibit the activity of the virus to a certain extent, the outbreak in winter has brought huge disasters to the residents of all countries due to the unfavorable supervision of the pandemic in European countries. The aggravation of the pandemic situation in European countries in winter also shows that the pandemic is no longer a disaster for one country but a disaster for the whole world. The impact of the spillover effect of the pandemic on neighboring countries is inestimable, and the pandemic should be jointly managed and controlled to prevent its spread. The first category of countries increased significantly in the early stage of the pandemic. It is very likely that this category includes mostly Western European countries with developed tourism, which led to the outbreak of the pandemic due to the flow of tourists in the early stage. After the inflection point in May 2020, it surged after November 2020, but the severity was weaker than that of the second category of countries. It is speculated that the first category of countries had already dealt with the outbreak in the early stage of the pandemic, and so the pandemic control during the second outbreak was more in place than the second category of countries. The second category of countries had relatively mild pandemics before September 2020 and concentrated outbreaks from October 2020 to May 2021, with a sharp increase after October 2021. These countries paid more attention to pandemic supervision in the early stage of the pandemic, such that there was no large-scale outbreak of the pandemic. However, the pandemic was repeated. Owing to the mitigation of pandemic control by the first type of countries, the pandemic broke out in October 2020. At the same time, the countries quickly implemented pandemic supervision and prevention policies such that the COVID-19 pandemic severity gradually decreases after peaking in March 2021. 3.2. Comparison of public health security among countries Clustering analysis was performed on the PHS indicators of each country with the time period as a variable to judge the similarity of the degree of impact of the COVID-19 pandemic among countries as well as analyze whether the similarity is related to geographical distance. These indicators are clustered using the silhouette coefficient method and K-means clustering method. Using the silhouette coefficient method, it is known that the optimal number of classifications is divided into two categories. The classification results are Austria, Belgium, Denmark, Estonia, Finland, France, Germany, Sweden, Iceland, Italy, the Netherlands, Portugal, Spain, and Greece for the first category; and the Czech Republic, Hungary, Poland, Slovakia, and Slovenia for the second category. As shown in Figure 2, the second type of countries are relatively close in geographical Since the EU proposed to completely lift the lockdown in February 2022 and proposed new regulations on February 1, tourists only need to carry proof of vaccination, or proof of recovery or proof of negative test to travel unimpeded among the 27 OECD EU countries without isolation or additional coronavirus testing. Therefore, this study selects PHS and CLI before and after the lifting of the lockdown to conduct a preliminary analysis of the rationality 06 frontiersin.org Fu et al. 10.3389/fpubh.2023.1090436 FIGURE 2 Geographical location map of two kinds of countries. FIGURE 3 Twenty one months PHS line chart for two types of countries. FIGURE 2 Geographical location map of two kinds of countries. FIGURE 2 Geographical location map of two kinds of countries. FIGURE 2 Geographical location map of two kinds of countries. FIGURE 3 Twenty one months PHS line chart for two types of countries. of its policy. It selects the PHS of 19 countries from December 2021 to March 2022 for K-means clustering, as shown in Figure 4. The countries are clustered into two categories, and the CLI of these two categories of countries are calculated. From this, it can be concluded that the first type of countries (14 countries, including Austria, Berlin, and Denmark) originally had a relatively low burden of disease but were negatively affected by the lift lockdown policy, with public health security issues minor increase. The second type of countries (5 countries, including the Czech Republic and Sweden) has a relatively serious disease burden, and so they have not been negatively affected by the lift lockdown policy. Frontiers in Public Health 3.2. Comparison of public health security among countries Instead, due to the open-lift lockdown policy, the domestic pandemic has spilled over to neighboring countries. Governments should continue to be concerned about the spillover effects of public health security issues. On the other hand, the lift lockdown policy has not promoted economic recovery, but rather the CLI has declined in these two types of countries. The economic growth of the first type of countries with a lighter disease burden is better than that of the 07 frontiersin.org Fu et al. 10.3389/fpubh.2023.1090436 FIGURE 4 The line chart of changes in PHS and CLI before and after the lift lockdown of the two types of countries. FIGURE 4 The line chart of changes in PHS and CLI before and after the lift lockdown of the two types of countries. time lag effect of the degree of economic prosperity, is more reasonable and reflects economic and social development. Public health security issues have no significant impact on the future economic growth of the country. Although the coefficient of the multiplication term of ML and PHS shown in Table 6 is greater than the coefficient of PHS, it is not significant. In terms of spatial spillover effects, public health security issues will have a significant negative effect on the future economic growth of neighboring countries, indicating that a country’s public health security issues will have a negative impact on the future economic growth of neighboring countries. The coefficient of the multiplication term of ML and PHS is significantly positive in terms of spatial spillover, indicating that the improvement of medical level can have a certain mitigation effect on the negative impact of public health security issues in neighboring countries and regions on the degree of future economic prosperity. The coefficient of RR is significantly negative, but there is a significant positive spillover effect. It shows that the increase in RR of the COVID-19 pandemic will aggravate the pandemic and reduce people’s confidence in the future economic climate. Therefore, it will inhibit the future economic climate, but will not be negatively affected by the virus RR of neighboring countries. Similarly, the reason why the PRS coefficient is positive but not significant is most likely that the pandemic is serious and the state has responded positively to it. At the same time, the impact of COVID-19 on residents and the strictness of policy response offset each other, so PRS is not significant. 3.2. Comparison of public health security among countries second type of countries with a heavy disease burden. It shows from the side that lifting the lockdown has not immediately led to economic recovery, and the expected ability to lead to economic recovery has some lag. Improving medical care and reducing national disease burdens can, to some extent, promote economic growth. 3.3. Correlation test In order to measure the correlation between variables, this paper conducted a correlation analysis on explanatory variables and explained variables. In Figure 5, the variables of public health security and the degree of economic prosperity are negatively correlated, indicating that public health security issues significantly inhibit economic development. The specific relationship between the variables requires further judgment. Frontiers in Public Health frontiersin.org 3.4. Analysis of spatial linkage mechanism To obtain more robust results, this study adds the time lag term and spatial effect of the degree of economic prosperity to the panel model and uses the dynamic Durbin model to analyze the problems described in the article. To judge which fixed effects the dynamic Durbin model adopts, the study establishes models, respectively. When testing the variable CLI, the log-likelihood value is the maximum when selecting the individual effect model, so it chooses the dynamic Durbin model of the individual effect. When testing the variable GDP, the log-likelihood value is the maximum when selecting the individual effect model, so it chooses the dynamic Durbin model of the individual effect (Table 5). fi In Table 7, the impact of PHS on GDP is significantly negative and the spatial spillover effect is negative, indicating that increasingly serious public health security issues will inhibit the development of the degree of economic prosperity and have a negative impact on the economic growth of neighboring countries. Similarly, the coefficient of the multiplicative term of ML and PHS has increased and the spillover effect is significantly positive, indicating that the improvement of the medical level can bring a certain mitigation effect to the negative impact of domestic public health security issues on economic climate. Additionally, it can significantly alleviate the negative impact of public health security problems in neighboring countries on economic prosperity. The coefficient of RR is significantly The time lag item Yt−1 of the economic climate in Table 6 is very significant, proving that the above economic climate has the characteristics of time path dependence. The dynamic spatial Durbin model, which takes into account the endogeneity problem and the 08 frontiersin.org Fu et al. 10.3389/fpubh.2023.1090436 FIGURE 5 Correlation coefficient chart. TABLE 5 Test results of log-likelihood of the fixed-effect dynamic spatial Durbin model. explanatory variable (PHS) with the COVID-19 mortality rate (PHS-death) for retesting.fi Durbin model. Variable name Individual effect Time effect Two-way fixed effects CLI −323.5015 −860.8148 −992.2393 GDP 616.7979 −997.6296 −830.0706 From the perspective of coefficient changes, the medical level can also alleviate the negative impact of the death rate of the COVID-19 pandemic on the future and current economic climates. A spillover effect is observed. 3.4. Analysis of spatial linkage mechanism Within a country, the increase in mortality does not significantly affect people’s prospects for future economic development, but it will have a significant negative impact on the current economic climate and negatively affect the degree of future economic prosperity of neighboring countries (Table 8). Similar to the above results and weaker than the above, the findings further show that the selection of the DALY of the COVID-19 pandemic as a PHS indicator is more appropriate than the COVID-19 pandemic mortality rate. It also shows that the results of the dynamic spatial Durbin model constructed above are credible and robust. fi effects CLI −323.5015 −860.8148 −992.2393 GDP 616.7979 −997.6296 −830.0706 negative, but there is a significant positive spillover effect, indicating that the increase in RR of the COVID-19 pandemic will inhibit economic growth at present but will not necessarily have a negative impact on the economic growth of neighboring countries. Similarly, the coefficient of PRS is positive and has a significant negative spillover effect. It may be that the current response of the government to the pandemic will have a positive impact on economic growth but not on the economic development of neighboring countries. 3.6. Analysis of the impact mechanism of economic growth on the COVID-19 pandemic Combining the results in Tables 6, 7, the multiplicative coefficients of ML and PHS are significantly positive in both models in terms of spatial spillover, indicating that the improvement of medical level can bring a certain mitigation effect to the negative impact of domestic public health security issues on economic climate. Hypothesis 1 is confirmed. The GDP of coefficient is significantly positive (Table 9), indicating that the faster the economic development, the more serious the public health security issues become. Economic growth cannot alleviate public health security issues. The previous section illustrated that public health security issues can impede economic growth. The two results together confirm Hypothesis 2. The current economy develops faster the virus RR becomes faster. Economic development requires exchange contact, which will become a breeding ground for virus reproduction. But the CLI of the coefficient is significantly negative, indicating that the higher the future economic prosperity degree, the lower the virus RR will be. Due to reasonable and scientific government control measures, people no longer panic about the impending pandemic and are full of confidence in the anticipated economic climate. Therefore, the future economic climate will Frontiers in Public Health frontiersin.org 4. Discussion We have deeply realized the impact of COVID-19 on human daily life. In the context of economic globalization, public health security is not only a problem for individual countries but also a problem for the whole world. Therefore, besides the negative impact on the economy, is there any spillover effect of the COVID-19 pandemic? To better analyze the spillover effects of medical level and public health security issues on the economic climate, this study selects EU countries with close economic ties and geographical distance as the research object. This paper establishes a dynamic Durbin model under the theoretical framework of a moderating effect and then studies the impact of public health security on the current and future economic climate of the region and the moderating effect of the medical level. Furthermore, the research perspective is expanded to the spatial dimension to study the spatial spillover effect. The medical level can adjust the negative impact of COVID-19 on the economy and alleviate the negative impact of public health security issues on the economy of neighboring countries. The degree of economic prosperity will affect public health security issues to a certain extent. Our study found that the effect of the medical level on the pandemic and the economy is highly significant. In contrast, the impact of pandemic prevention policies on the pandemic and the economy is not fully significant. Pandemic prevention policies can only lead to economic recovery by indirectly influencing people’s confidence in the future economic climate. Thus, the negative impact of a strict pandemic prevention policy on the economy during the latter part of a pandemic is greater than its positive impact on pandemic prevention and control. In the event of COVID-19, because the impact of the COVID-19 pandemic on European countries is asymmetric, the challenges brought by this asymmetry greatly reduce the effect of joint measures taken at the EU level. Therefore, many scholars proposed that the EU should formulate flexible plans to combat the pandemic (59). For example, EU member states reached a 540 billion Euro rescue measure in early April 2020 and approved a 1.85 trillion Euro budget stimulus plan on December 10, 2020 (60). 3.5. Robustness test Although the COVID-19 DALY used above can accurately measure the severity of regional public health security problems, the increase in the death rate of COVID-19 is likely to increase people’s panic and accelerate the spread of public health security problems. Therefore, a new column of new variables, the COVID-19 death rate (PHS-death), is added below, which is the number of COVID-19 deaths per 100,000 people. Given this, to test the robustness of the dynamic spatial Durbin model, this research replaces the core 09 frontiersin.org 10.3389/fpubh.2023.1090436 Fu et al. Fu et al. TABLE 8 Influence of PHS on CLI and GDP–robustness test of the moderating effect of ML. Dependent variable Independent variable Main effect Spillover effect CLI GDP PHS-death −0.042 −0.108 PHS-death × ML 0.021 −0.246* Yt−1 0.663* 0.663* RR −0.121* 0.075 PRS 0.054 0.042 PHS-death −0.007 −0.012 PHS-death × ML −0.001 0.048* Yt−1 0.442* 0.442* RR −0.018* 0.018* PRS 0.021* −0.037* 5% significance level, *1% significance level. TABLE 8 Influence of PHS on CLI and GDP–robustness test of the moderating effect of ML. TABLE 6 The impact of PHS on CLI − the moderating effect of ML. CLI Main effect Spillover effect PHS −0.041 −0.107 PHS × ML 0.021 0.244* Yt−1 0.664* 0.664* RR −0.121* 0.075 PRS 0.054 0.042 5% significance level, **1% significance level. TABLE 7 The impact of PHS on GDP − the moderating effect of ML. be improved on this basis, which will inhibit the RR of COVID-19, so that the pandemic can be under control. TABLE 7 The impact of PHS on GDP − the moderating effect of ML. GDP Main effect Spillover effect PHS −0.007 −0.012 PHS × ML 0.001 0.048* Yt−1 0.443* 0.443* RR −0.018* 0.019* PRS 0.021* −0.038*** 10% significance level, 1% significance level. TABLE 9 Fixed effect regression model of the impact of economic climate on the COVID-19. Variable name Coefficient CLI −0.206* GDP 0.600* PHS −0.145*** PRS 0.042 10% significance level, **1% significance level. be improved on this basis, which will inhibit the RR of COVID-19, so that the pandemic can be under control. be improved on this basis, which will inhibit the RR of COVID-19, so that the pandemic can be under control. be improved on this basis, which will inhibit the RR of COVID-19, so that the pandemic can be under control. Frontiers in Public Health 5.1. Theoretical contributions At the moment of the pandemic, countries with high medical levels can better cope with the medical burden brought by the pandemic and effectively alleviate the negative effects of the pandemic. After reading the relevant literature, we found a few articles on the impact of the severity of the COVID-19 pandemic in a country on its neighboring countries, but this was very important in the context of economic globalization. The EU is a political and economic community. It has significant outbreak spillover effects. Therefore, this paper chooses the EU, a region with strong economic cooperation, for research. The 19 countries cited in this article are all from the EU. As a political and economic community, the EU promotes the development of countries in the EU through the implementation of treaties and plans. There are close political and economic exchanges between countries, so it is easier to transmit public health security issues. In order to study the spatial linkage problems encountered in adjacent regions when facing public health and safety problems, this paper introduces the moderating effect theory into the dynamic spatial Durbin model to explain the spillover effect of interregional public health security issues and the moderating role of the medical level. The aggravation of the pandemic in one country is very likely to cause pandemic burdens to neighboring countries. Similarly, the high level of national medical care can provide medical assistance to more patients, reducing the scope of public health security problems in the region. At the same time, it provides medical assistance to neighboring countries to alleviate the negative impact on economic growth trends of neighboring countries due to the spread of the public health security issues pandemic. Previous studies have often discussed the direct effect of the pandemic on the economy and other aspects. However, few studies have studied the spillover effect of the pandemic on the economy and the direct and indirect moderating effects of the medical level. In the process of globalization, countries connect more In this study, 19 countries in the EU were selected as subjects. When selecting other countries, it is necessary to consider the similarity of their political and economic systems. The presence of large political and economic differences among neighboring countries will most likely lead to weaker spillover effects in neighboring countries than in non-neighboring countries. 5.2. Recommendations Our research shows that the impact of a public health security problem is extensive, which will affect surrounding countries due to spillover effects. The improvement of medical level can help the country and even neighboring countries resist the attack of public health security problems. It is clear that the medical level is very important in any public health security issue, and therefore the government needs to maintain the medical system in such issues. For example, enhancing the health protection of healthcare workers. Some countries can provide medical assistance, such as medical supplies, personnel, and programs, to neighboring countries when the pandemic is controllable in their own. Our study finds that policy response strictness has a positive but insignificant effect on public health security and the economy throughout the outbreak period. It is because strict pandemic prevention policies are more effective in controlling the spread of the pandemic when the virus is virulent in the early stages of COVID-19. However, as the virus species mutates, the lethality rate of the pandemic decreases significantly, and strict pandemic prevention policies are less effective in controlling the pandemic but harm socioeconomic recovery. For example, the public health security issues do not intensify after the implementation of the lift lockdown policy in the EU. The intensity of pandemic control needs to base on the severity of the public health security issues and the intensity of pandemic transmission. Therefore, in the face of public health security issues, we recommend strict control in the early stages when the virus is strong. In the later period, control measures should be gradually relaxed. Eventually, economic recovery will be achieved. 5.1. Theoretical contributions Therefore, it is necessary to take into account the inter-country differences in the geographic distance and economic level matrix when considering the public health security issues’ spillover effects among countries with large political and economic differences. When discussing the impact of the COVID-19 pandemic on the economy, this study introduced policy response strictness and virus RR as control variables. However, in real life, there are still many variables that can affect the severity of the COVID-19 pandemic and economic growth, such as psychological factors (i.e., people’s fear of the COVID-19 pandemic). These variables are difficult to quantify, so more precise frameworks need to be constructed to measure people’s psychological factors. There is still room for improvement in the selection of control variables in this study. 5. Conclusion Our research shows that public health security issues in one country have spillover effects on the economic development of neighboring countries. Severe public health security issues can hurt the economic development of neighboring countries. At the same time, the development of the medical level can not only alleviate the negative effect of the pandemic on the economy in one country but also alleviate the negative effect of the pandemic on the economy in neighboring countries. 4. Discussion On April 15, 2020, the president of the European Council and the president of the European Commission jointly proposed the “EU road map” to gradually eliminate restrictive measures in pandemic prevention and control, flexibly control and gradually restore normality to residents’ lives, and restore strict control measures when the infection rate of COVID-19 surges (61). Most scholars agree on the need for a greater policy focus on economic recovery, which is consistent with our findings. Our study confirms that policy response strictness has a positive but insignificant effect on pandemic control. At a time when viruses are weaker, economic recovery is even more The global economy has been significantly affected by the COVID-19 pandemic. Some studies show that COVID-19 has spillover effects on the global economy. First, the spread of the virus has limited people’s social distance and closed economic activity venues. The national economy is developing slowly. Second, the virus spreads exponentially, which causes consumers and investors to lack confidence in future economic development and makes it difficult for the economy to recover steadily (58). It is consistent with the conclusion of the present study. Public health security issues affect not only the current economic situation but also the future economic development trend. 10 frontiersin.org Fu et al. Fu et al. 10.3389/fpubh.2023.1090436 important. At present, all countries have an open attitude toward COVID-19, and this practically confirms that strict prevention and control policies are no longer appropriate for implementation. closely, and the spillover effects will become stronger. This research provides a new perspective and method of interregional linkages for future research on public health security. Our study observes a relationship between the COVID-19 pandemic and the degree of economic prosperity. The aggravation of the pandemic will slow down economic growth. If we want to improve the economic climate, we must first improve people’s confidence in the degree of future economic prosperity, to focus on economic recovery in the post-pandemic era. Taking COVID-19 as an example, we must take a compromise between economic growth and public health security issues control. It will improve the future economic climate, control current public health security issues with a long-term perspective, and achieve long-term economic growth. Frontiers in Public Health frontiersin.org Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 7. Işık O, Tengilimoğlu D, Tosun N, Zekioğlu A, Tengilimoğlu O. Evaluating the factors (stress, anxiety and depression) affecting the mental health condition of nurses during the COVID-19 pandemic. Eval Health Prof. (2022) 45:86–96. doi: 10.1177/01632787211062660 Ethics statement Ethical review and approval was not required for the study on human participants in accordance with the local legislation and Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability statement Publicly available datasets were analyzed in this study. This data can be found here: https://github.com/owid/covid-19-data/tree/ master/public/data. Acknowledgments The authors are highly appreciative of the invaluable comments and advice from the editor and the reviewers. Funding This research was funded by the National Social Science Fund of China, grant number 20BTJ005. 9. Casaroto E, Generoso JR, Tofaneto BM, Bariani LM, Auler MA, Xavier N, et al. Hand hygiene performance in an intensive care unit before and during the COVID-19 pandemic. Am J Infect Control. (2022) 50:585–7. doi: 10.1016/J.AJIC.2022.01.018 5.3. Future research perspectives In terms of research methodology, the spatial Durbin model relies on the spatial matrix, and the study of other factors that affect the spatial matrix can make the spatial Durbin model more accurate. At the same time, more control variables should be introduced in future studies to improve the credibility of the model results. In terms of Frontiers in Public Health 11 frontiersin.org Fu et al. Fu et al. 10.3389/fpubh.2023.1090436 research content, the COVID-19 of the outbreak impact on all types of economies varies in degree. For the tourism, catering, and retail industries, COVID-19 deals a severe blow. The travel companies, hotels, airports, and train stations have taken a hit large due to a drastic decrease in travel demand. The restaurant companies are facing reduced patronage and operational difficulties due to the ongoing pandemic. The brick-and-mortar stores in the retail industry have seen a decrease in patronage and sales due to the ongoing pandemic. For the healthcare and online economies, the pandemic is both a hardship and an opportunity. During the pandemic, sales in these industries grow as people spent most of their time at home, leading to an increase in demand for home entertainment and digital products. Many gaming industries experience significant economic benefits from the pandemic situation. In addition, the healthcare industry also sees growth due to the ongoing pandemic. Therefore, in future research, we can analyze the economic development of different industries under the background of the COVID-19 pandemic. COVID-19 has significantly impacted the global unemployment rate. Once a country’s unemployment rate becomes too high, the society is likely to become unstable. To cope with the effects of COVID-19, the government must take measures, and the purpose of our study is to provide suggestions to address the negative impacts of the COVID-19 crisis. It can even extend the pandemic to global public health and security events, enabling scholars to find the commonalities of economic development under various public health and security events. General conclusions can be drawn in the face of global changes, and future public health and security events must be prepared in advance. institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. 6. Nurchis MC, Pascucci D, Sapienza M, Villani L, D’Ambrosio F, Castrini F, et al. 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https://openalex.org/W2106523371	https://almob.biomedcentral.com/counter/pdf/10.1186/1748-7188-1-2	English	null	Finding the region of pseudo-periodic tandem repeats in biological sequences	Algorithms for molecular biology	2,006	cc-by	6,005	Open Acc Research Finding the region of pseudo-periodic tandem repeats in biological sequences Xiaowen Liu* and Lusheng Wang* Open Access Address: Department of Computer Science, City University of Hong Kong, Kowloon, Hong Kong Email: Xiaowen Liu* - liuxw@cs.cityu.edu.hk; Lusheng Wang* - lwang@cs.cityu.edu.hk * Corresponding authors Received: 23 February 2006 Accepted: 28 February 2006 Received: 23 February 2006 Accepted: 28 February 2006 © 2006Liu and Wang; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Summary: The genomes of many species are dominated by short sequences repeated consecutively. It is estimated that over 10% of the human genome consists of tandemly repeated sequences. Finding repeated regions in long sequences is important in sequence analysis. We develop a software, LocRepeat, that finds regions of pseudo-periodic repeats in a long sequence. We use the definition of Li et al. [1] for the pseudo-periodic partition of a region and extend the algorithm that can select the repeated region from a given long sequence and give the pseudo-periodic partition of the region. Availability: LocRepeat is available at http://www.cs.cityu.edu.hk/~lwang/software/LocRepeat This tandem repeat consists of 35 approximate copies of the repeated pattern CCT. Background be partitioned as x = p1p2...pk and is mini- mized. Here d(p, pl) is the relative edit distance which is the edit distance, where L = (\|p\| + \|pl\|)/2 is the average length of the two strings p and pl. Note that, the normali- zation of the edit distance is important for finding repeated patterns since otherwise, one can give a partition in which each pattern has one letter and the edit distance is at most 1 (small). The problem in general is NP-hard [7]. When the repeated pattern p is assumed to be a sub- string of x, The problem can be solved in O(\|x\|4) time. Note that the second measure is more general than the first since it allows insertions and deletions. Both meas- ures in [7] and [6] use the bottleneck function that finds the repeated pattern p and assumes that each copy pi in the long string is close to the repeated pattern p, i.e., d(pi, p) ≤ δ and δ is minimized. However, in biological sequences, copies of the repeated patterns may change gradually so that some repeats in the region may have very little in max ( , ) l k l d p p =1 1 L × Measures for finding repeats Three measures can be used to give partitions of repeated regions. Background g Finding pseudo-periodic repeats (or tandem repeats) is an important task in biological sequence analysis [1-3]. The genomes of many species are dominated by short sequences repeated consecutively. It is estimated that over 10% of the human genome consists of tandemly repeated sequences. About 10–25% of all known proteins have some form of repeated structure ranging from simple homopolymers to multiple duplications of entire globu- lar domains. An instance (originally from Jaitly et al. [2]) of a human tandem repeat appears below (Gen- bank:10120313): Variation in the pseudo-periodic repeats demonstrates biologically important information. Sensitive tools for finding those regions containing pseudo-periodic repeats are required in practice. Repeats occur frequently in bio- logical sequences, but they may not be exact in many cases. If the repeats are exact, the problem can be easily solved from computation point of view. However, repeats are seldom exact in biological sequences. The errors in those repeats make it difficult to find regions of those repeats. Many measures and algorithms have been pro- posed. CCTCCTCCTCCACCTCCTCCTCCTCCTCCTCCTCCTC- CGCCTTCTCATCCTCCTCCACTT Landau and Schmidt [4] studied the problem of finding the two consecutive copies in a sequence of length n such that the edit distance (a match costs 0 and a mismatch/ indel costs 1) between the two copies is at most k. The run- ning time of the algorithm is O(kn log k log(n/kL)). CCTCCTCCTCCTCCTCCTCCCCTTCTCATCCTCCTC- CTCTTCATCTACCC CCTCCTCCTCCTCCTCCTCCCCTTCTCATCCTCCTC- CTCTTCATCTACCC Page 1 of 8 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:2 http://www.almob.org/content/1/1/2 Table 1: Pseudo periodic repeats of 1SRY (matrix:blosum62, gap penalty: -4) Unit Pseudo-periodic unit Length Similarity with previous unit 1 MVDLKRLR 8 2 QEPEVFHR 8 -5 3 AIREKGVA 8 -10 4 LDLEALLA 8 -1 5 LDREVQEL 8 7 6 KKRLQEVQ 8 6 7 TERNQVA 7 6 8 KRVPKAP 7 -4 9 PEEKEAL 7 -2 10 IARGKAL 7 3 11 GEEAKRL 7 3 12 EEALRE 6 10 13 KEARLE 6 12 14 ALLLQV 6 -6 15 PLPP 4 -8 Table 1: Pseudo periodic repeats of 1SRY (matrix:blosum62, gap penalty: -4) Schmidt [5] used weighted grid digraphs for finding all non-overlapping pairs of substrings (not necessarily con- secutive) with the highest scores in a given string of length n. The algorithm can handle any score scheme. It requires O(n2 log n) time and Θ(n2) space. In both [4] and [5], only two copies of the pattern are considered. Quasiperiodicty Wan and Song proposed a measure in which all the repeated copies (except the last one) have the same length [6]. For this measure, a linear time and space algorithm was given [6]. Pseudo-periodic Partition common. For example, it is well-known that the N-termi- nal non-globular region of Thermus thermophilus seryl- tRNA synthetase (PDB:1SRY) [1,8] has weak 7-residue repeats. See Table 1. The similarity score between two con- secutive patterns is calculated using Blosum62 matrix and the gap penalty is set to be -4. The repeated patterns grad- ually changes from the 4-th unit LDLEALLA to the 13-th unit KEARLE. The average similarity score for the nine pairs of consecutive patterns is 4.56. But the similarity score between the 4-th unit and the 8-th unit is -11. In this case, the algorithms based on the bottleneck function may fail to find the multiple repeats. Let s = a1a2...an be a string of length n. A partition π(s) = {s1, s2, ..., sk} of s is a set of substrings of s such that s = s1s2...sk (si's are also called repeats). When s is clear, we use π instead of π(s). Π(s) denotes the set of all partitions of s. Let si and si+1 be two strings. The similarity measure µ(si, si+1) between si and si+1 is the maximum alignment value for si and si+1. For any two letters (possibly spaces) x and y, µ(x, y) is the similarity score between the two letters. For exam- ple, one can use the following score scheme I: a match costs 1, a mismatch costs -1, and an insertion or deletion costs -1. Here we choose to use maximization version since for protein sequences, there are popular similarity matrices, e.g., PAM matrix. Definitions h In this section, we first give a definition of the pseudo-peri- odic partition of a string that is originally proposed in Li et al. [1]. We then give a definition of the local pseudo-periodic partition of a string. The alignment for π(sA) Figure 1 The alignment for π(sA). The alignment for π(sA) Figure 1 The alignment for π(sA). Approximate periods Sim et al. [7] introduced a notion of approximation peri- ods (approximate period) using edit distance or relative edit distance. The problem in general is defined as follows: given a string x, find a repeated pattern p such that x can Table 2: Pseudo periodic repeats of LPXA_ECOLI (matrix:blosum62, gap penalty: -4) Unit Pseudo-periodic unit Length Similarity with previous unit 1 MIDKSAFVHPTAIVEEGA 18 2 SIGANAHIGPFCIVGPHV 18 14 3 EIGEGTVLKSHVVVNGHT 18 16 4 KIGRDNEIYQFASI 14 -7 5 GEVNQDLKYAGEPTR 15 -3 6 VEIGDRNRIRESVTI 15 2 7 HRGTVQGGGL 10 -14 8 TKVGSDNLLMINAHIAHD 18 -24 9 CTVGNRCILANNATLAGH 18 20 10 VSVDDFAIIGGMTAVHQF 18 4 11 CIIGAHVMVGGCSGV 15 4 Table 2: Pseudo periodic repeats of LPXA_ECOLI (matrix:blosum62, gap penalty: -4) Page 2 of 8 (page number not for citation purposes) http://www.almob.org/content/1/1/2 Algorithms for Molecular Biology 2006, 1:2 pseudo-periodic partition and develop an algorithm that solves the local pseudo-periodic problem in O(n2) time, where n is the length of the input sequence s. Pseudo-periodic repeats Li et al. [1] gave the first measure that allows gradual changes of patterns and changes of pattern lengths in the region. The repeats they defined are called the pseudo-peri- odic repeats. Given a repeated region (a string) x and a par- tition X = s1s2...sk, the pseudo periodic score is Page 3 o (page number not for citation purpos The alignment for π(sB) Figure 2 The alignment for π(sB). The alignment for π(sB) Figure 2 The alignment for π(sB). If there is a gap at c 2 Dynamic programming algorithm and local alignment for s = CAGAGT Figure 3 Dynamic programming algorithm and local alignment for s = CAGAGT. For a partition π of s and a fixed i, 1 ≤ i ≤ \|se\|, let V(π, c, i) be the value of the self-alignment such that s1 is mapped to spaces with score each, sj is optimally aligned with sj+1 for j = 1, 2, ..., k -2, pre(se, \|se\| - i) is scored according to the above two cases, and the last i letters in se are mapped to spaces with score each. We have c 2 c 2 V c i s s pre s s i s c s i j j e e k j k ( , , ) ( , ) ( ( , ), ) ( ) π µ µ = + − + × + + = − ∑ 1 1 1 2 2 if i s s s x s i c s i i s k j j k j k k ≥ + × − + × + <    + = − ∑ ; ( , ) ( , ) ( ) ( ) . µ µ 1 1 1 1 2 ∆ if     In V(π, c, i), the alignment between s1s2...sk-2pre(se, \|se\| - i) and s2s3...sk is called the middle alignment. The value of the self-alignment of π is defined as . V c V c i i se ( , ) max ( , , ) π π = =1 In V(π, c, i), the alignment between s1s2...sk-2pre(se, \|se\| - i) and s2s3...sk is called the middle alignment. The value of the self-alignment of π is defined as . V c V c i i se ( , ) max ( , , ) π π = =1 Dynamic p CAGAGT Figure 3 For example, let sB = s1s2s3 and π(sB) = {s1, s2, s3}, where s1 = aaaa, s2 = aaat and s3 = aaa. We use score scheme I and c = -1. The valid value of i is 1, 2, ..., 7 since \|s2s3\| = 7. For i Dynamic programming algorithm and local alignment for s = CAGAGT Figure 3 Dynamic programming algorithm and local alignment for s = CAGAGT. The alignment for π(sB) Figure 2 The alignment for π(sB). Denote by se the suffix sk-1sk of s. We can designate that only the last i letters in se are mapped to spaces with score each, for 1 ≤ i ≤ \|se\|. Now let us consider the remain part pre(se, \|se\| - i) of se. There are two cases: (1) If i ≥ \|sk\|, pre(se, \|se\| - i) is a prefix of sk-1 and is optimally aligned with sk. (2) If i < \|sk\|, pre(se, \|se\| - i) contains sk-1 and a prefix of sk. In this case, sk-1 is optimally aligned with sk and the letters in the prefix of sk are scored as µ(x, ∆) each. c 2 Let s = s1s2...sk be the string and π(s) = {s1, s2, ..., sk}. \|s\| denotes the length of the string. pre(s, i) is the length-i pre- fix of s and suf(s, i) is the length-i suffix of s. Note that the gap at the right end of the self-alignment of s only appears in the segments sk-1 and sk. Denote by se the suffix sk-1sk of s. We can designate that only the last i letters in se are mapped to spaces with score each, for 1 ≤ i ≤ \|se\|. Now c 2 Let us consider the following example. sA = s1s2s3s4s5 and π(sA) = {s1, s2, s3, s4, s5}, where s1 = aaa, s2 = aat, s3 = att, s4 = ttt and s5 = tta. The self-alignment of this partition is show in Figure 1. The value of the self-alignment is , where \|s1\| and \|s5\| are the penalty scores for the two segments s1 and s5 aligned to spaces. µ( , ) ( ) s s c s s i i i + = + + ∑ 1 1 4 1 5 2 c 2 c 2 let us consider the remain part pre(se, \|se\| - i) of se. There are two cases: (1) If i ≥ \|sk\|, pre(se, \|se\| - i) is a prefix of sk-1 and is optimally aligned with sk. (2) If i < \|sk\|, pre(se, \|se\| - i) contains sk-1 and a prefix of sk. In this case, sk-1 is optimally aligned with sk and the letters in the prefix of sk are scored as µ(x, ∆) each. Note that the score for insertion and deletion would be different from the granularity factor . The alignment for π(sB) Figure 2 The alignment for π(sB). d s s c s s i i k i k ( , ) ( ), + = − + × + ∑ 1 1 1 1 where d(·) is the edit distance, \|si\| is the length of si, and c is a factor that control the penalty of the two ends of the partition. Li et al. [1] gave a O(\|x\|2) algorithm to compute an optimal partition of a given repeated region x. It was shown that the pseudo-periodic score can accurately give partitions for tandem repeated regions, where the repeated patterns are weakly similar. Example: The example is from [1]. The sequence of the LbH domain of members of the LpxA family consists of the imperfect tandem repetition of hexapeptide units [9- 11]. These imperfect tandem repeats (partitions) have been accurately detected by the algorithm using the pseudo periodic score [1]. (See Table 2). In sequence analysis, we may have a long sequence s and only a substring t (or a few substrings) of s contains the consecutive repeats. The problem here is to find out the substring t and give an optimal pseudo-periodic partition. We call this problem the local pseudo-periodic problem. In this paper, we define the maximization version of the Algorithms for Molecular Biology 2006, 1:2 http://www.almob.org/content/1/1/2 Table 3: Results for the speed test of LocRepeat Length 2000 4000 6000 8000 10000 DNA 0.5s 2.2s 4.8s 7.0s 10.8s PROTEIN 1.1s 4.3s 10.0s 17.7s 26.9s Table 3: Results for the speed test of LocRepeat Table 3: Results for the speed test of LocRepeat the right end of the alignment between s4 and s5, there is ambiguity in the calculation of the self-alignment value. Therefore, we need a more precise definition for the value of the self-alignment corresponding to a partition. Let c be a negative constant. We call the granularity fac- tor. Let ∆ denotes a space in an alignment. In this paper, we assume that > µ(x, ∆) for any letter x in the given sequence. c 2 c 2 Let s = s1s2...sk be the string and π(s) = {s1, s2, ..., sk}. \|s\| denotes the length of the string. pre(s, i) is the length-i pre- fix of s and suf(s, i) is the length-i suffix of s. Note that the gap at the right end of the self-alignment of s only appears in the segments sk-1 and sk. The alignment for π(sB) Figure 2 The alignment for π(sB). Page 4 of 8 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:2 http://www.almob.org/content/1/1/2 Table 4: Local optimal pseudo-periodic region for PRNP Unit Position Length Unit 1 215–238 24 ggtggtggctgggggcagcctcat 2 239–262 24 ggtggtggctgggggcagcctcat 3 263–286 24 ggtggtggctgggggcagccccat 4 287–310 24 ggtggtggctggggacagcctcat 5 311–327 17 ggtggtggctggggtca Table 4: Local optimal pseudo-periodic region for PRNP = 5 ≥ \|s3\|, pre(s2, 2) is optimally aligned with s3, s1 and suf(s2s3, 5) is scored as (Figure 2(a)). So V(π(sB), c, 5) = µ(s1, s2) + µ(pre(s2, 2), s3) + × (\|s1\| + 5) = . For i = 2 < \|s3\|, s2 is optimally aligned with s3, pre(s3, 1) is scored as µ(x, ∆) and suf(s3, 2) is scored as (Figure 2(b)). In this c 2 c 2 −3 2 c 2 c Let Π(s) be the set of all possible partitions of s. Bc(s) = maxπ∈Π(s) V(π, c) is the optimal V(·) value of partitions. A partition πq = {s1, s2, ..., sk} of s is called the pseudo-peri- odic partition of s if Bc(s) = V(πq, c). In Li et al. [1], it was demonstrated that the numerical measure Bc(s) (in fact, the minimization version) is sensitive for partitioning s into repeats that allow the gradual changes of patterns and changes of pattern lengths. < \|s3\|, s2 is optimally aligned with s3, pre(s3, 1) is scored as µ(x, ∆) and suf(s3, 2) is scored as (Figure 2(b)). In this c 2 In practice, we are given a long string s. We want to find a region (substring) t of s that contains pseudo-periodic repeats. Once the region t is found, we want to get the pseudo-periodic partition of t. The mathematical problem is defined as follows: case, V(π(sB), c, 2) = µ(s1, s2) + µ(s2, s3) + µ(x, ∆) × 1 + c 2 Local pseudo-periodic partition problem Given a string s, find a substring t (the local optimal pseudo-periodic region) of s such that B t B t c t Sub s c ( ) max ( ), ( ) = ′ ′∈ B t B t c t Sub s c ( ) max ( ), ( ) = ′ ′∈ where Sub(s) is the subset of all substrings of s. The algorithm Let s be the given string. We want to find a substring t of s with the maximum self-alignment value . Let s[1, j] be the substring of s that consists of the first j letters. Informally, we use w(i, j) to denote the maximum self-alignment value of a suffix tj of s[1, j] such that there are i letters at the right end of the self-alignment of tj that are aligned with spaces and scored as . Note that, the right end of the self-alignment of tj could contain more than i spaces. However, only the last i spaces are scores as each and the rest of them are scored as the score for µ(x, ∆). c 2 c 2 LocRepeat interface Figure 4 LocRepeat interface. Let Tj be the set of all suffixes of s[1, j]. For a substring t of s and an integer i, Π(t, i) = {π(t)\|π(t) ∈ Π(t) and \|tk-1tk\| ≥ i}, where tk-1, tk are the last two repeats in π(t). We define LocRepeat Figure 4 p g LocRepeat interface. p g LocRepeat interface. w i j V t c i t T t t i j ( , ) max ( ( ), , ) & ( ) ( , ) = ( ) ∈ ∈∏ π π 1 ( ) 1 ( ) 1 Page 5 of 8 (page number not for citation purposes) (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:2 http://www.almob.org/content/1/1/2 Table 5: Local optimal pseudo-periodic region for LGR6 Unit Position Length Unit 1 30–53 24 LSMNNLTELQPGLFHHLRFLEELR 2 54–77 24 LSGNHLSHIPGQAFSGLYSLKILM 3 78–101 24 LQNNQLGGIPAEALWELPSLQSL D 4 102–124 23 LNYNKLQEFPVAIRTLGRLQELG 5 125–148 24 FHNNNIKAIPEKAFMGNPLLQTI H 6 149–172 24 FYDNPIQFVGRSAFQYLPKLHTLS 7 173–195 23 LNGAMDIQEFPDLKGTTSLEILT 8 196–219 24 LTRAGIRLLPSGMCQQLPRLRVLE 9 220–241 22 LSHNQIEELPSLHRCQKLEEIG 10 242–265 24 LQHNRIWEIGADTFSQLSSLQAL D 11 266–289 24 LSWNAIRSIHPEAFSTLHSLVKLD 12 290–311 22 LTDNQLTTLPLAGLGGLMHLKL Table 5: Local optimal pseudo-periodic region for LGR6 to be the maximum V(·, c, i) value of all the partitions in Π(t, i), where t is a substring in Tj. To compute w(i, j) using dynamic programming method, we first consider the boundary values of w(i, j). We set w(0, j) = -∞ since we do not allow suf(tk-1tk, i) to be empty. Note that, by defini- tion, i ≤ j. The algorithm w i j c i w i j s j i s j w i j s j ( , ) max , ( , ) ( [ ], [ ]), ( , ) ( , [ ]) = ⋅ − + − − − + 1 1 1 µ µ ∆ + + + − −          ( ) c w i j s j i c 2 1 2 3 , ( , ) ( [ ], ) . µ ∆ ( ) 3 Lemma 1 For a sequence s of length n, w(j, j) = c·j for 1 ≤ j ≤ n. Proof. Consider the partition π(t) = {t1, t2, ..., tk} such that t ∈ Tj, π(t) ∈ Π(t, i) and V(π(t), c, i) = w(i, j). We analyze the value of V(π(t), c, i) based on different cases. Proof. For a partition π(t) = {t1, t2, ..., tk} satisfying t ∈ Tj and π(t) ∈ Π(t, j), from the definition of Π(t, j), \|tk-1tk\| ≥ j. Since t is a suffix of s[1, j] and \|t\| ≥ \|tk-1tk\| ≥ j, we have t = s[1, j] and 1 ≤ k ≤ 2. Consider the self alignment of π(t) such that the last j letters in t are mapped to spaces with score . Two cases arise. Case 1: k = 1 and t = t1 = s[1, j]. In this case, the middle alignment is empty. Thus, V(π(t), c, j) = × (\|t1\| + j) = c·j. Case 2: k = 2 and t = t1t2 = s[1, j]. In this case, the middle alignment is the alignment between \|t2\| spaces and t2. By the assumption that > µ(∆, x), V(π(t), c, j) = \|t2\| × µ(∆, x) + × (\|t1\| + j) <c·j. c 2 c 2 c 2 c 2 case 1. π(t) has only one repeat. We have \|t\| = \|t1\| = i and the middle alignment is empty. Therefore V(π(t), c, i) = × (\|t1\| + i) = c·i. c 2 × (\|t1\| + i) = c·i. case 2. π(t) has k ≥ 2 repeats. In this case, the middle align- ment is not empty since it contains tk. The algorithm c 2 c 2 c 2 c 2 c 2 the end of the self alignment of π'(t'), there are only i - 1 letters mapped to spaces with score , and there is one more letter in the self alignment of π(t) mapped to spaces with score . Thus, V(π(t), c, i) = w(i - 1, j - 1) + µ(∆, s[j]) + . For sub-case (c), π(t) ∈ Π(t, i + 1). We can impose that the alignment of the letter s[j - i] and the space is scored as , not µ(s[j - i], ∆). From V(π(t), c, i + 1) = w(i + 1, j), we have V(π(t), c, i) = w(i + 1, j) + µ(s[j - i], ∆) - . c 2 c 2 c 2 c 2 c 2 alignment, it is easy to get the partition of π(t) = {AG, AG}. The space complexity required is also O(n2) if we are not careful. However, we can release the space whenever they are no longer useful. Thus, only two columns, are required for the computation. For each of the two columns, we use two arrays: one array stores the value of w(i, j) and the other array stores the starting position of the subsequence t that maximizes w(i, j). Therefore, the space complexity is O(n) for computing all w(i, j)'s. After w(i, j)'s are com- puted, we know the substring t that leads to the optimal w value. Therefore, we can reconstruct the alignment for t in + . For sub-case (c), π(t) ∈ Π(t, i + 1). We can impose that the alignment of the letter s[j - i] and the space is scored as , not µ(s[j - i], ∆). From V(π(t), c, i + 1) = w(i + 1, j), we have V(π(t), c, i) = w(i + 1, j) + µ(s[j - i], ∆) - . c 2 c 2 c 2 time and space, where n1 is the length of t, the repeated region. If n1 is still big, we can use the standard technique in [12] to reduce the space to O(n1) by dou- bling the computation time for reconstructing the align- ment of t. O n ( ) 1 2 Based on Theorem 2, a dynamic programming algorithm is designed. Let n be the length of the input sequence s. Implementation We have implemented the algorithm using Visual C++ 6.0 and Windows XP. The software is called LocRepeat and has a user-friendly GUI (See Figure 4). Another version without GUI that works for Linux is also available. The following example illustrates the algorithm. Let s = CAGAGT. We set c = -2 and use the following score scheme: a match costs 10, a mismatch costs -10, and an insertion or a deletion costs -10. The table constructed by using the dynamic programming algorithm is shown in Figure 3. The table is constructed in from the top to the bottom. For every row in the table, the w(i, j)'s are com- puted from left to right. From the table, it is easy to see that the maximum value of w(i, j) is w(2, 5) = 16. From the maximum value w(2, 5) = 16, we know that the local opti- mal pseudo-periodic region t is a suffix of s[1, 5] = CAGAG and there are 2 letters aligned with spaces and scored as c 2 LocRepeat accepts three kinds of sequence: DNA, RNA and Protein. The user can either click 'New Data' button to directly input the sequence at the input area, or click 'Input Data from File' button to input a sequence from a file. The user can click 'Set Parameters' button to set parameters, such as granularity factor, gap penalty and similarity score matrix. After the sequence is input and the parameters are set, click the 'Start' button to begin the computation. for j = 1 to n do for j = 1 to n do In practice, a sequence may contain more than one repeated region. To find all the repeated regions, we can select the best k values of w(i, j)'s for some pre-defined value k. Each backtracking gives a repeated region. Another way to set a threshold for the value of w(i, j) and select all w(i, j)'s with value greater than the threshold. for i = j downto 1 do for i = j downto 1 do for i = j downto 1 do compute w(i, j) based on Theorem 2. Obviously, the time complexity is O(n2), where n is the length of the whole string. A standard backtracking proc- ess allows us to find the local optimal pseudo-periodic region t. Experiment results We have done experiments to test the speed and sensitiv- ity of the software. at the right end of the self alignment of the local optimal pseudo-periodic region t. From w(2, 5), we can backtrack w(2, 5) → w(2, 4) → w(2, 3) and stop at w(2, 3) since w(2,3) gets its value from c·i indicating the first segment of the partition of t ends at 3-th letter in s and the length of the segment is 2. Thus, we get t = AGAG. From the self The algorithm The last column in the middle alignment has three configurations: (a) the last column contains two letters s[j - i] and s[j], (b) the last col- umn contains a space and the letter s[j], (c) the last col- umn contains the letter s[j - i] and a space. For sub-case (a), if we take away the last letter s[j] from the self align- ment of π(t), we can get a self alignment of π'(t'), where t' ∈ Tj-1, π'(t') ∈ Π(t', i) and V(π'(t'), c, i) = w(i, j - 1). By com- paring the two self alignment, we have V(π(t , c, i = V(π'(t'), c, i) + µ(s[j - i], s[j]) = w(i, j - 1) + µ(s[j - i], s[j]). For sub-case (b), if we take away the last letter s[j] and space aligned with s[j] from the self alignment of π(t), we can get a self alignment of π'(t'), where t' ∈ Tj-1, π'(t') ∈ Π(t', i - 1) and V(π'(t'), c, i) = w(i - 1, j - 1). Notice that in From the above analysis, the initial and boundary values are From the above analysis, the initial and boundary values are w(0, j) = -∞, w(j, j) = c·j (2) w(0, j) = -∞, w(j, j) = c·j (2) Theorem 2 For a sequence s of length n and 2 ≤ j ≤ n, 1 ≤ i <j, Theorem 2 For a sequence s of length n and 2 ≤ j ≤ n, 1 ≤ i <j, Page 6 of 8 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:2 http://www.almob.org/content/1/1/2 the end of the self alignment of π'(t'), there are only i - 1 letters mapped to spaces with score , and there is one more letter in the self alignment of π(t) mapped to spaces with score . Thus, V(π(t), c, i) = w(i - 1, j - 1) + µ(∆, s[j]) + . For sub-case (c), π(t) ∈ Π(t, i + 1). We can impose that the alignment of the letter s[j - i] and the space is scored as , not µ(s[j - i], ∆). From V(π(t), c, i + 1) = w(i + 1, j), we have V(π(t), c, i) = w(i + 1, j) + µ(s[j - i], ∆) - . The algorithm We compute w(i, j) in the order shown below: or j = 1 to n do for i = j downto 1 do compute w(i, j) based on Theorem 2. Sensitivity testing using real data We applied LocRepeat to the DNA sequence gene PRNP which contains tandem repeats (GenBank:M13667). The length of the sequence is 2420. We find the local optimal pseudo-periodic region [215,327], that contains 5 pseudo-periodic units (Table 4). The pseudo-periodic region misses the first several sites of the tandem repeats, but the region and the partitions show the tandem repeats correctly. We also applied LocRepeat to the protein sequence LGR6 (Swiss-Prot: Q9HBX8). The length of the sequence is 828. We use PAM120 as the similarity score matrix and find the local units (Table 5). In conclusion, the algorithm presented in this paper offers the possibility to find regions of pseudo-periodic repeats in a long sequence. Speed Testing The time complexity of the algorithm is O(n2). To test the speed in practice, we use arbitrarily generated DNA and protein sequences. We ran our software on a PC with Pen- tium 4 3.4G CPU and 1GB memory, the result is shown in Table 3. We can see that for long DNA and protein Page 7 of 8 (page number not for citation purposes) Page 7 of 8 (page number not for citation purposes) Algorithms for Molecular Biology 2006, 1:2 http://www.almob.org/content/1/1/2 sequences, our software can get the result in short time. For example, if the length of the sequence is 10000, it takes about 10.8 seconds and 26.9 seconds for DNA sequences and protein sequences, respectively. In some real applications, the length of sequences could be much longer than 10000. In this case, one can cut the long sequence into several short pieces and find out the repeated regions for each piece. If a region covers two pieces, then we can re-cut that segment to get that region. 10. Vuorio R, Harkonen T, Tolvanen M, Vaara M: The novel hexapep- tide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria. FEBS Let- ter 1994, 337:289-292. Acknowledgements We thank the referees for their helpful suggestions. This work is fully sup- ported by a grant from the Research Grants Council of the Hong Kong Spe- cial Administrative Region, China [Project No. CityU 1070/02E]. 12. Myers EW, Miller W: Optimal alignments in linear space. Com- puter Applications in the Biosciences 1988, 4:11-17. 11. Raetz CRH, Roderick SL: A left-handed parallel beta helix in the structure of UDP-N -acetylglucosamine acyltransferase. Sci- ence 1995, 270:997-1000. References 1. Li L, Jin R, Kok P, Wan H: Pseudo-periodic partitions of biologi- cal sequences. Bioinformatics 2004, 20:295-306. 1. Li L, Jin R, Kok P, Wan H: Pseudo-periodic partitions of biologi- cal sequences. Bioinformatics 2004, 20:295-306. q f 2. Jaitly D, Kearney PE, Lin G, Ma B: Methods for reconstructing the history of tandem repeats and their application to the human genome. Journal of Computer and System Sciences 2002, 65(3):494-507. ( ) 3. Tang M, Waterman M, Yooseph S: Zinc finger gene clusters and tandem gene duplication. In Proceedings of the Fifth Annual Interna- tional Conference on Computational Biology, April 22–25 2001 Montreal, Canada, ACM; 2001:297-304. 4. Landau GM, Schimidt JP: An algorithm for approximate tandem repeats. In Proceedings of the Fourth Annual Symposium on Combinato- rial Pattern Matching New York, LNCS 684, Springer-Verlag; 1993:120-133. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 5. Schmidt JP: All highest scoring paths in weighted grid graphs and their application to finding all repeats in strings. SIAM Journal on Computing 1998, 27:972-992. 5. Schmidt JP: All highest scoring paths in weighted grid graphs and their application to finding all repeats in strings. SIAM Journal on Computing 1998, 27:972-992. J p g 6. Wan H, Song E: Quasiperiodic biosequences and modulo inci- dence matrices. Proceedings of the 16th International Parallel and Dis- tributed Processing Symposium 2002:280. g y p 7. References Sim JS, Iliopoulos CS, Park K, Smyth WF: Approximate period of strings. Theoretical Computer Science 2001, 262:557-568. Å g p , 8. Biou V, Yaremchuk A, Tykalo M, Cusack MS: The 2.9 Å crystal structure of T. thermophilus seryl-tRNA synthetase com- plexed with tRNA(Ser). Science 1994, 263:1404-1410. p ( ) 9. Vaara M: Eight bacterial proteins, including UDP-N-acetylglu- cosamine acyltransferase (LpxA) and three other trans- ferases of Escherichia coli, consist of a six-residue periodicity theme. FEMS Microbiology Letter 1992, 76:249-254. 10. Vuorio R, Harkonen T, Tolvanen M, Vaara M: The novel hexapep- tide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria. FEBS Let- ter 1994, 337:289-292. 11. Raetz CRH, Roderick SL: A left-handed parallel beta helix in the structure of UDP-N -acetylglucosamine acyltransferase. Sci- ence 1995, 270:997-1000. 12. Myers EW, Miller W: Optimal alignments in linear space. Com- puter Applications in the Biosciences 1988, 4:11-17.
https://openalex.org/W2147045282	https://healthandjusticejournal.biomedcentral.com/track/pdf/10.1186/2194-7899-1-2	English	null	Prior criminal charges and outcomes among individuals initiating office-based buprenorphine treatment	Health & justice	2,013	cc-by	5,595	RESEARCH ARTICLE Open Access Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Harris et al. Health and Justice 2013, 1:2 Abstract Background: There is little data on the impact of prior criminal activity on the treatment of opioid dependence with office-based buprenorphine. The goal of this study was to investigate the association between prior criminal charges and treatment outcomes in a cohort of patients initiating buprenorphine treatment in a primary care practice. Methods: This was a retrospective study of 252 consecutive patients with opioid dependence who were given at least one prescription for buprenorphine in a primary care practice. A public database was used to collect data on criminal charges prior to enrollment. For every month after initiation of treatment, patients who remained in treatment were classified as “opioid-positive” or “opioid-negative” based on urine drug test results, patient report and clinician assessment. The primary outcomes of interest were treatment retention at one year and achieving ≥6 “opioid negative” months. Results: Most (80%) of the subjects had at least one prior criminal charge. Those with prior criminal charges were more likely to have Medicare or Medicaid insurance, to be unemployed, to use cocaine and to have injected drugs. In the year after initiation of buprenorphine treatment, these subjects had significantly less opioid-negative months than those without criminal charges (5.1 months vs. 6.4 months; p = 0.030), and were less likely to have ≥6 opioid-negative months (43.2% vs. 58.4%; p = 0.048). While there was no difference in treatment retention at one-year for those who had any prior history (55.4% vs. 52.0%; p = 0.854), having recent charges (in the previous two years) was associated with poorer treatment retention and drug outcomes. On the other hand, having only distant charges (more than two years prior to treatment initiation) was not associated with poorer outcomes. Using multivariate analysis, recent criminal charges was the only factor significantly associated with treatment retention at one year and achieving ≥6 opioid-negative months. Conclusions: Subjects with recent criminal charges had poorer treatment retention and opioid-abstinence outcomes after initiating office-based buprenorphine treatment. These individuals may benefit from more intensive treatment than is typically offered in a primary care setting. Keywords: Opioid-related disorders; Crime; Buprenorphine; Primary health care Prior criminal charges and outcomes among individuals initiating office-based buprenorphine treatment Elizabeth E Harris1, Janet S Jacapraro2 and Darius A Rastegar3* © Harris et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2013 © Harris et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproductio in any medium, provided the original work is properly cited. 2013 * Correspondence: drasteg1@jhmi.edu 3Johns Hopkins School of Medicine, Division of Chemical Dependence, Johns Hopkins Bayview Medical Center, 5200 Eastern Avenue, Baltimore, MD 21224, USA * Correspondence: drasteg1@jhmi.edu Background 2004, 53% of state and 45% of federal prisoners met cri- teria for drug dependence or abuse, and 13.1% and 9.2%, respectively, regularly abused opioids, but only 0.3% of state and 0.4% of federal prisoners were managed with opioid replacement therapy (Mumola and Karberg 2006). Given the prevalence of substance use in the criminal justice population and the current lack of access to pharmacologic treatment for opioid dependence, as well as the high rates of recidivism associated with substance use disorders (Hankansson and Berglund, 2012), there is a tremendous need for effective treatments. Incarceration Substance dependence is a common medical problem and is associated with criminal activity (Ball et al., 1983). While there is wide variability of rates of substance use disorders in prison populations, the rates are consistently much higher than the general population (Fazal et al., 2006). In a survey by Bureau of the Justice System in SA ull list of author information is available at the end of the article Full list of author information is available at the end of the article Data collection As part of a previously performed study (Soeffing et al., 2009), a database of all patients who received at least one prescription for buprenorphine during this period of time had already been created. Data were collected retrospectively from the patient medical records. Demo- graphic information recorded included age, gender, type of insurance and employment status. Substance abuse history collected included substances used and history of injection drug use. Also recorded were relevant comorbidities (hepatitis C, HIV, chronic pain and chronic psychiatric illness). While a criminal history may be associated with poorer treatment retention in methadone maintenance therapy (Magura et al., 1998, Villafranca et al., 2006, Kelly et al., 2011, Cox et al., 2012), there is little data on the treatment outcomes associated with office-based buprenorphine; one recent study found that a history of incarceration was not associated with poorer outcomes in office-based buprenorphine treatment (Wang et al., 2010). The time period after receiving the first prescription was divided into twelve monthly blocks for the purposes of this analysis. Patients were considered to be in treat- ment for each block in which they were prescribed buprenorphine at any point. Patients were considered to be in treatment during the time period covered by their prescriptions and were considered to be out of treatment if there was a gap in the period covered by their prescriptions. For those who dropped out of treatment, they were considered to be in treatment for the duration of time covered by their most recent prescription. There was no fixed protocol for collection of urine drug tests and patients sometimes did not have urine collected when they admitted to recent nonprescribed opioid use or when they had sequential negative urine tests, so for each month in which the patient was receiving treatment, patients were classified as “opioid-positive” or “opioid-negative”. Patients were classified as “opioid-positive” if any of their urine drug tests during that month were positive for opioids (other than those prescribed), they reported using other non-prescribed opioids, or a urine drug test was not collected and their most recent test was positive (practitioners would sometimes not collect urine if the patient reported recent substance use or patients may have left without leaving a urine specimen). Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Page 2 of 6 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Page 2 of 6 Data collection Patients were classified as “opioid-negative” if all urine drug tests col- lected during that month were negative for opioids (other than those prescribed) or if the provider decided not to test the urine and their most recent test was negative. The goal of this study is to evaluate the impact of having prior criminal charges on treatment outcomes among patients initiating office-based buprenorphine treatment. We hypothesized that subjects with prior charges would not do as well as those without prior criminal charges and that this association would be strongest for those with recent charges. Subjects may be an opportunity to initiate treatment for opioid dependence and improve rates of recidivism (Chandler et al., 2009). For prisoners with opioid dependence, metha- done maintenance treatment after release from prison has been associated with a reduction in mortality and re-incarceration (Dolan et al., 2005, Larney et al. 2011). Studies suggest that buprenorphine is also a feasible treatment post-release (Springer et al., 2010), treatment retention is associated with a reduction in opiate use and crime (Garcia et al., 2007). and treatment can im- prove other medical conditions (Springer et al., 2012). Buprenorphine maintenance therapy may be more accept- able than methadone to criminal offenders released from prison (Magura et al., 2009). The study included all patients who had been given at least one prescription for sublingual formulation of buprenorphine from August 2003 to September 1, 2007. The study included all patients who had been given at least one prescription for sublingual formulation of buprenorphine from August 2003 to September 1, 2007. Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Statistical analysis Th d The data was analyzed to see if having prior criminal charges was associated with retention and treatment outcomes. We also divided the cohort with prior charges into those with “recent charges” (in the two years prior to the initial visit) and those with only “distant charges” (more than two years prior to the initial visit), as well as those with more than or less than the median number of prior charges. Specific outcomes were retention in treatment, defined as being in treatment 12 months after the initial prescription (including those who may have dropped out temporarily during that year), “opioid- negative” months, and successful treatment, defined as achieving ≥6 “opioid-negative” months in a 12-month period (not necessarily consecutive months). Table 1 Characteristics of subjects with and without prior criminal charges Characteristic Any prior No prior p value criminal charges criminal charges (N = 202) (N = 50) Mean Age (SD) 40.2 (9.2) 37.9 (10.8) 0.132 Sex Male 112 (55.4%) 30 (60.0%) 0.561 Female 90 (44.6%) 20 (40.0%) Insurance Commercial 71 (35.1%) 33 (66.0%) <0.001 Medicaid 78 (38.6%) 11 (22.0%) 0.028 Medicare 47 (23.3%) 4 (8.0%) 0.016 None 6 (3.0%) 2 (4.0%) 0.710 Employment Status Employed 75 (37.1%) 38 (76.0%) <0.001 Unemployed 66 (32.7%) 8 (16.0%) 0.020 Disabled 61 (30.2%) 4 (8.0%) 0.001 Abused Substances Heroin 176 (87.1%) 33 (66.0%) <0.001 Opioid Rx 47 (23.3%) 25 (50.0%) <0.001 Cocaine 116 (57.4%) 18 (36.0%) 0.007 Alcohol 32 (15.8%) 10 (20.0%) 0.480 Benzodiazepines 18 (8.9%) 5 (10.0%) 0.811 IDU 128 (63.4%) 24 (48.0%) 0.047 Co-morbidities HIV 29 (14.4%) 7 (14.0%) 0.949 HCV 112 (55.4%) 13 (26.0%) <0.001 Psychiatric 103 (51.0%) 22 (44.0%) 0.376 Chronic Pain 35 (17.3%) 11 (22.0%) 0.444 O i id R i ti i id IDU i j ti d HIV h Table 1 Characteristics of subjects with and without prior criminal charges Bivariate analysis was used to compare demographic factors and outcomes among subjects with and without prior criminal charges. Chi-square tests were used to analyze categorical variables and t tests for continuous variables. P values less than 0.05 were considered statisti- cally significant. Multivariate logistic regression was used to evaluate factors associated with being in treatment at 12 months and achieving ≥6 “opioid-negative” months. Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Page 3 of 6 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Page 3 of 6 the state of Maryland since 1991. The database provides the defendant name, city and state, date of birth, trial date, charge, and case disposition. We searched this database by each patient’s name and birthdate, and recorded whether the subject had ever had criminal charges prior to initiating treatment. We tabulated the total number of cases during their lifetime and in the two years prior to the date of the first prescription for buprenorphine. We used “cases” as our unit of measure- ment rather than “charges” because subjects often had multiple individual charges associated with each case. We defined “recent charges” as those within two years of initiation of treatment and “distant charges” as those more than two years prior to initiation of treatment. Prior criminal charges and demographic factors Prior criminal charges and demographic factors The number of subjects with criminal charges prior to initiating treatment was 202 (80.2%). Among the 202 subjects with prior criminal history, the number of prior cases ranged from 1 to 43 and the median number was 6; 108 (42.9%) subjects had charges in the two years prior to initiation of treatment. Table 1 provides demographic and outcome data on subjects with and without prior criminal charges. A number of characteristics were as- sociated with having prior criminal charges. Subjects with prior charges were more likely to be unemployed or disabled; they were more also more likely to be uninsured or publicly-insured. Those who reported a history of heroin and cocaine use were more likely to have had prior criminal charges, while those who reported prescription drug abuse were less likely to have had prior criminal charges. Hepatitis C virus (HCV) infection and Statistical analysis Th d Factors that were associated with these outcomes at a p value ≤0.1 were included in the multivariate model; when there was a high collinearity between factors (correl- ation coefficient >0.4), only one factor was entered into the model. Analysis was performed using PASW software (version 18). This study was approved by the Johns Hopkins Institutional Review Board. Opioid Rx: prescription opioids, IDU: injection drug use, HIV: human immunodeficiency syndrome, HCV: hepatitis C antibody positive. Opioid Rx: prescription opioids, IDU: injection drug use, HIV: human immunodeficiency syndrome, HCV: hepatitis C antibody positive. Methods Setting The Comprehensive Care Practice is a primary care clinic on the Johns Hopkins Bayview Medical Center campus, which is staffed by 5 internists, a nurse practitioner, and 3 residents who share a panel of patients. The practitioners provide general primary care, with a focus of serving patients with HIV infection and/or substance use disorders. Visits for opioid dependence occured as routine primary care visits. There was no uniform protocol or dosing regi- men, with buprenorphine doses that ranged from 2 to 32 mg, with most patients on 8–16 mg daily. Patients were typically given a prescription for a one-week supply of buprenorphine initially and induction occurred at home or in the office; follow-up occurred weekly to monthly, based on the provider’s discretion; patients were usually seen more frequently early in treatment or when there was continued substance use. Treatment was continued or discontinued based on the provider’s discretion. The practice did not provide any additional onsite psychosocial services and patients were referred to community resources. A more detailed description of this clinic’s treatment prac- tices can be found in Soeffing et al., 2009. We utilized the Maryland Judiciary Case Search website (http://casesearch.courts.state.md.us/inquiry/inquiry-index. jsp) to collect data on prior criminal charges. “Criminal charges” are defined in this study as a written accusation charging that an individual named has been accused of committing an act or omitted to do something that is punishable by law. We did not include traffic offenses. This database includes data on all criminal charges in Results h The study included 252 patients who had been given at least one prescription for sublingual formulation of buprenorphine from August 2003 to September 1, 2007. Three subjects from the original cohort were not included because they could not be identified due to gaps in record-keeping. Page 4 of 6 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Page 4 of 6 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 injection drug use (IDU) were also associated with prior criminal charges. significantly better than those with recent charges and their outcomes were not significantly different from those with no prior charges. Association between criminal charges and treatment outcomes Bivariate and multivariate analysis of factors associated with remaining in treatment at 12 months and achieving ≥6 opioid negative months is shown in Table 3. Subjects age 40 or less and those with recent charges were less likely to remain in treatment, while those with prior co- caine use were more likely. In multivariate analysis, co- caine use was significantly associated with remaining in treatment at 12 months, while having recent charges was associated with a lower likelihood of remaining in treatment. For the outcome of achieving ≥6 opioid nega- tive months, subjects with recent charges were less likely to achieve this outcome, as were those who used heroin. On multivariate analysis, both heroin use and recent charges were significantly associated with a lower prob- ability of achieving ≥6 opioid negative months. Recent criminal charges was the only factor that was signifi- cantly associated with both outcomes. The association between prior criminal charges and the main treatment outcomes is summarized in table 2. Over- all, subjects with prior criminal charges were as likely to be in treatment at 12 months as those without (55.4% vs. 52.0%; p = 0.854). However, subjects with criminal charges were less likely to achieve ≥6 opioid negative months (43.1% vs. 60.0%; p = 0.032) and had significantly fewer opioid-negative months than those without prior criminal charges (mean 5.1 months vs. 6.7 months; p = 0.028). There appeared to be a dose–response relationship be- tween number of prior cases and outcomes; subjects with six or fewer prior cases (at or below the median) did not have significantly poorer outcomes than those with no prior history, while those with seven or more prior cases had significantly fewer opioid-negative months and were less likely to achieve ≥6 opioid negative months. Both groups were as likely to remain in treatment as those with no prior charges. “Recent charges” are those that occurred in the 2 years prior to treatment initiation, “distant charges” occurred more than 2 years prior to treatment iniation. ap value for difference with subjects with no prior charges. bp value for difference with subjects with no recent charges. cp value for difference with subjects with recent charges. “Recent charges” are those that occurred in the 2 years prior to treatment initiation, “distant charges” occurred more than 2 years pri ap value for difference with subjects with no prior charges e that occurred in the 2 years prior to treatment initiation, “distant charges” occurred more than 2 years prior to treatment iniation. h subjects with no prior charges. Conclusions In a previous analysis, we found that prior charges, particularly recent charges, were highly correlated with subsequent charges (Harris et al., 2012); it is possible that some of the poorer outcomes could have simply been due to incarceration. charges may be a better predictive factor, particularly when the charges have occurred recently. Our findings may be relevant to the development of treatment strategies for recently-incarcerated persons who are opioid dependent. While we did not look at incarceration per se, our findings suggest that recent criminal activity is associated with poorer outcomes. These individuals may benefit from more intensive treatment and support than can be offered in a typical primary care practice. While many may express a preference for office-based buprenorphine treatment, it is possible that they would do better in a program (for example, a traditional methadone maintenance program) that offers closer supervision and more psychosocial support than is typically available in a primary care setting. On the other hand, previous studies suggest that buprenophine main- tenance is effective for recently incarcerated individuals (Garcia et al., Magura et al., 2009) and may be preferred over methadone by incarcerated individuals (Awgu et al., 2010). It should be noted that in our study, many of those with recent criminal activity did stay in treatment (over 50% at 12 months) and many were abstinent from opioids. Our finding that having any prior criminal charges was not associated with poorer treatment retention is consist- ent with a recent study of a cohort of subjects receiving office-based buprenorphine treatment, which found that a history of incarceration was not associated with treatment retention (Wang et al., 2010). However, Wang et al. re- ported that a history of incarceration was likewise not associated with increased drug use after initiation of office-based treatment, while we found poorer treatment outcomes among those with a history of criminal charges, particularly those with recent charges. The disparity in our findings may be due to one or more of the differences between the two studies. Unlike our study, Wang et al. excluded individuals who were dependent on other sub- stances, or if they did not complete the 2-week induction period, thus perhaps selecting for patients who would tend to do better in treatment. Additionally, the subjects in their study received more intensive counseling and moni- toring than that provided in our primary care setting. Conclusions Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Page 5 of 6 Page 5 of 6 Table 3 Bivariate and multivariable analysis of factors associated with being in treatment in 12 months and achieving ≥6 opioid negative months Variable In treatment at 12 months ≥6 opioid negative months OR (CI) AOR (CI) OR (CI) AOR (CI) Age ≤40 0.58 (0.36 - 0.97) 0.61 (0.37 – 1.02) 0.96 (0.59 – 1.58) p = 0.036 p = 0.062 p = 0.960 Female 0.69 (0.42 - 1.14) 1.00 (0.60 – 1.64) p = 0.148 p = 0.985 Employed 1.04 (0.63 - 1.72) 1.43 (0.87 – 2.36) p = 0.864 p = 0.160 Heroin abuse 0.97 (0.50 – 1.88) 0.40 (0.20 – 0.79) 0.43 (0.21 – 0.86) p = 0.924 p = 0.008 p = 0.016 Cocaine abuse 1.80 (1.09 – 2.98) 1.75 (1.05 – 2.92) 1.20 (0.73 – 1.97) p = 0.022 p = 0.032 p = 0.481 Recent Charges 0.53 (0.32 – 0.88) 0.54 (0.32 – 0.90) 0.51 (0.31 – 0.85) 0.54 (0.32 – 0.91) (past 2 years) p = 0.015 p = 0.019 p = 0.010 p = 0.021 Statistically significant associations are shown in bold. The following factors were excluded due to high correlation with another factor (indicated in parenthesis): commercial insurance (employed), prescription opioid abuse (heroin use), injection drug use (heroin use), any prior charges and ≥7 prior cases (recent charges). Table 3 Bivariate and multivariable analysis of factors associated with being in treatment in 12 m achieving ≥6 opioid negative months nd multivariable analysis of factors associated with being in treatment in 12 months and d negative months ociations are shown in bold. The following factors were excluded due to high correlation with another factor (indicated in parenthesis) mployed), prescription opioid abuse (heroin use), injection drug use (heroin use), any prior charges and ≥7 prior cases (recent charges). Statistically significant associations are shown in bold. The following factors were excluded due to high correlation with another facto commercial insurance (employed), prescription opioid abuse (heroin use), injection drug use (heroin use), any prior charges and ≥7 p For example, Cox and colleagues (2011) reported that having six or more lifetime arrests was associated with an approximately 3-fold increase in risk of voluntary or involuntary discharge from a methadone program. Those awaiting charges, trial or sentence likewise had a higher risk of voluntary or involuntary discharge. Conclusions In summary, we found that 80% of patients with opioid dependence initiating buprenorphine maintenance therapy in this primary care setting had prior criminal charges. While a history of criminal charges did not negatively affect treatment retention, those with prior charges had significantly fewer opioid-negative months and were less likely to have ≥6 opioid-negative months. Those with more prior criminal cases or recent charges did worse than those with fewer or more distant charges. As shown in Table 2, 108 (42.9%) had criminal charges within two years of initiating treatment. Recent charges appeared to have the most significant association with treatment outcomes. When compared to those who did not have recent charges (including those with no prior charges), subjects with charges in the two years prior to initiation of treatment were significantly less likely to remain in treatment or to achieve ≥6 opioid negative months and had significantly fewer opioid-negative months. On the other hand, those with only distant charges did Our findings are consistent with a number of studies conducted with subjects on methadone maintenance. Table 2 Relationship between prior criminal charges, treatment retention and drug treatment outcomes Prior charges In treatment at 12 Mean number of opioid- ≥6 opioid-negative month months (%) negative months (%) None 27 (52.0%) 6.68 30 (60.0%) (n = 50) Any charges 112 (55.4%) 5.12 87 (43.1%) (n = 202) p = 0.854a p = 0.028a p = 0.032a 1-6 cases 57 (54.3%) 5.36 47 (44.8%) (n = 105 ) p = 0.973a p = 0.103a p = 0.076a ≥7 cases 55 (56.7%) 4.87 40 (41.2%) (n = 97) p = 0.755a p = 0.015a p = 0.031a Recent charges 58 (53.7) 4.50 40 (37.0%) (n = 108) p = 0.014b p = 0.004b p = 0.010b Distant charges 62 (66.0%) 5.84 47 (50.0%) (n = 94) p =0.160a/0.005c p = 0.252a/0.031c p = 0.252a/0.063c “Recent charges” are those that occurred in the 2 years prior to treatment initiation, “distant charges” occurred more than 2 years prior to treatment iniation. ap value for difference with subjects with no prior charges. bp value for difference with subjects with no recent charges. cp value for difference with subjects with recent charges. Relationship between prior criminal charges, treatment retention and drug treatment outcomes Table 2 Relationship between prior criminal charges, treatment retention and drug t Harris et al. Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Page 6 of 6 Page 6 of 6 measures. A final limitation is that we only looked at criminal charges recorded in Maryland, and could not include charges in other states. On the other hand, un- like many other studies, we used an “intention-to-treat” model and included everyone who had received at least one prescription for buprenorphine, not just those who remained in treatment; so our findings are probably more representative of the outcomes of all who enter treatment, not just those who remain in treatment. Also, we used an objective measure, rather than patient report. Garcia, CA, Correa, GC, Viver, AD, Kinlock, TW, Gordon, MS, Avila, CA, Reyes, IC, & Schwartz, RP. (2007). Buprenorphine-naloxone treatment for pre-release opioid-dependent inmates in Puerto Rico. Journal of Addiction Medicine, 1(3), 126–32. Hankansson, A, & Berglund, M. (2012). Risk factors for criminal recidivism – a prospective follow-up study in prisoners with substance abuse. BMC Psychiatry, 12(111). doi:10.1186/1471-244×-12-111. Harris, EE, Jacapraro, JS, & Rastegar, DA. (2012). Criminal charges prior to and after initiation of office-based Buprenorphine treatment. Substance Abuse, Treatment, Prevention, and Policy, 7(10). doi:10.1186/1747-597X-7-10. Kelly, SM, O’Grady, KE, Mitchell, SG, Brown, BS, & Schwartz, RP. (2011). Predictors of methadone treatment retention from a multi-site study: a survival analysis. Drug and Alcohol Dependence, 117, 170–175. of methadone treatment retention from a multi-site study: a survival analysis. Drug and Alcohol Dependence, 117, 170–175. Larney, S, Toson, B, Burns, L, & Dolan, K. (2011). Effect of prison-based opioid substitution treatment and post-release retention in treatment on risk of re-incarceration. Addiction, 107, 372–80. In summary, our study found that subjects with crim- inal charges tended to do worse than those with no prior charges, but the difference appears to be limited to those with recent criminal charges. Subjects with distant prior charges (none in the two years before initiation of treatment) did as well as those with no prior charges. Although some of the individuals with recent criminal charges appeared to do well with office-based bupre- norphine treatment, many may benefit from more inten- sive treatment and monitoring than can be provided in a typical primary care setting. Further research is needed to determine the most effective treatment approach for these individuals. Magura, S, Nwakeze, PC, & Demsky, SY. (1998). Pre- and in-treatment predictors of retention in methadone treatment using survival analysis. Addiction, 93(1), 51–60. Author details 1 1Albert Einstein College of Medicine/Montefiore Medical Center, 1621 Eastchester Road Bronx, NY 10461, USA. 2Health Care for the Homeless, 421 Fallsway, Baltimore, MD 21202, USA. 3Johns Hopkins School of Medicine, Division of Chemical Dependence, Johns Hopkins Bayview Medical Center, 5200 Eastern Avenue, Baltimore, MD 21224, USA. Division of Chemical Dependence, Johns Hopkins Bayview Medical Center, 5200 Eastern Avenue, Baltimore, MD 21224, USA. Competing interests Competing interests The authors declare that they have no competing interests. Villafranca, SW, McKellar, JD, Trafton, JA, & Humphreys, K. (2006). Predictors of retention in methadone programs: a signal detection analysis. Drug and Alcohol Dependence, 83, 218–224. Authors’ contributions EEH ti i t d i th d EEH participated in the design of the study, data collection and the drafting of the manuscript. JSM participated in the design of the study and data collection. DAR participated in the design of the study, data analysis and drafting of the manuscript. All authors have read and approved the final manuscript. Wang, EA, Moore, BA, Sullivan, LE, & Fiellin, DA. (2010). Effect of incarceration history on outcomes of primary care office-based Buprenorphine/naloxone Journal of General Internal Medicine, 25(7), 670–4. Cite this article as: Harris et al.: Prior criminal charges and outcomes among individuals initiating office-based buprenorphine treatment. Health and Justice 10.1186/2194-7899-1-2 2013, 1:2 Cite this article as: Harris et al.: Prior criminal charges and outcomes among individuals initiating office-based buprenorphine treatment. Health and Justice 10.1186/2194-7899-1-2 2013, 1:2 Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 Magura, S, Lee, JD, Hershberger, J, Joseph, H, Marsch, L, Shropshire, C, & Rosenblum, A. (2009). Buprenorphine and methadone maintenance in jail and post-release: a randomized clinical trial. Drug and Alcohol Dependence, 99(1–3), 222–230. Mumola, CJ, & Karberg, JC. (2006). Drug use and dependence, state and federal prisoners, 2004. Washington, DC: U.S. Department of Justice, Office of Justice Programs, Bureau of Justice Statistics. Soeffing, JM, Martin, LD, Fingerhood, MI, Jasinski, DR, Rastegar, DA. (2009). Buprenorphine maintenance treatment in a primary care setting: outcomes at 1 year. Journal of Substance Abuse Treatment, 37(4), 426–430. Soeffing, JM, Martin, LD, Fingerhood, MI, Jasinski, DR, Rastegar, DA. (2009). Buprenorphine maintenance treatment in a primary care setting: outcomes at 1 year. Journal of Substance Abuse Treatment, 37(4), 426–430. Springer, SA, Chen, S, & Altice, FL. (2010). Improved HIV and substance abuse treatment outcomes for released HIV-infected prisoners: the impact of Buprenorphine treatment. Journal of Urban Health, 87(4), 592–602. Springer, SA, Qiu, J, Saber-Tehrani, AS, & Altice, FL. (2012). Retention on Buprenorphine is associated with high levels of maximal viral suppression among HIV-infected opioid dependent releases prisoners. PLoS ONE, Springer, SA, Chen, S, & Altice, FL. (2010). Improved HIV and substance abuse treatment outcomes for released HIV-infected prisoners: the impact of Buprenorphine treatment. Journal of Urban Health, 87(4), 592–602. Springer, SA, Qiu, J, Saber-Tehrani, AS, & Altice, FL. (2012). Retention on Buprenorphine is associated with high levels of maximal viral suppression among HIV-infected opioid dependent releases prisoners. PLoS ONE, 7(5), e38335. Conclusions Moreover, many of their subjects who reported never being incarcerated had a criminal history and they did not distinguish between recent incarceration and incarceration in the distant past; our findings suggest that criminal A major limitation of our study is that we looked only at criminal charges; not all criminal activity leads to criminal charges and not all charges are necessarily indicative of criminal activity (i.e., some may have been wrongly charged). On the other hand, criminal charges are an objective measure and likely correlate with criminal activity. Another limitation is that we looked at the out- comes of a cohort treated at a single practice in Baltimore; this may not reflect the outcomes of subjects treated at other sites or in other localities. We also relied on chart review and fairly infrequent drug testing for our outcome Harris et al. Health and Justice 2013, 1:2 http://www.healthandjusticejournal.com/content/1/1/2 References Awgu, E, Magura, S, & Rosenblum, A. (2010). Heroin-dependent inmates’ experiences with buprenorphine or methadone maintenance. Journal of Psychoactive Drugs, 42(3), 339–346. Awgu, E, Magura, S, & Rosenblum, A. (2010). Heroin-dependent inmates’ experiences with buprenorphine or methadone maintenance. Journal of Psychoactive Drugs, 42(3), 339–346. Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com experiences with buprenorphine or methadone maintenance. Journal of Psychoactive Drugs, 42(3), 339–346. Ball, JC, Shaffer, JW, & Nurco, DN. (1983). The day-to-day criminality of heroin addicts in Baltimore-a study in the continuity of offence rates. Drug and Alcohol Dependence, 12(2), 119–142. Chandler, RK, Fletcher, BW, & Volkow, ND. (2009). Treating drug abuse and addiction in the criminal justice system: improving public health and safety. Journal of the American Medical Association, 301(2), 183–190. Cox, J, Allard, R, Maurais, E, Haley, N, & Small, C. (2013). Predictors of methadone program non-retention for opioid analgesic dependent patients. Journal of Substance Abuse Treatment, 44(1), 52–60. Dolan, KA, Shearer, J, White, B, Zhou, J, Kaldor, J, & Wodak, AD. (2005). Four-year follow-up of imprisoned male heroin users and methadone treatment: mortality, re-incarceration and hepatitis C infection. Addiction, 100, 820–28. Fazal, S, Bains, P, & Doll, H. (2006). Substance abuse and dependence in prisoners: a systematic review. Addiction, 101, 181–91. mortality, re-incarceration and hepatitis C infection. Addiction, 100, 820–28. Fazal, S, Bains, P, & Doll, H. (2006). Substance abuse and dependence in prisoners: a systematic review. Addiction, 101, 181–91. Cox, J, Allard, R, Maurais, E, Haley, N, & Small, C. (2013). Predictors of methadone program non-retention for opioid analgesic dependent patients. Journal of Substance Abuse Treatment, 44(1), 52–60. Submit your manuscript to a journal and beneﬁ t from: Ball, JC, Shaffer, JW, & Nurco, DN. (1983). The day-to-day criminality of heroin addicts in Baltimore-a study in the continuity of offence rates. Drug and Alcohol Dependence, 12(2), 119–142. Ball, JC, Shaffer, JW, & Nurco, DN. (1983). The day-to-day criminality of heroin addicts in Baltimore-a study in the continuity of offence rates. Drug and Alcohol Dependence, 12(2), 119–142. Chandler, RK, Fletcher, BW, & Volkow, ND. (2009). Treating drug abuse and addiction in the criminal justice system: improving public health and safety. Journal of the American Medical Association, 301(2), 183–190.
W2170594794.txt	https://koedoe.co.za/index.php/koedoe/article/download/296/281	en		Anuran fauna of the Lesotho Highlands in the Khatse Dam catchment area and Jorodane River region	Koedoe	1,996	cc-by	0
https://openalex.org/W4308318536	https://link.springer.com/content/pdf/10.1007/s11098-022-01894-3.pdf	English	null	Acting for reasons and the metaphysics of time	Philosophical studies	2,022	cc-by	12,530	Philosophical Studies https://doi.org/10.1007/s11098-022-01894-3 Philosophical Studies https://doi.org/10.1007/s11098-022-01894-3 https://doi.org/10.1007/s11098-022-01894-3 Acting for reasons and the metaphysics of time Olley Pearson1 Accepted: 30 September 2022 © The Author(s) 2022 1 Philosophy, School of History and Heritage, University of Lincoln, Brayford Pool, LN6 7TS Lincoln, Lincolnshire, United Kingdom Olley Pearson opearson@lincoln.ac.uk Abstract This paper concerns acting for reasons and how this can inform debates about the metaphysics of time. Storrs-Fox (2021) has argued against the A-theory of time on the grounds that it cannot adequately account for the explanation of actions. Storrs- Fox assumes that explanation is forever. He argues that this is incompatible with the A-theory because the reasons people act for are the explanantia of their actions, though according to the A-theory these reasons, that is facts, often do not obtain forever and therefore nor do the explanations. I will argue that many particular explanatory relations are not forever because reasons for actions must often fade as a person cannot now have a reason to do something at an earlier time. Generally, people cannot affect the past and therefore cannot have rational obligations to do so. Therefore, the A-theory does not face the problem Storrs-Fox suggests. In fact, the A-theory says what ought to be said and accounts for the wider phenomena of responding to reasons better than the B-theory. It is often the case that an agent acts rationally only if that agent acts on the basis of a present tensed belief. This implies that present tensed beliefs capture facts that no tenseless beliefs do, because the ra tionality of an action is determined by the reasons an agent acts for, not the beliefs through which the agent is aware of these. However, the B-theory, unlike the A- theory, denies there are any facts thus uniquely captured by present tensed beliefs. Keywords Reasons · Time · A-theory · B-theory In a recent paper Storrs-Fox (2021) has argued against the A-theory of time on the grounds that it cannot adequately account for the explanation of actions. Storrs-Fox’s argument makes use of the idea that explanation is forever. He argues that this is 1 3 1 3 O. Pearson incompatible with the A-theory because the reasons people act for are the explanantia of their actions, though according to the A-theory, these reasons, that is facts, often do not obtain forever and therefore nor do the explanations. I will argue that particular explanatory relations clearly are not forever because reasons for actions must often fade as a person cannot now have a reason to do something at an earlier time. Gener ally, people cannot affect the past and therefore cannot have rational obligations to do so. Abstract This means that anyone who, on the basis of an instance of acting for a reason, infers that at later times that reason is still an explanans is making an inference that involves two gaps: because that particular explanatory relation of acting for a reason does not occur at those later times and because that particular reason does not occur at those later times. This shows that the A-theorist can coherently refuse to accept that inference and hence deny that explanation is forever, thus avoiding Storrs-Fox’s problem. In fact, the A-theory says what ought to be said and arguably accounts for the wider phenomena of action explanation better than the B-theory.i In the first section of this paper I will outline Storrs-Fox’s argument against the A-theory which focuses on explaining an action of Anna’s. In Sects. 2 and 3 I will highlight two particular explanatory relations that arise in Anna’s case and show that neither supports Storrs-Fox’s claim that explanation by Anna’s reason is forever. In Sect. 4 I will respond to a semantic concern Storrs-Fox raises arguing it is more of a problem for the B-theory than A-theory. In Sect. 5 I will point out that there is one sense in which explanation is forever, explanations at one time give rise to different explanations at later times, though this does not mean reasons/explanantia must exist forever and so is not problem for the A-theory. Finally, in Sect. 6, I will outline an argument for thinking that the A-theory is in fact better placed than the B-theory to account for the explanation of actions as it is better placed to account for the rational requirement that agents have tensed beliefs when acting. It is often the case that an agent acts rationally only if that agent acts on the basis of a present tensed belief. This implies that present tensed beliefs capture facts that no tenseless beliefs do, because the rationality of an action is determined solely by the reasons, that is facts, an agent acts for, not the beliefs through which the agent is aware of these. However, the B-theory, unlike the A-theory, denies there are any facts thus uniquely captured by present tensed beliefs. 1 As Storrs-Fox notes, not all proponents in the debate adopt this terminology or presentation of the position, but, thus described, A-theorists include e.g. Cameron (2015) and Deasy (2015), and B-theoris include e.g. Mellor (1998) and Maclaurin and Dyke (2002). 2 Storrs-Fox does not actually speak of days or dates, merely a time and a later time. My argument on requires a time and a later time, but I include the days for explanatory ease. 1 Storrs-Fox’s argument Storrs-Fox gives his argument as an argument against the A-theory of time, which he is understanding as a theory committed to two points, factual-futurism and proposi tional temporalism. According to factual futurism there are facts that obtain but that at some point will not obtain (temporary facts). According to propositional tempo ralism, there are propositions that are true but not always true (temporary proposi tions). For example, it is currently sunny so according to this A-theory the fact that it is sunny currently obtains and the proposition that it is sunny is currently true, but later, when the clouds roll in, the fact will no longer obtain and the proposition will no longer be true. 1 3 Acting for reasons and the metaphysics of time The A-theory is contrasted with the B-theory which involves the denial of both factual futurism and propositional temporalism. According to this B-theory, there will not be a temporary fact of it being sunny now, though, given it is now noon on the 20th of May 2022, there will be a permanent fact of it being sunny at noon on the 20th of May 2022. Likewise, although there will not be the temporary proposition of it being sunny now, there will be the permanently true proposition that it is sunny at noon on the 20th of May 2022. According to this B-theory, our tensed utterances will express such unchanging facts and propositions, not, as one might initially think, temporary facts or propositions.1 Storrs-Fox gives his argument by considering an example of an action by Anna. The example is broadly as follows. On Monday2 it is raining and Anna, on her way to a meeting, hails a taxi. Her friend Bernard truthfully utters S1. S1, “Anna’s reason for hailing a taxi is that it is raining”. On Tuesday, it is no longer raining and Bernard, thinking back, utters S2 and subse quently S3 and S4. On Tuesday, it is no longer raining and Bernard, thinking back, utters S2 and subse quently S3 and S4. S2, “Anna’s reason for hailing the taxi was that it was raining”. S2, “Anna’s reason for hailing the taxi was that it was raining”. S3, “If Anna’s reason for hailing the taxi was that it was raining, then the fact that it was raining explains why she was hailing the taxi”. 1 As Storrs-Fox notes, not all proponents in the debate adopt this terminology or presentation of their position, but, thus described, A-theorists include e.g. Cameron (2015) and Deasy (2015), and B-theorists include e.g. Mellor (1998) and Maclaurin and Dyke (2002). 2 Storrs-Fox does not actually speak of days or dates, merely a time and a later time. My argument only requires a time and a later time, but I include the days for explanatory ease. 2 The motivation explanatory relation Storrs-Fox’s argument seems to commit the A-theory to a contradiction: R both does and does not obtain on Tuesday. It does not because it is a temporary fact and it does because on Tuesday it is an explanans in an explanatory relation that obtains on Tuesday. I suggest that the A-theory ought to respond to this argument by denying that R is an explanans that obtains on Tuesday. Crucially, R needn’t be an explanans that obtains on Tuesday, because S4 does not capture an explanation with R as explanans that obtains on Tuesday. Storrs-Fox’s argument takes reasons to be facts. In order to clarify this point and my reply, it is helpful to get a little more clear about reasons. Reasons are commonly distinguished into two kinds, normative reasons and motivating reasons. Normative reasons are reasons that promote or justify an action, they are considerations that stand in favour of acting. Motivating reasons are reasons that agents act for, they are reasons that motivate actions.3 To talk of two kinds of reasons here can be potentially misleading, insofar as it is not being suggested that normative reasons are different kinds of entities from motivating reasons. Rather, both kinds of reasons can be taken to be facts.4 The difference concerns the different roles that a fact can play. If a fact justifies an action, then it is a normative reason for that action. If someone becomes aware of that reason and hence becomes motivated by it to perform the action, then the fact is also a motivating reason for the action. Often normative reasons and moti vating reasons will coincide in this way. Though, the two can come apart, for exam ple, if a fact is a normative reason to perform an action, but an agent is not aware of it and so not motivated by it to act (the fact that my toast is burning is a normative reason for me to turn off the toaster, but unaware of that fact, I will not perform that act and the fact will not be a motivating reason). Now, Storrs-Fox’s argument is that R is a motivating reason for Anna to act to hail a taxi on Monday. Anna is motivated by R to hail a taxi on Monday. Plausibly, how ever, R is also a normative reason for Anna to hail a taxi on Monday. 1 Storrs-Fox’s argument The B-theory therefore does not face the same problem and is hence indirectly supported by Storrs-Fox’s argument. 1 Storrs-Fox’s argument S4, “Therefore, the fact that it was raining explains why Anna was hailing the taxi”. S3, “If Anna’s reason for hailing the taxi was that it was raining, then the fact that it was raining explains why she was hailing the taxi”. S3, If Anna s reason for hailing the taxi was that it was raining, then the fact that it was raining explains why she was hailing the taxi”. S4, “Therefore, the fact that it was raining explains why Anna was hailing the taxi”. S4, “Therefore, the fact that it was raining explains why Anna was hailing the taxi”. Storrs-Fox assumes, as I agree, that the reasons people act for, the reasons that moti vate them to act, are facts. In this instance, the reason/fact that Anna is motivated by is supposed to be picked out by the clause ‘it is raining’ in S1. According to the A-theory, this clause in S1 will pick out a temporary fact, a fact that no longer obtains on Tuesday. Storrs-Fox refers to this as fact R. Storrs-Fox suggests that on Tuesday Bernard ought to be able to refer to R using the clause ‘it was raining’ in S2 and hence in S3 and S4. He concludes from this, that if we accept S3 and S4 as true, then on Tuesday R explains why Anna was hailing the taxi. However, Storrs-Fox argues that explanation is factive, by which he means that if x explains y, then x and y must obtain. Therefore, R must obtain on Tuesday because it is then an explanans for the explanation picked out by S4, an explanation that obtains on Tuesday. Therefore, the A-theory must be wrong to say R does not obtain on Tuesday and, more generally, it must be wrong to take facts like R to be temporary because cases like Anna’s are common and various. On the other hand, the B-theory will not take a temporary fact like R to be Anna’s reason. Instead, it will take Anna’s reason to be a fact such as the fact that it rains on 1 3 O. Pearson Monday, 12th of July, 2021. Let us refer to this fact as RB. RB will always obtain according to the B-theory and so can be both Anna’s reason on Monday and the explanans on Tuesday. 3 Cf, e.g. Alvarez (2009) for this distinction between kinds of reasons. 4 It is currently widely accepted that normative reasons are facts. Some who adopt this position argue that motivating reasons belong to a different ontological kind, cf. e.g. Audi (2001). Though, cf. e.g. Dancy (2000) and Alvarez (2010) for arguments to the effect that both kinds of reasons are facts. 4 It is currently widely accepted that normative reasons are facts. Some who adopt this position argue th motivating reasons belong to a different ontological kind, cf. e.g. Audi (2001). Though, cf. e.g. Danc (2000) and Alvarez (2010) for arguments to the effect that both kinds of reasons are facts. 3 Cf, e.g. Alvarez (2009) for this distinction between kinds of reasons. 5 Note, Storrs-Fox specifically says he is concerned with explanation as a relation between entities lik facts, not as some sort of linguistic practice (2021: 4246). We might say his concern is with metaphysic as opposed to epistemological explanation. M seems to be such a metaphysically explanatory relation. 5 Note, Storrs-Fox specifically says he is concerned with explanation as a relation between entities like facts, not as some sort of linguistic practice (2021: 4246). We might say his concern is with metaphysical as opposed to epistemological explanation. M seems to be such a metaphysically explanatory relation. 6 Even if backwards time travel and effecting the past are possible, which is questionable (cf. e.g. Le Poidevin (2003: ch. 10)), Anna is supposed to be an everyday individual in an everyday circumstance and as such clearly it is not possible for her to effect the past. 7 This point is linked to the well known principle that ought implies can. For a defence of this point, cf. e.g. Streumer (2007). 8 There may be unusual circumstances in which an agent is motivated to try to do something it is impos sible for them to do, but Anna does not appear to be in such an unusual circumstance. 2 The motivation explanatory relation In fact, plausibly it is in part because R is a normative reason for the action that it motivates Anna to perform the action: it is a motivating reason in part because it is a normative reason. Given R motivates Anna’s action on Monday, it is plausible to say that R explains Anna’s action on Monday. Crucially, the explanatory relation here appears to be realised by the motivation relation: the relation of motivation is an explanatory rela 1 3 Acting for reasons and the metaphysics of time tion. (I will refer to this motivation relation as M. M is a relation that holds between the fact/reason R, and Anna’s action.)5 tion. (I will refer to this motivation relation as M. M is a relation that holds between the fact/reason R, and Anna’s action.)5 Turning to Tuesday, it is important to note something. Anna cannot perform the action talked of earlier in this section on Tuesday. That is, on Tuesday Anna cannot perform the action of hailing a taxi on Monday.6 Because Anna cannot perform that action on Tuesday, Anna also cannot on Tuesday have a normative reason to perform that action. One cannot have a normative reason to perform an action that it is impos sible for one to perform.7 Following from this, it is also implausible that Anna would be motivated on Tuesday to perform the action of hailing a taxi on Monday.8 Thus, on Tuesday Anna also does not have a motivating reason to perform the action. Thus, it is clear that whilst relation M occurs on Monday, it does not occur on Tuesday. This shows that there is an important sense in which explanation is not for ever: a particular explanatory relation can occur at one time and not others. Let us now return to Bernard and his utterances. M, our motivating/explanatory relation, holds on Monday. So, S1 is plausibly true. Bernard would also speak the truth if on Monday he uttered S1. S1, “That it is raining explains Anna’s hailing a taxi”. M does not occur on Tuesday. So, what should we make of S2? Well, S2 does not seem to say that M occurs on Tuesday. S2 is in the past tense, the implication of it is that at a time earlier than Tuesday Anna was motivated by a reason. S2 is hence true. M does not occur on Tuesday. 8 There may be unusual circumstances in which an agent is motivated to try to do something it is impo sible for them to do, but Anna does not appear to be in such an unusual circumstance. 6 Even if backwards time travel and effecting the past are possible, which is questionable (cf. e.g. L Poidevin (2003: ch. 10)), Anna is supposed to be an everyday individual in an everyday circumstance an as such clearly it is not possible for her to effect the past. p p p g p p , e.g. Streumer (2007). 8 There may be unusual circumstances in which an agent is motivated to try to do something it is impos sible for them to do, but Anna does not appear to be in such an unusual circumstance. 7 This point is linked to the well known principle that ought implies can. For a defence of this point, c e.g. Streumer (2007). 2 The motivation explanatory relation So, what should we make of S2? Well, S2 does not seem to say that M occurs on Tuesday. S2 is in the past tense, the implication of it is that at a time earlier than Tuesday Anna was motivated by a reason. S2 is hence true. However, S3 and S4 occur on Tuesday and speak of explanation in the present tense. So far, we have been given no explicit reason for taking S3 and S4 to be true: all we have established is that an explanatory relation, M, occurs on Monday and not Tuesday. Nevertheless, if Bernard had made two closely related utterances, S3* and S4* on Tuesday, these would have been true. S3, “If Anna’s reason for hailing the taxi was that it was raining, then the fact that it was raining explained why she was hailing the taxi”. S3, “If Anna’s reason for hailing the taxi was that it was raining, then the fact that it was raining explained why she was hailing the taxi”. S4, “Therefore, the fact that it was raining explained why Anna was hailing the taxi”. S4, “Therefore, the fact that it was raining explained why Anna was hailing the taxi”. S3* and S4* concern an explanatory relation that obtains on Monday, not Tues day. (They are utterances concerning an explanation that is in the past on Tuesday.) Because the truth of S3* and S4* does not commit us to there being an explanatory relation that obtains on Tuesday, it also does not commit us to the explanans of this S3* and S4* concern an explanatory relation that obtains on Monday, not Tues day. (They are utterances concerning an explanation that is in the past on Tuesday.) Because the truth of S3* and S4* does not commit us to there being an explanatory relation that obtains on Tuesday, it also does not commit us to the explanans of this 1 3 3 O. Pearson relation obtaining on Tuesday. Hence, we are also not committed by them to taking R to obtain on Tuesday, pace Storrs-Fox. A careful consideration of Anna’s acting for a reason gives us a ground for think ing R obtains on Monday but not on Tuesday, because the motivation relation it is a relatum of is an explanatory relation that occurs on Monday but not Tuesday. 2 The motivation explanatory relation Storrs-Fox does not anticipate this argument, however, he does anticipate the kind of response it supports, that is, that on Tuesday it is not true to say that R explains Anna’s action, only that it explained it (2021: 4249). Storrs-Fox provides a positive and a negative comment on such a response. The positive comment, as related to the specifics of my argument, is that the close link between S3* and S4* on the one hand, and S3 and S4 on the other, provides an error theory for why one might have mis takenly thought S3 and S4 accurately portray R as an explanans on Tuesday. I accept and would also wish to press this positive point. After giving the positive comment, Storrs-Fox goes on to give the negative one. “Of course, there are other truths in the vicinity—most notably, that if Anna’s reason for hailing the taxi was R, then R explained (at the time of action) why she was hailing the taxi. But the futurist also needs to say something about what later explains (at the time of (S3)’s utterance) why Anna was hailing the taxi. It would be very odd if there were no longer any explanation of her action, or if the explanation no longer includes facts about the rain.” (2021: 4249) Thus, Storrs-Fox is supposing that it would be odd if on Tuesday there were no longer an explanation of Anna’s action. The challenge to a reply like mine is that it is stuck with this oddness. However, I would deny that there is in fact anything odd here at all. It might be odd if Anna’s action was devoid of explanation, but that is not the case. The action obtained on Monday, the explanans/motivating reason obtained on Mon day, and the explanatory/motivating relation obtained on Monday. Anna’s action was explained. On Tuesday, however, the reason no longer obtains, the action no longer obtains, the motivation/explanatory relation no longer obtains.9 Rather, I have argued that it would be odd to suppose that the explanation does obtain on Tuesday. Anna can’t perform the action on Tuesday, and so cannot have a normative reason to perform it on Tuesday, and so does not have a motivation or motivational reason to perform it on Tuesday. Therefore, it would be odd to take there to be an explanation on Tuesday on the basis of a consideration of Anna’s motivation. 9 This response thus accepts that explanation is factive in an A-theoretic sense: if x previously explained y, then x previously obtained; if x currently explains y, then x currently obtains; and if x will subsequently explain y, then x will subsequently obtain. (Note, Storrs-Fox’s arguments to establish that explanation is factive, are compatible with factivity understood in this way.) 10 Note also in the third line of the quotation given two above and in the second and third lines of the quotation given one above Storrs-Fox speaks about explaining on Tuesday why Anna was hailing a taxi. Prima facie this is a comment about explaining a Tuesday fact about the past, not a Monday fact about the present. 2 The motivation explanatory relation If there is an oddity here, it is one that is faced by Storrs-Fox, not the A-theorist. Before closing this section we should note that Storrs-Fox does provide an argu ment to try to further motivate the idea that it would be odd to deny that Anna’s action is explained on Tuesday. He invites us to consider that Anna’s phone was stolen whilst she was hailing the taxi and that the thief later put the phone on his dresser where it remained on Tuesday. Storrs-Fox says the following: 1 3 Acting for reasons and the metaphysics of time “It seems that the fact that it was raining explains … why Anna’s phone is later on the thief’s dresser. And the fact that it was raining explains the phone’s posi tion because it explains the fact that Anna was hailing the taxi …It is hard to see how the fact that it was raining could explain (at the later time) why the phone is on the thief’s dresser, unless that fact explains (at the later time) why Anna was hailing the taxi.” (2021: 4249) In the following section I will account for this case in a way that makes it clear that it is not odd to deny that R is an explanans on Tuesday. In the following section I will account for this case in a way that makes it clear that it is not odd to deny that R is an explanans on Tuesday. 3 The historical explanatory relation 1 1 3 O. Pearson Crucially, H is not M and R* is not R. Therefore, the A-theorist can admit that there is an explanation that obtains on Tuesday. Though, the explanans of this expla nation is R, not R, so it is R, not R, that must in light of this obtain on Tuesday.f Storrs-Fox anticipates that an A-theorist may respond to his argument by offer ing an alternative fact to R as explanans on Tuesday. He suggests that this form of response would land the A-theory with an additional cost and so the A-theory is still cast in a negative light by the discussion. He brings out the problem in the following way. “Either R* was among Anna’s reasons for hailing the taxi, or it was not. Sup pose firstly that it was not. In that case, it appears that the explanation of Anna’s action at the later time of Bernard’s utterances is rather different in structure from the explanation at the time of her action. At the time of her action, the explanation includes her reason for acting (i.e., R). But at the later time, this reason drops out of the explanation and is replaced by a fact that was not her reason (i.e., R). The later explanation therefore seems to miss something.” (2021: 4249–4250) The reply I am outlining here is thus supposed to arrive at a problem, because R is not amongst Anna’s reasons for acting. However, I would push back against the idea that this is a problem, or at least a problem for the A-theory. The reply I am outlining here is thus supposed to arrive at a problem, because R* is not amongst Anna’s reasons for acting. However, I would push back against the idea that this is a problem, or at least a problem for the A-theory. Above I have argued that Anna cannot on Tuesday have a normative or motivating reason for her action. Therefore, any explanans that obtains on Tuesday must fail to be a reason for her action. This means that there must be a gap between the motivat ing reason obtaining on Monday, and the explanans obtaining on Tuesday. If this gap is a problem, it is a problem that must face all theories (including Storrs-Fox’s B-the ory) that take there to be an explanatory relation and explanans obtaining on Tuesday. 3 The historical explanatory relation Storrs-Fox’s motivation for speaking of explanation in Anna’s case seems to funda mentally lie with the fact that Anna’s action was motivated by a reason. He speaks of explanation because of the motivating role of this reason. However, we have just seen that a careful examination of this motivation in fact gives us no direct ground for thinking that Anna’s reason does any explaining at a later time. The reason and the explanatory relation that is the motivation relation do not occur on Tuesday. Nev ertheless, one might wonder if there are distinct explanatory relations that occur on Tuesday because the motivation relation occurred on Monday. I will now move to considering such a suggestion. Whilst pressing the idea that there is explanation obtaining on Tuesday, Storrs-Fox says the following. “At the time of Bernard’s utterances of (S2) and (S3), R explains why Anna was (earlier) hailing the taxi.” (2021: 4245).10 One point of note here, is that what is being proposed as explanandum is the fact, on Tuesday, that Anna was earlier hailing a taxi. This explanandum is distinct from Anna’s action. The explanan dum here concerns something about Tuesday, namely that Anna was hailing a taxi earlier than then. We might put a more clear A-theory twist on this by describing it as a fact of the form ‘Anna was, earlier than now [Tuesday], hailing a taxi’. I will refer to this explanandum as AP and I will refer to the explanandum of M as AN. Given AP concerns Tuesday I suggest that it is appropriate to think that the explan ans for this explanandum also concerns Tuesday. In particular, I propose the fact that it was raining earlier than then: Anna is hailing earlier than Tuesday because it is rain ing earlier than Tuesday. Or, to put an A-theory twist on this, I propose the explanans is a fact of the form ‘it was, earlier than now [Tuesday], raining’. This is the sort of temporary fact that an A-theorist might expect to be picked out by an utterance, on Tuesday, of ‘it was raining’. I will refer to this fact as R. This explanatory relation that occurs on Tuesday thus plausibly involves facts about the past, and hence I will refer to it as a historical relation, relation H. H is a historical relation that is an explanatory relation and that occurs on Tuesday. 3 3 The historical explanatory relation Further, Storrs-Fox is here simply assuming that on Tuesday Anna’s action is an explanandum, or a relatum of an explanatory relation that obtains on Tuesday. How ever, so far our examination of Anna’s case has not led us to this conclusion. Rather, we have uncovered two relations that are explanatory, M and H. M has Anna’s action, AN, as explanandum, but occurs on Monday not Tuesday. H occurs on Tuesday but does not have Anna’s action as explanandum (rather, the explanandum of H is the fact that Anna was earlier acting, AP). So, it is not the case that on Tuesday we shift to explaining Anna’s action in terms of something other than a reason for her action. We are now in a better position to deal with the case of Anna’s phone raised at the end of the last section. Storrs-Fox’s argument seems to be that Anna’s action must have an explanation on Tuesday as her phone’s being on the dresser has an explana tion on Tuesday. But, what explains her phone’s being on the dresser on Tuesday is not its being stolen then, but specifically its being stolen earlier than Tuesday, and relatedly, what explains it being stolen earlier than Tuesday is Anna’s hailing earlier than Tuesday, and what explains Anna’s hailing earlier than Tuesday is that it is rain ing earlier than Tuesday. We are again dealing with a form of historical explanation. In more A-theoretical terms, the explanantia on Tuesday are that it was earlier than now [Tuesday] stolen, because Anna was earlier than now [Tuesday] hailing, because 1 3 3 Acting for reasons and the metaphysics of time it was earlier than now [Tuesday] raining. That is, on Tuesday it is R, not R, that is an explanans and the explanation of the phone’s location involves H, not M. it was earlier than now [Tuesday] raining. That is, on Tuesday it is R, not R, that is an explanans and the explanation of the phone’s location involves H, not M. 4 Semantics Let us now address a semantic concern that Storrs-Fox raises for the A-theorist. Storrs-Fox says that the B-theorist can render the semantics of S2-4 in something like the following way. S-SIMPLE-B: S2: Anna’s reason for hailing the taxi was RB. S3: If Anna’s reason for hailing the taxi was RB, then RB explains why she was hailing the taxi. S2: Anna’s reason for hailing the taxi was RB. S3: If Anna’s reason for hailing the taxi was RB, then RB explains why she was hailing the taxi. S4: Therefore, RB explains why Anna was hailing the taxi. Storrs-Fox says the A-theorist, however, will have to render it in something like the following way. S-ALTERNATIVE: S2: Anna’s reason for hailing the taxi was R. S2: Anna’s reason for hailing the taxi was R. S3: If Anna’s reason for hailing the taxi was R, then R explains why she was hailing the taxi. S3: If Anna’s reason for hailing the taxi was R, then R* explains why she was hailing the taxi. S4: Therefore, R* explains why Anna was hailing the taxi. S4: Therefore, R* explains why Anna was hailing the taxi. Storrs-Fox argues that the semantics of S-SIMPLE-B is more simple than the seman tics of S-ALTERNATIVE. It is more simple because RB occurs throughout S-SIM PLE-B, making the inference straightforward, whilst there is a shift from R to R* in S-ALTERNATIVE. This is therefore a cost for the A-theorist that takes this line of response. I wish to respond to this by denying that the A-theorist is saddled with S-ALTER NATIVE. Rather, following the argument of Sect. 2, the A-theorist can adopt S-SIM PLE-A and deny that S3 and S4 are true (they are not true as S2 refers to M, and M occurs on Monday and not Tuesday). In addition, they can offer S3* and S4* which are true, the semantics of which may look something like the following. S-SIMPLE-A: S-SIMPLE-A: S2: Anna’s reason for hailing the taxi was R. S3: If Anna’s reason for hailing the taxi was R, then R explains why she was hailing the taxi. S4: Therefore, R explains why Anna was hailing the taxi. S-SIMPLE: S2: Anna’s reason for hailing the taxi was R. S3: If Anna’s reason for hailing the taxi was R, then R explained why she was hailing the taxi. S4: Therefore, R explained why Anna was hailing the taxi. S2: Anna’s reason for hailing the taxi was R. S3: If Anna’s reason for hailing the taxi was R, then R explains why she was hailing the taxi. S3: If Anna’s reason for hailing the taxi was R, then R explains why she was hailing the taxi. S4: Therefore, R explains why Anna was hailing the taxi. S-SIMPLE: S2: Anna’s reason for hailing the taxi was R. S3: If Anna’s reason for hailing the taxi was R, then R explained why she was hailing the taxi. S4: Therefore, R explained why Anna was hailing the taxi. 1 3 O. Pearson The semantics of S-SIMPLE-A and S-SIMPLE* are at least as simple as that of S-SIMPLE-B, and therefore the A-theorist has no cost here. The explanatory relation uncovered in Sect. 3 is the historical relation H. H and M have distinct explananda, namely AP and AN respectively. It is of note that this distinction between AP and AN is one that can be recognised by both A-theorists and B-theorists. For an A-theorist, AN is a hailing by Anna that (presently) occurs on Monday, whilst AP is a Tuesday temporary fact that Anna was hailing. For a B-theorist AN is a hailing by Anna that (tenselessly/permanently) occurs on Monday, whilst AP is the permanent fact that Anna hailed earlier than Tuesday. (I thus use the labels ‘AN’ and ‘AP’ in a way that allows for an A-theory or B-theory interpretation, allowing context to settle which is at issue if just one is.) I now wish to turn to Storrs-Fox’s semantics and consider it with AP and AN thus clearly distinguished. Storrs-Fox seems to be concerned with AN as an explanandum on Tuesday (though, we have seen it is not clear AN, as opposed to AP, actually is an explanandum on Tuesday). With this in mind the B-Theory semantics of S2-S4 can be clarified as S-SIMPLE-B-AN. S-SIMPLE-B-AN: S2: Anna’s reason for AN was RB. 3 S-SIMPLE-A: S3: If Anna’s reason for AN was RB, then RB explains AN. S4: Therefore, RB explains AN. 11 Storrs-Fox focuses on motivating reasons and related explanation of action, though he suggests that he believes the general form of his argument will also apply to other forms of explanation. I will not discuss other cases in detail, as he does not, but I do want to highlight that these two points have parallels that could ground similar replies concerning different forms of explanation. For example, it seems true that it is impossible (in everyday settings) for a cause at one time to have an effect at an earlier time, and so there cannot be a cause at one time with an effect at an earlier time. S-SIMPLE-B-AN: S3: If Anna’s reason for AN was RB, then RB explains AN. S3: If Anna’s reason for AN was RB, then RB explains AN. S4: Therefore, RB explains AN. If we also clarify the A-theory semantics of S-SIMPLE* in this way we get S-SIMPLE-AN. S-SIMPLE-AN: S2: Anna’s reason for AN was R. S3: If Anna’s reason for AN was R, then R explained AN. S4: Therefore, R explained AN. S-SIMPLE-AN: S2: Anna’s reason for AN was R. S3: If Anna’s reason for AN was R, then R explained AN. S4: Therefore, R explained AN. S4: Therefore, R explained AN. At this stage, it is in fact the B-Theory semantics, not the A-theory semantics that comes at a cost, contra Storrs-Fox. Assuming that a motivation relation can be an explanatory relation, the inference in S-SIMPLE-AN is straightforward. M gives us this. However, the inference in S-SIMPLE-B-AN is not. In the latter inference S3 contains a crucial shift from RB being a reason for AN on Monday, to RB being an explanans of AN on Tuesday. This shift opens up two important gaps, one concerning reasons and one concerning explanatory relations. As stressed above, Anna can’t on Tuesday perform the action at issue in AN. She did earlier perform it, but can’t then. So, on Tuesday, Anna cannot have a reason to perform it. Thus, if RB occurs on Tuesday, it cannot do so as a reason for that action. Perhaps it was on Monday a reason for it, but it is no longer a reason for it on Tues day. In this way, Storrs-Fox’s semantics seems to suffer from a form of the problem that we saw he himself raises in Sect. 3. The explanatory relation that occurs on Tuesday must be of a different form to that which holds on Monday. This is the first gap passed over in S3 and the inference for the B-theorist. 1 3 Acting for reasons and the metaphysics of time The second gap concerns the explanatory relations themselves. M is an explana tory motivating relation with AN as explanandum. M is at issue in S2 and the ante cedent of S3. However, M occurs on Monday and not Tuesday. Therefore, M is not at issue in the consequent of S3 or S4. S-SIMPLE-B-AN: Storrs-Fox is simply assuming that because one explanatory relation holds on Monday a different one with AN as explanandum will hold on Tuesday (this is his assumption that explanation is forever). But, this is not something he has argued for. Nor is it something we have directly observed as occurring in assessing Anna’s case. The gaps just highlighted for the B-theorist do not disprove the B-theory position. There might be a way to justify the inference. However, it has been shown that the B-theorist is saddled with a cost that the A-theorist is not. (These gaps in the B-theory inference have analogues in the inference given in S-SIMPLE-A. The gaps in this latter inference also give the A-theorist grounds to deny its validity, and hence deny that R is an explanans on Tuesday.) 5 The A-theory reply The A-theory form is given by S-MIX-A and the B-theory form is given by S-MIX-B. S-MIX-A: S2: Anna’s reason for AN was R. S3: If Anna’s reason for AN was R, then R explains AP. S4: Therefore, R* explains AP. S-MIX-B: S2: Anna’s reason for AN was RB. S3: If Anna’s reason for AN was RB, then RB explains AP. S4: Therefore, RB explains AP. S3: If Anna’s reason for AN was R, then R* explains AP. S2: Anna’s reason for AN was RB. S3: If Anna’s reason for AN was RB, then RB explains AP. S4: Therefore, RB explains AP. Both of these inferences clearly involve gaps. Though, I suggest that it is not obvious the gaps are more problematic in one case than the other, or hence, that these infer ences preference either the A-theory or B-theory. Both of these inferences clearly involve gaps. Though, I suggest that it is not obvious the gaps are more problematic in one case than the other, or hence, that these infer ences preference either the A-theory or B-theory. Both involve a shift from an explanatory relation occurring at one time (namely M) to a different explanatory relation (such as H) with a different explanandum (namely AP) occurring at a later time. S-MIX-B, unlike S-MIX-A has the same fact as reason in the motivating relation and as explanans in the later explanatory relation. I suggest it is not clear that this is an advantage. Given general features of the A-theory of time R and R* will be fun damentally linked: unless time ends facts such as R are necessarily followed by facts such as R* due to the flow of time.12 The B-theory inference has the fundamental tie of identity where the A-theory inference has a fundamental tie of necessary covari ance. (In neither case can you have the reason on Monday without the explanans on Tuesday. And, in both cases, given the passing of time, the existence of the entity that is reason on Monday is followed by the existence of the entity that is explanans on Tuesday.) It is true that the lack of identity means that R, unlike R, is not a reason for AN. 12 Note, this is not an ad hoc point, rather it is a general feature of A-theories. 13 Note, we should also deny RB is a reason for AP on Tuesday. One way to motivate this, is to note AP concerns a Monday action, so not one Anna can perform or have a reason to perform on Tuesday. But, a second way to motivate this is to note that AP itself is not an action at all, but rather a fact concerning an action. Thus, it is not something that anyone can ever do, or hence have a reason to do. 5 The A-theory reply The A-theory reply to Storrs-Fox’s argument that I propose combines the observa tions of the last three sections. The point is essentially this. If we take S3 and S4 to concern R as explanans, then we ought to take them to be false, though replaceable by the true S3 and S4* (along the lines of Sect. 2). On the other hand, if we take there to be explanation occurring on Tuesday, we ought to take the explanans to be some thing other than R (along the lines of Sect. 3). Crucially, this might be understood to vary with context, so that in some contexts we are focused on R (and for example M) and in others on current explanation (and for example H). In neither case are we committed to taking there to be an explanatory relation that occurs on Tuesday with R as explanans. I would like to emphasise that the reply that I have given, whilst coinciding with the metaphysics of temporary facts adopted by the A-theory, is in fact motivated inde pendently of this metaphysics. The key motivation, throughout, has stemmed from two points, that one cannot have a reason to do something that it is impossible for one to do and that one cannot possibly at one time perform an action at an earlier time.11 These two points have a plausibility independently of the A-theory. They motivate my reply by making it clear that the A-theorist can coherently deny that there is an explanation obtaining on Tuesday with R as explanans. The general nature of these points also means that the form of reply that I have here outlined concerning Anna’s case would also apply to other cases. Before closing this section I would like to say a little more about the relation between M and H. In the last section we considered the explanation of AN. Neverthe 1 1 3 O. Pearson less, plausibly, as with H, Anna’s case does give us grounds for thinking AP can be explained. This gives us two further forms of the inference as captured in S2 to S4. The A-theory form is given by S-MIX-A and the B-theory form is given by S-MIX-B. less, plausibly, as with H, Anna’s case does give us grounds for thinking AP can be explained. This gives us two further forms of the inference as captured in S2 to S4. 12 Note, this is not an ad hoc point, rather it is a general feature of A-theories. 13 15 With Perry’s essential indexicals highlighted, one might wonder if the argument I will outline in this section concerning temporal indexicals has analogues concerning first-personal indexicals and spatial indexicals. I cannot discuss these points or the self and space in detail here. But, it is of note that an analogous argument concerning the self can be accommodated by a view of the self that denies reductive physicalism and denies some interpretations of Wittgenstein’s private language argument. Whilst a parallel argument concerning space fails to go through because spatial indexicals such as ‘here’ are not essential, rather they can be replaced with temporal first personal ones such as ‘the place where I am now’. For discussion of these points see Pearson (2015) and (2018). 5 The A-theory reply However, as noted above, RB cannot on Tuesday, and hence in the explanatory rela tion at issue here, be a reason for AN either (even if it was earlier one).13 Both infer ences hence involve a similar shift from a motivation relation concerning a reason, to an explanatory relation the explanans of which is not a reason. The A-theory reply I have given does make use of explanatory relations like M and H, relations that are no doubt linked. However, this is not a cost that the A-theory has that the B-theory lacks. Storrs-Fox’s argument assumes that explanation is forever. I have approached this claim by focusing on specific explanatory relations (something Storrs-Fox does not do). Looking at the explanatory relation M, we have seen that there is an important 1 3 Acting for reasons and the metaphysics of time sense in which the claim that explanation is forever is false. Some explanatory rela tions occur at one time and not at another. Focusing on the link between the explana tory relations M and H, we have seen that there is another different sense in which the claim is true. Given the explanatory relation M occurs on Monday, the related explanatory relation H occurs on Tuesday (and relatedly for later times). However, neither of these points give us what Storrs-Fox needs, namely that R explains on Monday and at later times. (Storrs-Fox’s criticism of the A-theory relies on commit ting the A-theory to R being an explanans on Tuesday. What I have argued, is that the A-theory is not committed to this.) 14 For clarity in this section I will pick out phrases that capture beliefs with single inverted commas. By “present tensed belief”, I mean a belief one would naturally express using a present tensed utterance, and so on for other beliefs. 14 For clarity in this section I will pick out phrases that capture beliefs with single inverted commas. By “present tensed belief”, I mean a belief one would naturally express using a present tensed utterance, and so on for other beliefs. 15 With Perry’s essential indexicals highlighted, one might wonder if the argument I will outline in this section concerning temporal indexicals has analogues concerning first-personal indexicals and spatial 6 A problem for the B-theory Or, at least, he would be irra tional if he did so without also forming a present tensed belief such as that ‘it is now 11:55am’. Bilal can act on a tenseless belief, even a relevant tenseless belief, but if he does not also act on a relevant present tensed belief, he will act irrationally.16 Why then must Bilal have a present tensed belief in order to act rationally? office. But, again, he would be irrational if he did so. Or, at least, he would be irra tional if he did so without also forming a present tensed belief such as that ‘it is now 11:55am’. Bilal can act on a tenseless belief, even a relevant tenseless belief, but if he does not also act on a relevant present tensed belief, he will act irrationally.16 Why then must Bilal have a present tensed belief in order to act rationally? I will here assume that reasons are facts (as opposed, for example, to beliefs or desires). Here is not the place to defend this theory of reasons, but it is of note that such a theory has been forcefully defended, is a live option in the literature, and appears to be adopted by Storrs-Fox.17 I will also assume that the demands of rationality stem from reasons: what one rationally ought to do, is what one has normative reason to do. Again, here is not the place to defend an account of rationality (it would take us too far away from the central point and take too much space). However, it is again of note that such a theory has been forcefully defended and is a live option in the literature.18 Bringing these points together,19 we can say, broadly, one is rational if one acts for reasons that justify one’s action, that is, if one’s motivating reasons are normative reasons, that is, facts that justify the action.20 So, the demand that rationality places on Bilal is that he acts for relevant normative reasons, that the facts that motivate his action justify his action. However, reasons are facts and in order to be able to respond to a fact Bilal must be aware of it. 16 Note, we can and often do recognise people acting irrationally and on the basis of beliefs/reasons inap propriate for justifying the action (cf. Wason (1966) for a well-known example). 17 Cf. e.g. Dancy (2000) and Lowe (2008) for a defence of this idea. 18 Cf. e.g. Kolodny (2005) and Raz (2005) for a defence of this idea. (It is of note that this is argued to be the case quite generally, including specific arguments to show that so called structural rationality (roughly, psychological coherence) is also determined by responsiveness to reasons.) 19 Note Kolodny (2005) speaks of reasons as facts, and for an explicit combing of this view of reasons and rationality see Pearson (2018). 20 This needs to be made a little more complicated to accommodate instances in which an agent has false beliefs about what normative reasons obtain, e.g. one might amend it to say an agent is rational if, had their beliefs been true, their motivating reasons would have been normative reasons (cf. e.g. Kolodny (2005)). But, this complexity is not crucial to the argument here. 6 A problem for the B-theory Having argued that responding to reasons does not pose a particular problem for the A-theory, in this section I will present an argument to show that it does present a problem for the B-theory. Bilal has a meeting today. He knows that the meeting is at midday on the 20th of May 2022 and he fully intends on going to the meeting. However, as the meeting nears, Bilal remains in his office tidying his papers. Suddenly, Bilal stops what he is doing and heads for the meeting. It seems that Bilal’s action of heading to the meeting must be explained by his forming a present tensed belief, such as the belief that ‘it is now 11:55am’.14 It seems no related tenseless belief could account for his action, because he could have such a tenseless belief all morning without being moved to act. For example, he could believe all morning ‘the meeting occurs at 12pm, 20th of May 2022’ and ‘it takes 5 minutes to travel from my office to the meeting room’. This is, of course, an instance of one of Perry’s (1979) cases of the essential index ical.15 Something that it is important to note regarding such cases, however, is that the essentiality here stems from rationality. Bilal must have the present tensed belief in order to act rationally. It is not, for example, a physiological demand. We could imagine Bilal forming a tenseless belief and then suddenly because of this heading for the meeting. But, Bilal would be irrational if he acted in this way. Bilal does not require the present tensed belief to act, he requires it in order to act rationally. For example, we could imagine Bilal forming the tenseless belief ’11:55am is 11:55am’, and on the basis of this heading to his meeting. But, he would be irrational if he did so. Alternatively, we could imagine Bilal suddenly forming the belief that ‘the meeting occurs at 12pm, 20th of May 2022’ and on the basis of this leaving his 14 For clarity in this section I will pick out phrases that capture beliefs with single inverted commas. By “present tensed belief”, I mean a belief one would naturally express using a present tensed utterance, and so on for other beliefs. 1 3 O. Pearson office. But, again, he would be irrational if he did so. 21 Cf. e.g. Alvarez (2008). 16 Note, we can and often do recognise people acting irrationally and on the basis of beliefs/reasons inap propriate for justifying the action (cf. Wason (1966) for a well-known example). 17 Cf. e.g. Dancy (2000) and Lowe (2008) for a defence of this idea. 6 A problem for the B-theory That is, in order for Bilal to have a normative reason as his motivating reason, he must have a belief with that fact as content.21 The rational demand that Bilal acts for relevant normative reasons therefore gives rise to the demand that he acts on beliefs with those facts as content. Therefore, if there is a belief or type of belief that rationality demands Bilal to act in light of, then that is because there is a normative reason that rationality demands Bilal acts for and only that belief or that type of belief has that normative reason, that is fact, as content. Rationality demands that Bilal acts because of his belief that ‘it is now 11:55am’ (or for a present tensed belief of a related type, such as the belief ‘the time is currently 11:55am’). It demands this because it demands that he acts because of the fact that is the content of this belief (or the content of this type of belief). If Bilal does not act for 1 3 Acting for reasons and the metaphysics of time that normative reason, he does not act for facts that justify his action, and therefore his action is not rational. Thus, if Bilal must have a present tensed belief such as the belief that ‘it is now 11:55am’ to act rationally in heading for the meeting, then it must be that such a pres ent tensed belief captures a fact not captured by any other non-present tensed beliefs. This point is readily accommodated by the A-theory, because according to the A-the ory present tensed beliefs will capture temporary facts that other tenseless beliefs do not (such as the fact that it is now 11:55am). The B-theory, however, cannot account for this. According to the B-theory any present tensed belief will have a permanent fact as content. Permanent facts, however, can also be the content of tenseless beliefs.fi We can bring out the difficulty here by considering Bilal’s belief that ‘it is now 11:55am’. There are two prominent B-theory accounts of the truthmakers of such tensed beliefs. According to the first, date account, Bilal’s belief would be made true by the permanent fact that 11:55am is 11:55am. 24 Even if Bilal had not noticed the time, and hence formed no belief like belief B and stayed tidying his office, it would still have been the case that he ought to have attended the meeting. On the other hand, Bilal could have failed to form belief B and still have rationally left for the meeting (for example, if he had a belief that was a different token of the same type as belief B, or if he had a token belief of a different type, such as of the type ‘the meeting is 5 minutes from now’). 22 Cf. e.g. Mellor (1998) who mentions both options, arguing for the former option in opposition to his earlier adoption of the latter option in his (1981). Maclaurin and Dyke (2002: 279) seem to present both options without explicitly distinguishing them. 23 Perhaps Bilal could act rationally on the latter belief, but only if his act was also performed on the basis of a present tensed belief such as the belief that ‘B is occurring now’ and therefore the issue simply arises again. 25 Compare Storrs-Fox’s suggestion that Anna’s reason for hailing is the permanent fact that rain occurs at a specific time. 22 Cf. e.g. Mellor (1998) who mentions both options, arguing for the former option in opposition to his earlier adoption of the latter option in his (1981). Maclaurin and Dyke (2002: 279) seem to present both options without explicitly distinguishing them. 23 Perhaps Bilal could act rationally on the latter belief, but only if his act was also performed on the basis 6 A problem for the B-theory According to the second, token reflexive account, it would be made true by the permanent fact that Bilal’s belief occurs at 11:55am.22 Both of these permanent facts can be the contents of tenseless beliefs, such as the belief ‘11:55am is 11:55am’ and the belief ‘B occurs at 11:55am’ (where ‘B’ is a name for that belief). However, Bilal would be irrational if he acted on the basis of either of these tenseless beliefs.23 Further, the truthmakers proposed by the B-theory for Bilal’s present tensed belief do not pick out normative reasons essential for justifying his action. The fact that 11:55am is 11:55am is not a reason for him to attend his meeting. All entities are self- identical whether or not Bilal ought to attend the meeting. The fact that a particular belief of Bilal’s occurs at 11:55am is also not an essential reason for him to attend the meeting. He ought to attend even if he does not have that particular belief.24 The tenseless truthmakers proposed by the B-theory are not normative reasons for Bilal’s action, and tenseless beliefs with these facts as contents also do not make his action rational. The truthmakers proposed by the B-theory for Bilal’s present tensed belief therefore play no role in explaining why rationality would demand that he had such a present tensed belief. On the other hand, let us consider permanent facts that a B-theorist might propose as essential normative reasons for Bilal’s action. These might include the fact that the meeting occurs at 12pm, 20th of May 2022 and the fact that it takes 5 min to travel from Bilal’s office to the meeting room.25 Both of these facts could be the contents of 24 Even if Bilal had not noticed the time, and hence formed no belief like belief B and stayed tidying his office, it would still have been the case that he ought to have attended the meeting. On the other hand, Bilal could have failed to form belief B and still have rationally left for the meeting (for example, if he had a belief that was a different token of the same type as belief B, or if he had a token belief of a different type, such as of the type ‘the meeting is 5 minutes from now’). 23 Perhaps Bilal could act rationally on the latter belief, but only if his act was also performed on the bas of a present tensed belief such as the belief that ‘B is occurring now’ and therefore the issue simply aris again. 22 Cf. e.g. Mellor (1998) who mentions both options, arguing for the former option in opposition to his earlier adoption of the latter option in his (1981). Maclaurin and Dyke (2002: 279) seem to present both options without explicitly distinguishing them. 23 Perhaps Bilal could act rationally on the latter belief, but only if his act was also performed on the basis of a present tensed belief such as the belief that ‘B is occurring now’ and therefore the issue simply arises again. 24 Even if Bilal had not noticed the time, and hence formed no belief like belief B and stayed tidying his office, it would still have been the case that he ought to have attended the meeting. On the other hand, Bilal could have failed to form belief B and still have rationally left for the meeting (for example, if he had a belief that was a different token of the same type as belief B, or if he had a token belief of a different type, such as of the type ‘the meeting is 5 minutes from now’). 25 Compare Storrs-Fox’s suggestion that Anna’s reason for hailing is the permanent fact that rain occurs at a specific time. 6 A problem for the B-theory 25 Compare Storrs-Fox’s suggestion that Anna’s reason for hailing is the permanent fact that rain occu at a specific time. 1 3 O. Pearson beliefs that Bilal has all morning. Furthermore, both of these beliefs could play a part in motivating Bilal to act.26 Nevertheless, rationality would still demand that Bilal must, in addition, have and act on a present tensed belief (such as the belief that ‘it is now 11:55am’). Therefore, whilst the facts the B-theorist picks out could be amongst Bilal’s motivating reasons, they are not sufficient normative reasons to make his act rational.27 Thus, there must be a normative reason for Bilal’s action beyond these per manent facts, a normative reason essential for justifying Bilal’s action. The B-theory appears to be at a loss to offer such a fact. The points just made can be summarised as follows. The demands of rationality are firstly that the motivating reasons for an action are normative reasons that justify it. The demands of rationality are hence, derivatively, that the beliefs we act on have as contents normative reasons that justify our actions. Therefore, if two different beliefs have as contents the same normative reason, rationality will not demand that an agent acts on one of these beliefs over the other. On the other hand, if rationality demands an agent acts on one belief instead of another, then that belief must have as content a normative reason the other fails to capture. Rationality demands that Bilal acts on a present tensed belief instead of or in addition to any tenseless beliefs. There fore, Bilal’s present tensed belief must capture a normative reason distinct from any normative reasons captured by tenseless beliefs. The A-theory can readily accom modate this point by taking the required normative reason to be a temporary fact. The B-theorist cannot readily accommodate this point, as all B-theory facts can be captured by tenseless beliefs in addition to tensed ones. The B-theory therefore faces a problem when we consider actions performed for reasons.28l Storrs-Fox does briefly mention essential indexical beliefs such as Bilal’s (2021: 4251). He says that a belief such as the present tensed belief ‘it is raining now’, will have as content a permanent fact. Further, this will be a permanent fact that might also be the content of a tenseless belief, perhaps, for example, the belief that ‘it is raining at 12pm, 20th of May 2022’. 26 It is of note that there are two elements to the rational demand for an agent to act on the basis of par ticular beliefs, firstly, that the beliefs have relevant normative reasons as contents and, secondly, that those beliefs enable the agent to act for those normative reasons. The permanent facts a B-theorist may offer as normative reasons are not only the contents of tenseless beliefs of Bilal’s but also beliefs that enable him to act for those reasons. Thus, it is right to say that the rational demand must be a demand for beliefs with different reasons as contents (not merely for beliefs that enable him to act for reasons which were already the contents of other beliefs that he could not act for). 27 This point could be taken in two ways. One might say the permanent facts are not normative reasons at all. Alternatively, one could say whilst they are normative reasons, they are inadequate ones. The notion of adequacy here stems from the fact that many normative reasons are pro tanto, that is they can be weighed with and against one another. Therefore, a fact may be a normative reason to perform an action, but alone may be outweighed by a normative reason not to perform the action. For example, the fact that I need some fresh air is a normative reason to go outside, but alone it is outweighed by the normative reason that I have work to finish. But, if it becomes the case that I need to go to the library to finish the work, then this normative reason will combine with the first to make it all things considered reasonable and rational for me to go outside. We often act for more than one reason and rationality demands that these normative reasons collectively justify the action (something that a normative reason in isolation may fail to do). 6 A problem for the B-theory Storrs-Fox suggests that the beliefs differ because they present that permanent fact under different guises. More generally, tensed beliefs and 27 This point could be taken in two ways. One might say the permanent facts are not normative reasons at all. Alternatively, one could say whilst they are normative reasons, they are inadequate ones. The notion of adequacy here stems from the fact that many normative reasons are pro tanto, that is they can be weighed with and against one another. Therefore, a fact may be a normative reason to perform an action, but alone may be outweighed by a normative reason not to perform the action. For example, the fact that I need some fresh air is a normative reason to go outside, but alone it is outweighed by the normative reason that I have work to finish. But, if it becomes the case that I need to go to the library to finish the work, then this normative reason will combine with the first to make it all things considered reasonable and rational for me to go outside. We often act for more than one reason and rationality demands that these normative reasons collectively justify the action (something that a normative reason in isolation may fail to do). 28 Cf. Pearson (2018) for a further defence of the points in this argument. 1 3 Acting for reasons and the metaphysics of time tenseless beliefs differ despite having the same facts as content, because they present those facts under a different guises. tenseless beliefs differ despite having the same facts as content, because they presen those facts under a different guises. It is not clear exactly what a guise is in this context. But, it is clear that according to this account tensed and tenseless beliefs have the same reasons as content. Storrs- Fox’s reference to guises therefore does not account for the rational demand that Bilal has a tensed belief. To put the point differently, rationality is concerned with what reasons Bilal has and acts for, not with what guises those reasons occur to him under. Therefore, a reference to guises does not account for the rational demands placed on Bilal (or other similar agents). The point just made makes it clear that the argument of this section is importantly different from arguments such as Prior’s (1959) thank goodness argument that have been extensively discussed in the literature. 29 Cf. e.g. Mellor (1998: Chap. 4) and Maclaurin and Dyke (2002). 6 A problem for the B-theory Prior’s argument establishes that tensed and tenseless beliefs can play different roles in our lives, so cannot have the same meaning (in some broad sense of the term). A prominent B-theorist reply to Prior’s argument is to suggest that tensed and tenseless beliefs differ in meaning but never theless concern the same facts.29 This reply relies on the observation that beliefs can be opaque, that is, that two beliefs might concern the same fact without a believer recognising this. Storrs-Fox’s reference to guises is no doubt linked to this claim, guises are supposed to account for such opacity. However, by focusing on rationality and the role of facts and motivating reasons, the argument of this section goes beyond Prior’s. My argument specifically focuses on the facts beliefs concern. (Where Prior’s argument highlights a difference in roles, my argument, via a consideration of reasons, specifically ties this to a difference in facts concerned.) Therefore, reference to meaning, opacity, or guises is not sufficient to provide a B-theory response to my argument (so long as it is assumed there is no difference in facts picked out). Authors’ contributions solo author. Authors’ contributions solo author. Funding Not applicable Funding Not applicable Data Availability not applicable. Data Availability not applicable. Code Availability not applicable. Conflicts of interest/Competing interests not applicable. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. Declarations Conflicts of interest/Competing interests not applicable. 7 Conclusion Storrs-Fox has argued that the A-theory is wrong, because it takes motivating reasons to often be temporary facts though motivating reasons can be explanantia and as such ought to obtain forever. However, I have argued that motivating reasons often do not obtain forever, because an agent cannot at one time have a reason to perform an action at an earlier time. The A-theory can therefore coherently deny explanation is forever and does not face the problem Storrs-Fox suggests. I have also argued that Storrs-Fox’s B-theory does face a problem related to act ing for a reason. It is common for there to be a rational demand on an agent to act on the basis of a tensed belief. The B-theory does not appear to be able to account for this, because the rational demand is a demand for beliefs with particular facts as con tents and according to the B-theory, tensed beliefs will share contents with tenseless beliefs and so will not be demanded in place of them. Storrs-Fox’s discussion has helped to make it clear that the manner in which peo ple respond to reasons, the rationality of people, is something that can inform the 1 3 3 O. Pearson debate between the A-theory and the B-theory of time. However, Storrs-Fox’s discus sion fails to establish that it is the latter theory that gains credence over the former in light of this. Authors’ contributions solo author. References Alvarez, M. (2008). Reasons and the ambiguity of ‘belief’. Philosophical Explorations, 11, 53–65 M. (2009). How many kinds of reasons? Philosophical Explorations, 12, 181–193 Alvarez, M. (2009). How many kinds of reasons? Philosophical Explorations, 12, 181–193 Alvarez, M. (2010). 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Philosophical Studies, 178(12), 4239–42 St B (2007) R d I ibilit Phil hi l St di 136(3) 351 384 orrs-Fox, D. (2021). Explanation and the A-theory. Philosophical Studies, 178(12), 4239–4259 reumer, B. (2007). Reasons and Impossibility. Philosophical Studies, 136(3), 351–384 Storrs-Fox, D. (2021). Explanation and the A-theory. Philosophical Studies, 178(12), 423 Streumer, B. (2007). Reasons and Impossibility. Philosophical Studies, 136(3), 351–384 Wason, P. C. (1966). Reasoning. In B. M. Foss (Ed.), New horizons in psychology, Vol. I. Penguin. References 1 3 Acting for reasons and the metaphysics of time Acting for reasons and the metaphysics of time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published map and institutional affiliations. 1 3
https://openalex.org/W2011147405	https://www.jstage.jst.go.jp/article/hrl/5/0/5_0_27/_pdf	English	null	Urbanization influences on stream temperature behavior within low-discharge headwater streams	Hydrological research letters	2,011	cc-by	3,807	Abstract: on water quality, especially in a warming climate. Stream temperature, in particular, is an important aspect of water quality and has wide-ranging implications for overall stream health (Bogan et al., 2003; LeBlanc et al., 1997; Constantz, 1998; Nelson and Palmer, 2007). Stream temperature is also a measure of water quality, which previous work has shown to be subject to influence by urbanization (Klein, 1979; LeBlanc et al., 1997; Nelson and Palmer, 2007; Pluhowski, 1970). Urbanization has compromised water quality globally, especially stream temperature, by reducing shading and converting natural landscapes to impermeable surface coverage (ISC). We analyze stream-air temperature relationships in a low-order, moderate-gradient urban stream using three years of stream temperature data collected at nine monitoring sites. At the sub-catchment scale, ISC increases from 13.7% to 24.3% among sites, causing mean summer temperatures to increase 4–5°C or 0.37°C for each 1% increase in ISC. ISC at these spatial scales influences stream-air temperature relationships at daily-, weekly-, and monthly-averaged time scales. ISC at smaller spatial scales within a 25-m buffer of the stream, which ranges from 1% to 75%, does not correlate with mean stream temperatures at any temporal scale; however, buffer ISC does correlate with short-term temperature surge events, which we define as an increase of at least 1°C within 15 minutes. Mean surge amplitudes range from 1.90°C to 3.27°C in areas with low and high buffer ISC, respectively. Our results show that ISC influences stream temperatures at stream buffer and sub- catchment spatial scales and daily, weekly, and monthly temporal scales, and may render the concept of equilibrium temperature obsolete for predicting stream temperatures, especially in low-order, headwater streams. Past work has shown that there is a strong correlation between stream temperature and air temperature. For example, models using air temperature as a predictor have been used to accurately forecast daily, weekly, and monthly stream temperatures with and without adjusting for time lag (Johnson, 1971; Crisp and Howson, 1982; Stefan and Preud’homme, 1993; Pilgrim et al., 1998; Erickson and Stefan, 1996). These models are particularly useful as they only require one input variable, air temperature (Mohseni and Stefan, 1999). The simplicity of these models makes them particularly attractive for studying the effects that a warming climate may have on stream temperatures (Mohseni and Stefan, 1999); however, the effects of urbanization may render stream-air temperature relationships obsolete. Abstract: As a result, it becomes prudent to examine the impact that urbanization may have on stream temperature, particularly in low-order urban streams whose low thermal inertia makes them sensitive to changes in the surrounding environment. KEYWORDS stream temperature; air temperature; equilibrium temperature; headwater; urbanization; impervious surface KEYWORDS stream temperature; air temperature; equilibrium temperature; headwater; urbanization; impervious surface g g While stream temperature is known to be influenced by many variables, several effects of urbanization play a large role and are readily quantifiable. Urbanization often results in an increase in impervious surfaces and reduced shading of land surfaces by vegetation (Herb et al., 2008). To examine the effect of these variables upon stream temperature, we use impervious surface coverage (ISC) as a general measure of urbanization within the study area catchment. ISC has been found to be a fairly accurate measure of urbanization and urban impacts on streams (McMahon and Cuffney, 2000; Schueler, 1994; Wang et al., 2003). Though previous work has shown that urbanization can influence stream temperature, there are relatively few studies in the literature that focus on long-term, in-stream temperature series in low-order urban streams. This is largely due to fact that maintaining temperature data loggers in such streams can be difficult as they have a tendency to get washed away, silted in, or buried (Nelson and Palmer, 2007). It is this lack of research focused specifically on low-order urban streams that provides the motivation for this study; Corresponding to: Joshua S. Rice, Environmental Science Program, College of Arts and Sciences, Appalachian State University, Boone, NC, 28608- 2021, USA. E-mail: ricejs@appstate.edu, ricejs1@gmail.com ©2011, Japan Society of Hydrology and Water Resources. Urbanization influences on stream temperature behavior within low-discharge headwater streams Joshua S. Rice1, William P. Anderson, Jr.2 and Christopher S. Thaxton3 1 Environmental Science Program, College of Arts and Sciences, Appalachian State University, Boone, NC, 28608-2021, USA Received 4 November, 2010 Accepted 2 April, 2011 Hydrological Research Letters 5, 27–31 (2011) Published online in J-STAGE (www.jstage.jst.go.jp/browse/HRL). DOI: 10.3178/HRL.5.27 Hydrological Research Letters 5, 27–31 (2011) Published online in J-STAGE (www.jstage.jst.go.jp/browse/HRL). DOI: 10.3178/HRL.5.27 Hydrological Research Letters 5, 27–31 (2011) Published online in J-STAGE (www.jstage.jst.go Temperature Data Temperature time-series were collected between January 1, 2007 and October 1, 2010. Stream temperature measurements for this period were collected from 9 sites in the study area (Figure 1) at 15-minute intervals using HOBO Water Temp Pro v2 loggers. Hourly air temperature averages and precipitation data from the nearest weather station (BOON-ECONET, ~4.5 km away from the midpoint of the study area) were obtained from the NC State Climate Office (requested from http://www.nc-climate.ncsu.edu/ on various dates). Temperature data loggers were submerged in the stream and fixed in place along the stream bed in a manner that allowed them to float freely in the water column while avoiding the direct influence of solar radiation. Stream temperature means were calculated using programs written Our analysis of approximately three years of stream and air temperature data along with quantitative measures of ISC within the study area demonstrates a significant relation between urbanization and stream temperature behavior at daily, weekly, and monthly time scales. Urbanization has an impact on stream temperature behavior at varying temporal scales and at the sub-catchment and 25 m buffer spatial scales. The relation between stream temperature and with increasing ISC is seen through (1) increases in average stream temperature; (2) short-term surges in stream temperature due to runoff that increases in amplitude as ISC increases; and (3) a downward trend in correlation of stream and air temperature as ISC increases. We expect these results based on previous work (Herb et al., 2008; Klein, 1979; LeBlanc et al., 1997; Nelson and Palmer, 2007; Pluhowski, 1970); however, unlike previous studies, the data of this study include a much higher level of spatial and temporal resolution and represent a headwater stream in an urban setting. Figure 1. Map of Boone Creek, ISC, study area watershed, and sites 1 and 2 catchments. in MATLAB as the dataset was updated. Land Coverage Quantification we aim to provide insight into the effects of increasing urbanization upon stream temperature behavior within low- order urban streams. Catchment boundaries of each study site were determined using the stock watershed delineation tools within the ArcHydro extension of ArcGIS v9.4 and a 3 m resolution, LIDAR-generated DEM obtained in 2008 covering the study area. The area of the Boone Creek drainage basin is 5.2 km2. ISC was quantified by differentiating between impervious and pervious surfaces within each study sites’ catchment using stock tools found within ArcGIS and a 15 cm resolution true-color orthorectified aerial photo from 2009 covering the study area. ISC was measured both as a percentage within the catchment draining to each monitoring site as well as within a buffer area that extended 25 m from each side of the channel. The 25 m buffer areas were created using tools within ArcGIS and were measured as the upstream area between one site and the next upstream site. The buffer area of the furthest upstream site (site 1) was extended 100 m upstream from the site to the point where the main trunk of the stream splits into a number of smaller tributaries. Site Description The study area (Figure 1) consists of a 1.8 km portion of Boone Creek, which flows through the Town of Boone, North Carolina, U.S.A., and the Appalachian State University campus. Boone Creek is a moderate-gradient urban stream that is influenced by high-gradient tributaries and is a tributary of the South Fork New River. The upper and lower reaches of the stream are separated by a 700 m culvert, effectively creating two streams within the study area (Anderson et al., 2010). While the majority of the study area experiences extensive urbanization, the upper segment has limited areas of riparian buffering and shading due to vegetation. In contrast, the lower section has no shading from riparian coverage and minimal vegetation that occurs in the form of non-native grasses as well as extensive artificial channelization and riprapping. J. S. RICE ET AL. J. S. RICE ET AL. INTRODUCTION As global population continues to grow, the demands placed on water resources by the growing population will increase as well. The forecasted growth in world population is also expected to be increasingly centered in urban areas (Paul and Meyer, 2001). Population growth in conjunction with increasing migration into urban areas will only exacerbate the stress placed on available water resources and further compromise water quality. This makes the activities that will result from these trends and that will negatively impact water quality, such as urbanization, vital to explore and understand. The increasing area of urbanization makes it important to understand and be able to accurately predict the impacts of increasing urbanization Received 4 November, 2010 Accepted 2 April, 2011 Received 4 November, 2010 Accepted 2 April, 2011 —27— URBANIZATION INFLUENCES ON STREAM TEMPERATURE URBANIZATION INFLUENCES ON STREAM TEMPERATURE Figure 2. Stream temperature versus air temperature with a fictional 1:1 temperature variable at daily, weekly, and monthly time scales. (a) Site 1. (b) Site 2. Figure 2. Stream temperature versus air temperature with a fictional 1:1 temperature variable at daily, weekly, and monthly time scales. (a) Site 1. (b) Site 2. emperature versus air temperature with a fictional 1:1 temperature variable at daily, weekly, and monthly te 1. (b) Site 2. Figure 3. ISC (%) of study site catchments versus correlation (R2) of stream temp. and air temp. for each site, linear best fit and correlation of data to linear best fit. At the catchment scale, ISC increases from 13.7% to 24.3% within the study area. As urbanization increases from catchment to catchment, average temperatures steadily increase during summer and winter months. Summer averages at monitoring sites with heavily urbanized catchments are as much as 4–5°C higher than the least urbanized catchment. This increase in summer stream temperature occurs in the daily, weekly, and monthly stream temperature averages. Urbanization also influences stream temperature averages during winter months. Winter stream temperature averages in heavily urbanized sub-catchments were as much as 3–4°C higher than less urbanized catchments. Rising average stream temperature with increasing urbanization agrees with the findings of Galli (1990), who suggests a 0.09°C increase in temperature for each 1% increase in ISC. In Boone Creek, however, we find that average stream temperature rises by 0.37°C for each 1% increase in ISC. We suspect that the large rise is a result of the low thermal inertia of Boone Creek and the combined influence of long-term and short-term temporal effects of increasing urbanization within the study area. This trend also strongly influences correlation between stream and air temperatures, as shown in Figure 3, which compares the correlation of stream versus air temperature (represented by R2) with percentage ISC within each catchment. In Figure 3 values for R2 are derived from plots of stream versus air temperature, such as what is shown in Figure 2, for each monitoring site. As Figure 3 clearly shows, there is a steady downward trend in the correlation of stream and air temperature as ISC within catchments increases. Values of R2 ranged from 0.70 to 0.42, 0.89 to 0.52, and from 0.89 to 0.52 at daily, weekly, and monthly time scales, respectively. URBANIZATION INFLUENCES ON STREAM TEMPERATURE The largest decrease in R2 of stream and air temperature was seen at the weekly and monthly scales; R2 decreased by 0.37 at both weekly and monthly scales. This, in addition to the increases seen in stream temperature averages, suggests that effects of urbanization within catchments have a more pronounced effect upon stream temperature behavior over long-term temporal scales rather than short-term. Figure 3. ISC (%) of study site catchments versus correlation (R2) of stream temp. and air temp. for each site, linear best fit and correlation of data to linear best fit. Catchment Effects Many of the effects that urbanization has on stream temperature behavior can be seen in Figure 2, which plots stream versus air temperature for the site with the least urbanized catchment (site 1) and a site with one of the most heavily urbanized catchments (site 2). Figure 2 also shows a variable representing a fictional 1:1 stream and air temperature series for reference. The data represented by Figure 2 portray increasing variation in stream temperature, increasing average stream temperature, and decreasing correlation between stream and air temperature as urbanization increases. They also show that groundwater has a strong influence on this gaining stream throughout the length of the reach. Similar plots for each site were constructed, but Figure 2 only includes plots for sites at the lower and upper range of catchment ISC among study sites. Figure 1. Map of Boone Creek, ISC, study area watershed, and sites 1 and 2 catchments. —28— DISCUSSION Previous studies have shown that with distance downstream, air and stream temperatures trend towards equilibrium (Mohseni and Stefan, 1999; Stefan and Preud’homme, 1993). Our results show that increasing urbanization within a watershed can cause this relationship to break down within low order, urban streams. Rising levels of ISC within a watershed relate to a downward trend in the correlation of stream versus air temperature which is seen at daily, weekly, and monthly time scales. The results of our analyses and previous work both suggest that this can be largely attributed to the effects of increased exposure to solar radiation as well as increased runoff from impervious surfaces (Beschta and Taylor, 1988; Brown, 1969; LeBlanc et al., 1997; Nelson and Palmer, 2007; Sinokrot and Stefan, 1993). Over short time scales (e.g., days to a week), the effects of runoff are more pronounced due to surges in stream temperature that occur during some storm events. Over longer periods (e.g., weeks to months), exposure to solar radiation due to the lack of shading is the dominant variable. Low-order urban streams are particularly susceptible to these influences due to their low thermal inertia. Analysis suggests that the cumulative influences of these short- and long-term variables within catchments as well as the immediate vicinity of a stream have a significant effect. Analysis also suggests that the impact of urbanization upon stream temperature within low-order urban streams can be seen at daily, weekly, and monthly temporal scales. Previous research suggests that similar results can be expected not only in other low- order streams, but also in larger discharge streams (Klein, 1979; LeBlanc et al., 1997; Nelson and Palmer, 2007; Pluhowski, 1970), though response times and the magnitude of the effects may be dampened as a result of higher thermal inertia. If urbanization, however, becomes increasingly widespread in the headwaters of larger streams, then the cumulative effects of urbanization may prove to be more significant than the dampening caused by the higher thermal inertia. ) Over short-term temporal scales ISC in the 25 m buffer influences stream temperature primarily through increased amplitude of stream temperature surges from storm runoff, as Figure 4 illustrates. This agrees with previous findings, though the increase we see in Boone Creek is more drastic than previous studies (Anderson et al., 2007; Herb et al., 2008; Nelson and Palmer, 2007). Buffer Area Effects ISC of the 25 m buffer areas was found to range from 1% to 75% for the study sites. A relation between stream temperature and increasing urbanization within these 25 m buffers was seen when comparing ISC and correlation of stream versus air temperature. As urbanization in the buffer area increases, correlation of air and stream temperature decreases, though the decrease is not as pronounced as what was observed at the catchment scale. This effect occurs at daily, weekly, and monthly temporal scales. A relation was not found between ISC in the buffer area and stream temperature averages. This is due to the low ISC within a 25 m buffer of each side of the channel in the downstream areas which, though vegetated, provide no shading and therefore have relatively low ISC but are still associated with increased stream temperature. The general lack of —29— J. S. RICE ET AL. of a threshold response, though additional data needs to be collected to support this. stream shading in these areas despite the presence of landscaped, non-native vegetation within the buffer areas of these sections suggests that in an urban setting, riparian vegetation that provides little shading has negligible effect on moderating long-term stream temperature changes in low- order urban streams. This suggests the importance that shading has on stream temperature and agrees with earlier work on this subject (Beschta and Taylor, 1988; Brown, 1969; LeBlanc et al., 1997; Nelson and Palmer, 2007; Sinokrot and Stefan, 1993). REFERENCES McMahon G, Cuffney TF. 2000. Quantifying urban intensity in drainage basins for assessing stream ecological conditions. Journal of the American Water Resources Association 36: 1247–1261. Anderson JL, Anderson WP, Thaxton CS, Babyak CM. 2007. Dynamics of episodic temperature variations in an urbanized, high-gradient stream. Geological Society of America Abstract with Program 39: GSA Southeast Section Meeting, Savannah, GA. Mohseni O, Stefan HG. 1999. Stream temperature/air temperature relationship: a physical interpretation. Journal of Hydrology 218: 128–141. Anderson WP, Anderson JL, Thaxton CS, Babyak CM. 2010. Changes in stream temperatures in response to restoration of groundwater discharge and solar heating in a culverted, urban stream. Journal of Hydrology 393: 309–320. Nelson KC, Palmer MA. 2007. Stream temperature surges under urbanization and climate change: data, models, and responses. Journal of the American Water Resources Association 43: 440–452. Paul MJ, Meyer JL. 2001. Streams in the urban landscape. Annual Review of Ecology & Systematics 32: 333–365. Beschta RL, Taylor RL. 1988. Stream temperature increases and land use in a forested Oregon watershed. Water Resources Bulletin 24: 19–25. Pilgrim JM, Fang X, Stefan HG. 1998. Stream temperature correlations with air temperature in Minnesota: implications for climate warming. Journal of the American Water Resources Association 34: 1109–1121. Bogan T, Mohseni O, Stefan HG. 2003. Stream temperature- equilibrium temperature relationship. Water Resources Research 39. Brown G. 1969. Predicting temperatures of small streams. Water Resources Research 5: 68–75. Pluhowski EJ. 1970. Urbanization and its effect on the temperature of streams on Long Island, New York. Geological Survey Professional Paper, 627-D. Constantz J. 1998. Interaction between stream temperature, streamflow, and groundwater exchanges in alpine streams. Water Resources Research 34: 1609–1615. Schueler TR. 1994. The importance of imperviousness. Watershed Protection Techniques 1: 100–111. Crisp DT, Howson G. 1982. Effect of air temperature upon mean water temperature in streams in the North Pennines and English Lake District. Freshwater Biology 12: 359–367. Sinokrot BA, Stefan HG. 1993. Stream temperature dynamics: measurements and modeling. Water Resources Research 29: 2299–2312. Erickson TR, Stefan HG. 1996. Correlations of Oklahoma stream temperatures with air temperatures. St. Anthony Falls Laboratory Project Report. Stefan HG, Preud’homme EB. 1993. Stream temperature estimation from air temperature. Water Resources Bulletin 29: 27–45. Wang L, Lyons J, Kanehl P. 2003. Impacts of urban land cover on trout streams in Wisconsin and Minnesota. Transactions of the American Fisheries Society 132: 825–839. Galli J. 1990. DISCUSSION During a portion of the monitoring period of this study (Spring and Summer 2010), 44 temperature surge events, which we define as a change of at least 1°C within 15 minutes of monitoring, occurred in Boone Creek. To avoid including cumulative effects of the downstream transport of thermal energy, temperature surge amplitudes were calculated for each monitoring site from simultaneous readings of the initial 15 minute temperature surge from each surge event. The surge events had amplitudes whose means ranged from 1.90°C to 3.27°C. The mean temperature surge duration of these events was above three hours, which is comparable to the findings of Nelson and Palmer (2007), and the maximum surge duration exceeded ten hours. While these durations last less than one day, there were several instances in the dataset in which storms occurred on multiple consecutive days or multiple times in a single day, rendering their influence to slightly longer temporal scales. Figure 4 plots the mean amplitude of these surges for each site versus 25 m buffer ISC and shows a significant increase in mean temperature surge amplitude as ISC increases. As ISC within the 25 m buffer increases, mean temperature surge amplitude quickly responds and peaks at 3.27°C for the site whose upstream section has 75% ISC. The sharp increase of mean temperature surge amplitudes above 40% ISC is suggestive Figure 4. ISC (%) within 25 m buffer area versus mean stream temperature surge amplitude. As world population and urban areas are both expected to expand in the future, it is likely that the impact of urbanization upon streams will also become more pronounced. Planning for urban effects may become more difficult because many existing models focus on, or at least heavily involve, the tendency of stream temperature to trend toward equilibrium with air temperature. The concept of equilibrium temperature is a relationship that appears to be negatively impacted by urbanization, and may become obsolete as a tool for predicting stream temperatures, especially in low-order, headwater streams. These issues highlight the need for careful urban planning that takes into account the need to protect urban waterways, not only from chemical pollution, but thermal pollution as well. It is our hope that this study demonstrates the need for focusing on the significant role that urbanization has on thermal water quality issues. Figure 4. ISC (%) within 25 m buffer area versus mean stream temperature surge amplitude. —30— URBANIZATION INFLUENCES ON STREAM TEMPERATURE Herb WR, Janke B, Mohseni O, Stefan HG. 2008. Thermal pollution of stream by runoff from paved surfaces. Hydrological Processes 22: 987–999. ACKNOWLEDGEMENTS The authors would like to thank the University Research Council of Appalachian State University (ASU), the ASU Office of Student Research, and the ASU Department of Geology and Department of Physics and Astronomy for funding portions of this research. This manuscript also benefitted from comments by Chuanhui Gu and Roy C. Sidle. The authors would like to thank the University Research Council of Appalachian State University (ASU), the ASU Office of Student Research, and the ASU Department of Geology and Department of Physics and Astronomy for funding portions of this research. This manuscript also benefitted from comments by Chuanhui Gu and Roy C. Sidle. Johnson FA. 1971. Stream temperatures in an alpine area. Journal of Hydrology 14: 322–336. Klein R. 1979. Urbanization and stream quality impairment. Water Resources Bulletin 15: 948–963. LeBlanc RT, Brown RD, FitzGibbon JE. 1997. Modeling the effects of land use change on the water temperature in unregulated urban streams. Journal of Environmental Management 49: 445–469. REFERENCES Thermal impacts associated with urbanization and stormwater best management practices. Metropolitan Washington Council of Governments: Washington, DC. —31—
W3084197144.txt	https://gredos.usal.es/bitstream/10366/147138/1/Rego%20et%20al.%20-%202020%20-%20Trains%20of%20attosecond%20pulses%20structured%20with%20time-o.pdf	en		Trains of attosecond pulses structured with time-ordered polarization states	Optics letters/Optics index	2,020	public-domain	3,871	5636 Letter Vol. 45, No. 20 / 15 October 2020 / Optics Letters Trains of attosecond pulses structured with time-ordered polarization states Laura Rego,* Julio San Román, Luis Plaja, AND Carlos Hernández-García Grupo de Investigación en Aplicaciones del Láser y Fotónica, Departamento de Física Aplicada, University of Salamanca, E-37008 Salamanca, Spain *Corresponding author: laura.rego@usal.es Received 17 August 2020; revised 4 September 2020; accepted 5 September 2020; posted 8 September 2020 (Doc. ID 404402); published 6 October 2020 Ultrafast laser pulses generated at the attosecond timescale represent a unique tool to explore the fastest dynamics in matter. An accurate control of their properties, such as polarization, is fundamental to shape three-dimensional laser-driven dynamics. We introduce a technique to generate attosecond pulse trains whose polarization state varies from pulse to pulse. This is accomplished by driving high-harmonic generation with two time-delayed bichromatic counter-rotating fields with proper orbital angular momentum (OAM) content. Our simulations show that the evolution of the polarization state along the train can be controlled via OAM, pulse duration, and time delay of the driving fields. We, thus, introduce an additional control into structured attosecond pulses that provides an alternative route to explore ultrafast dynamics with potential applications in chiral and magnetic materials. © 2020 Optical Society of America https://doi.org/10.1364/OL.404402 The most fundamental mechanisms in atoms, molecules, or solid systems are governed by dynamic interactions occurring at the attosecond timescale. The quest of obtaining light pulses at such short timescales, in order to drive, prove, and observe such dynamics, has driven the scientific community’s efforts towards the development of coherent high-frequency light sources during the last two decades. As a consequence of the uncertainty principle, ultrashort pulses must contain coherent radiation spanning into the extreme-ultraviolet (XUV) or x-ray regimes in order to reach the sub-femtosecond timescale. While recently it has been shown that high-energy sub-femtosecond pulses can be obtained from free-electron lasers, highly coherent light bursts emitted at the attosecond timescale can be obtained through high-order harmonic generation (HHG). Shortly after the first demonstration of the comb of high-order harmonics driven by intense lasers [1,2], Farkas and Tóth theoretically forewarned of their characteristic sub-femtosecond duration [3]. Since its experimental confirmation by several groups in the early 2000s [4,5], HHG stands as the most robust tool not only to generate pulses down to few tens of attoseconds, but also to gain control on the properties of XUV radiation with unprecedented accuracy. Attosecond pulse trains provide extraordinary 0146-9592/20/205636-04 Journal © 2020 Optical Society of America possibilities as quantum stroboscopes [6] for the time-control electron dynamics and the imaging of the electron’s quantum state. The success of HHG to produce attosecond pulses resides in its extraordinary coherence. At the microscopic level, the process is well understood from a semiclassical approach [7]. First, the driving laser field ionizes an electronic wavepacket, which is subsequently steered away from its parent ion. When the driving field reverses its sign, the wavepacket is driven back to the parent ion. Upon recombination, the kinetic energy acquired during the wavepacket excursion is released as high-order harmonics of the driving laser. This sub-femtosecond process is repeated at each half-cycle of the driving pulse, thus leading to the generation of a train of attosecond pulses. One of the properties of HHG attosecond pulses whose control has been elusive for many years is spin angular momentum (SAM), associated with polarization. The generation of high harmonics and attosecond pulses with different polarization states is of great interest as a tool to probe the fastest spin dynamics in materials, and to uncover fundamental understanding on spin scattering and transport on attosecond timescales. Until recently, the dynamics leading to HHG were restricted along the direction of the linear polarization of the driving field, thus limiting the harmonic photons to linearly polarized states. If driven elliptically, the recombination probability diminishes, drastically reducing the yield of the high-order harmonic emission [8]. Fortunately, different techniques have recently exploited the extremely coherent nature of HHG to generate high-order harmonics—and attosecond pulses—with polarization states from linear to circular [9]. The use of non-symmetric targets, such as molecules or solids [10,11], or the use of proper combinations of linearly polarized drivers with different frequencies [12] enable the generation of elliptically polarized harmonics. Other approaches made use of proper macroscopic configurations, such as the use of non-collinear counter-rotating driving beams with the same color [13]—which allowed for the production of circularly polarized attosecond pulses for the first time [14,15]—or the combination of two HHG sources [16,17]. The first technique that allowed the generation of circularly polarized high-order harmonics used a driving field composed of two-color, counter-rotating, circularly polarized laser fields— the so-called bi-circular driver [18–20], which can present Letter high ionization and recombination rates [21]. This technique stands nowadays as an efficient method to generate circularly polarized harmonics with brightness and energies comparable to single-color linearly polarized HHG [22,23]. The primary spectral feature of bi-circular HHG is the generation of pairs of circularly polarized harmonics with opposite helicity—right circular (RCP) and left-circular (LCP)—due to SAM conservation. The generation of both LCP and RCP harmonics from bi-circular HHG driving results in the emission of linearly polarized attosecond trains of pulses, where the polarization tilt angle rotates from pulse to pulse. Recently, the introduction of orbital angular momentum (OAM) in the driving pulses, has allowed us to not only to generate circularly polarized harmonic vortex beams, but also to control the polarization of the attosecond pulse trains in the bi-circular scheme [24]. However, up to now, SAM—or the polarization state— has been introduced at the attosecond timescale as a time-independent variable. To the best of our knowledge, there is no proposal—nor experimental demonstration— yet to produce attosecond pulse trains with controlled, well resolved, time-ordered polarization. We note, however, that some recent works have reported the generation of high harmonics whose polarization state slightly changes along the course of the pulse as a result of the highly nonlinear nature of HHG [11,25]. In this Letter, we introduce a technique to generate trains of attosecond pulses with different—and controlled—polarization states from pulse to pulse, thus presenting time-ordered SAM along the pulse train. The control knob lies on the proper choice of the combination of two time-delayed bi-circular driving fields, with different OAM content. Very recently, HHG has been proven to introduce time-dependent OAM into a light beam, denoted as self-torque [26]. In this work, we demonstrate that driving HHG with two time-delayed bi-circular vortex beams with properly chosen OAM content results in trains of attosecond pulses of the SAM state changing sequentially from pulse to pulse. Similar schemes that make use of time-delayed pulses with different SAM, instead of OAM, are now widely used, such as the polarization gating technique [27]. It is already known that introducing OAM in bi-circular driving results in high-order harmonics with opposite helicity, that can be spatially separated in the far-field into a double ring profile [24], as we schematically illustrate in Fig. 1(a). If we consider an OAM bi-circular field composed of two pulses with fundamental (ω1 ) and doubled frequency (ω2 = 2ω1 ), counterrotating (σ1 = −1 LCP, σ2 = 1 RCP) and with topological charge—or OAM content—of `1 and `2 , respectively, the difference in divergence in the far-field between the LCP and RCP harmonics is given by 1β ∝ (2`1 − `2 )\|`1 + `2 \|/(`1 + `2 ) [24]. As a general trend, the spatial separation between the LCP and RCP harmonic rings increases for larger differences between `1 and `2 , if `1 and `2 have opposite signs. The outer (inner) ring—with larger (smaller) divergence— exhibits the same helicity as that of the driving pulse with higher (lower) \|`\|. For the convention adopted in this work, if \|`1 \| > \|`2 \|, the far-field outer ring is composed of LCP harmonics, whereas if `2 \| > \|`1 \|, it is composed of RCP harmonics, as illustrated in Fig. 1(a). From the temporal point of view, by selecting the high-order harmonics emitted at each of the rings, attosecond pulse trains with LCP or RCP states are generated with a time-independent polarization state. Vol. 45, No. 20 / 15 October 2020 / Optics Letters 5637 Fig. 1. (a) Scheme of polarization control of HHG driven by an OAM bi-circular field (ω1 , `1 , LCP; 2ω1 , `2 , RCP). The HHG spectrum is composed of pairs of circularly polarized harmonics with opposite helicity, separated in divergence in the far-field. (b) Generation scheme of trains of attosecond pulses with time-ordered polarization states. In this case, HHG is driven by two time-delayed OAM bi-circular fields, A and B, where, for this example, we have chosen \|`1A \| > \|`2A \| and \|`1B \| < \|`2B \|. As a consequence, in the outer (inner) ring, the polarization of the harmonics—and the attosecond pulses—varies continuously in time from LCP (RCP) to RCP (LCP), as shown schematically in the right panel. In order to introduce a temporal dependency into the polarization state of the attosecond pulses within the train, we drive HHG with a combination of two time-delayed OAM bicircular beams with opposite topological charges [see Fig. 1(b)]. If we denote each of the OAM bi-circular beams as A and B, we require an OAM combination of \|`1A \| = \|`2B \| and \|`1B \| = \|`2A \|, maintaining `1 and `2 with opposite signs, as mentioned before. The temporal delay between the two OAM bi-circular beams, td , causes the emitted high-order harmonics in each of the rings to evolve from LCP (RCP) to RCP (LCP), as depicted schematically in Fig. 1(b). For this figure, we have selected \|`1A \| > \|`2A \| and \|`1B \| < \|`2B \|, and, thus, the polarization state of the emitted attosecond pulse trains will evolve from LCP (RCP) to RCP (LCP) at the outer (inner) harmonic ring. The pace of SAM changes can be controlled through the OAM of the driving pulses and their temporal properties (pulse duration and time delay). We have performed simulations of macroscopic HHG in an argon gas jet, including propagation. Our calculations combine the strong-field approximation with the electromagneticfield propagator [28]. This theoretical approach successfully describes the macroscopic HHG in several configurations involving SAM and/or OAM [13–16,24,26]. We model the driving pulses with a sin2 envelope, with central wavelengths of 800 nm (400 nm) for the ω1 (2ω1 ) driver and a peak intensity of 1.4 × 1014 W/cm2 each. The spatial structure of the laser beams is represented as a Laguerre–Gaussian beam propagating in the z direction, with topological charge ` and radial nodes index p. The beam waist, w0 , of each laser beam is chosen so 5638 Vol. 45, No. 20 / 15 October 2020 / Optics Letters Letter exhibits a rotation from pulse to pulse and also within each individual pulse. When driven by a single bichromatic counterrotating field, the tilt angle is known to rotate 120 deg from pulse to pulse. In contrast, in this scenario, the rotation is more complicated due to the overlap of the two time-delayed fields. The structure of the trains remains almost invariant within a divergence angle width of 0.5 mrad, approximately. Finally, we note thatqthe time-dependent degree of polarization, defined as Fig. 2. (a) Spatial intensity profile of the HHG beam obtained with an OAM bi-circular field with `1A = 3, `2A = −2 (and, thus, `1B = 2, `2B = −3). The helicity of the inner and outer rings changes along time. In panels (b) and (c), we show the HHG spectrum of the outer and inner rings, whereas in panels (d) and (e) we show the corresponding attosecond pulse train envelopes (purple), ellipticity (green), and tilt angle (orange). that the different OAM modes overlap at the rings of maximum intensity. In Fig. 2, we present the results of the simulations when considering `1A = 3, `2A = −2 (and thus `1B = 2, `2B = −3), all of them with p = 0. The pulse duration of each driver is 7.7 fs in full width at half-maximum (FWHM) in intensity, and the temporal delay between the A and B drivers is td = 7.7 fs. The waist of the driving beams, w0 , is 30 µm for ` = ±2 and 24.54 µm for ` = ±3. Looking at the spatial intensity profile of the HHG beam in Fig. 2(a), integrated over harmonic orders above the 10th, we distinguish two spatial rings. In Figs. 2(b) and 2(c), we show the far-field HHG spectrum of the outer ring (detected at 1.6 mrad) and that at the inner ring (detected at 0.6 mrad), respectively. In both HHG spectra, we can already distinguish the pairs of LCP (red line) and RCP (blue line) harmonics, a well-known result of SAM conservation in HHG driven by the bi-circular field [22]. Note that we observe a frequency shift in the harmonics, which is induced by the pulse envelope through the time-dependent variation of the intrinsic phase. Computing the inverse Fourier transform of the HHG spectra above the 10th harmonic order, we can retrieve the attosecond pulse trains shown in purple in Figs. 2(d) and 2(e), for the outer and inner rings, respectively. The polarization state of the attosecond pulse trains can be monitored through the temporal evolution of the Stokes parameters [29]. The time-dependent ellipticity can be thus described p as (t) = tan 0.5 arctan(S3 (t)/ S12 (t) + S22 (t)) , while the time-dependent tilt angle is θ (t) = 0.5 arctan[S2 (t)/S1 (t)]. In Figs. 2(d) and 2(e), we show the time-dependent ellipticity, (t), in green and the time-dependent tilt, θ (t), in orange for the outer and inner rings, together with the attosecond pulse train envelope in purple—which corresponds to the Stokes parameter S0 (t). We can see how the polarization state of the attosecond pulses evolves from LCP (RCP) to RCP (LCP) along the train for the outer (inner) spatial ring. The tilt angle P (t) = S12 (t) + S22 (t) + S32 (t)/S0 (t), is constant and equal to one for all of the cases presented in this work, as expected from the coherence of the HHG process. To have a better insight into the origin of the temporal structure of the attosecond pulse trains, we present in Fig. 3 the time-frequency analysis for the two cases depicted in Fig. 2. At the outer ring (left column), the LCP harmonics dominate at the front part of the pulse train, whereas RCP harmonics dominate at the rear part of the pulse. This behavior is reversed at the inner ring (right column). Note that the temporal delay between the OAM bi-circular drivers A and B is chosen to be equal to the FWHM of the driving pulses in order to obtain a homogeneous and linear variation of the ellipticity over time. A different choice of the time delay can be used to customize the temporal variation of the ellipticity of the attosecond pulse train. Analogously, the choice of the driving pulse lengths, and their OAM, allows for a custom control of the time-dependent polarization structure of the attosecond pulse train. To show the versatility of this technique, we have performed simulations with few-cycle driving laser pulses and a different OAM combination. In Fig. 4, we present the simulation results when considering Laguerre–Gauss beams with `1A = 2, `2A = −1 (and, thus, `1B = 1, `2B = −2), all of them with p = 0. The driving pulse length is 1.9 fs FWHM, and the temporal delay between the A and B drivers is td = 1.9 fs. The waist of the driving beams is w0 = 30 µm for ` = ±1 and w0 = 21.4 µm for ` = ±2. Now, the number of attosecond pulses in the train is reduced—as expected—, and, thus, the variation rate of the ellipticity in time increases. In this case, we present the attosecond pulse train emitted at an intermediate divergence angle (detected at 1.7 mrad) in order to illustrate the possibility of obtaining a polarization evolution from linear to highly elliptical, in contrast to the elliptical-to-elliptical polarization variation in the outer and inner rings. On the other hand, the Fig. 3. Time-frequency analysis for RCP (first row) and LCP (second row) projections of the harmonic emission at the outer (left column) and inner (right column) rings, depicted in Fig. 2. The outer (inner) ring LCP (RCP) harmonics precede temporally the RCP (LCP) ones. The spectral width of the Gaussian mask used to perform the time-frequency analysis is ω1 . Letter Fig. 4. Attosecond pulses—envelope (purple), ellipticity (green), and tilt angle (orange)—obtained with a few-cycle OAM bi-circular field with `1A = 2, `2A = −1 (and, thus, `1B = 1, `2B = −2) for an intermediate divergence angle (top) and inner ring (bottom) emission. The two-dimensional (2D) evolution of the attosecond electric fields is depicted in the right column. inner ring consists of two elliptically polarized attosecond pulses with opposite helicities (detected at 1 mrad). In conclusion, we have presented a scheme for the generation of attosecond pulse trains with time-ordered polarization states. By driving HHG with two time-delayed OAM bi-circular drivers, two spatially separated rings of harmonics are generated, each with polarization evolving from LCP to RCP and vice versa. The proper choice of the OAM, pulse length, and time delay of the driving pulses allows for custom control over the temporal evolution of the SAM of the pulses within the train. We, thus, introduce a novel structured ultrafast light source by adding a new degree of freedom, time-ordered SAM, or polarization into the quantum stroboscope scheme. Similarly, time-ordered OAM—or self-torque—was introduced recently into the family of structured light beams thanks to HHG [26]. By adding this temporal dependency into the SAM of attosecond EUV beams, this work opens the route towards new probing scenarios—such as the study of ultrafast dynamics in chiral systems, pump–probe experiments with polarization varying states, or time-dependent polarization x-ray magnetic circular dichroism—that provide an alternative route to probe the ultrafast dynamics of matter systems at the nanoscale. Funding. 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https://openalex.org/W3106158247	https://www.epj-conferences.org/articles/epjconf/pdf/2017/32/epjconf_fusion2017_00037.pdf	English	null	Interference effect between neutron direct and resonance capture reactions for neutron-rich nuclei	EPJ web of conferences	2,017	cc-by	4,585	1 Introduction incident neutron wave function smoothly connects with the interior wave function of A + 1 nucleus according to the R-matrix theory [1, 2]. For most nuclei, the com- pound process occupies its dominant part of the neutron capture reaction. This is because the number of resonances reaches to hundreds to millions at a capture state energy, E∗= E + S n, where E and S n are the incident neutron energy and the neuron threshold of A + 1 nucleus, respec- tively. As a consequence, the neutron capture cross section of the compound process ranges approximately from 10−1 to 105 barns at neutron resonance regions. On the other hand, typical neutron capture cross section of the direct process is the order of 0.1 to 1 mb. The neutron capture reaction is one of the simplest nu- clear reactions, but its importance is recognized in vari- ous ﬁelds. For example, the heavy elements existing in the nature are considered to be produced by a successive neutron capture in a neutron-rich environment (the leading candidates are the s-process and r-process). In the ﬁeld of accelerators and reactors, the neutron capture generates unstable nuclei in the structure materials used for conﬁn- ing neutrons and quantum beams in their long-term op- eration. The production of unstable nuclei by (secondary) neutrons cannot be ignored in terms of the radioactive pro- tection and the decommissioning process. Vice versa, the neutron capture has an potential to transmute a radioac- tive unstable one to stable nucleus in terms of reducing radioactive wastes produced in various nuclear sites, espe- cially in nuclear reactors. Moreover, using prompt γ-rays resulted from the neuron capture, non-destructive analysis in materials is also performed. It is therefore important to know accurate neutron capture cross sections systemat- ically for nuclei in the nuclear chart, in particular at low energy region where its cross section becomes large. However, it is not always the case that the compound process is larger than the direct one in case of light nuclei and magic nuclei, for which the number of resonances at the capture state energy is small. In this case, neutron cap- ture cross section of the compound process becomes small and the direct process comes to play a comparable role to the compound one. One of the examples is 12C [3] and 16O [4]. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Interference effect between neutron direct and resonance capture reactions for neutron-rich nuclei Futoshi Minato1,⋆and Tokuro Fukui1,2, 1Nuclear Data Center, JAEA, Tokai, 319-1195, Japan 2INFN, Napoli, 80126, Italy Abstract. Interference eﬀect of neutron capture cross section between the compound and direct processes is investigated. The compound process is calculated by resonance parameters and the direct process by the potential model. The interference eﬀect is tested for neutron-rich 82Ge and 134Sn nuclei relevant to r-process and light nucleus 13C which is neutron poison in the s-process and produces long-lived radioactive nucleus 14C (T1/2 = 5700 y). The interference eﬀects in those nuclei are signiﬁcant around resonances, and low energy region if s-wave neutron direct capture is possible. Maxwellian averaged cross sections at kT = 30 and 300 keV are also calculated, and the interference eﬀect changes the Maxwellian averaged capture cross section largely depending on resonance position. ⋆e-mail: minato.futoshi@jaea.go.jp DOI: 10.1051/epjconf/201716300037 DOI: 10.1051/epjconf/201716300037 EPJ Web of Conferences 163, 00037 (2017) FUSION17 1 Introduction The cross section has a maximum value at E = Eλ. The direct radiative neutron capture cross section can be calculated by It is then natural to think of whether the interference eﬀect also becomes signiﬁcant in neutron-rich nuclei. In fact, the compound and direct processes become compara- ble in the vicinity of the neutron magic number [7, 8], and therefore a clear interference eﬀect is expected to be found around that mass region. We choose three nuclei as test ones, that is 13C, 86Ge, and 134Sn. The ﬁrst nucleus locates next to the stability line in the nuclear chart, but it is im- portant for nuclear data evaluation because 13C is neutron poison in s-process and can be a generator of long-lived nucleus 14C (T1/2 = 5700 y). The latter two are typical neutron-rich nuclei which may be involved in r-process. σdir,i→f (E) = π k2 gJ\|Udir i→f \|2, (3) (3) where the collision matrix Udir is written as [10, 15] Udir i→f = 1 √ 2J + 1 ( 16 9ℏv )1/2 ¯e ∑ i k3/2 γ Q fi. (4) (4) The factor v−1/2 = √m/ℏk is coming from the assumption of a unit-ﬂux incoming wave in the entrance channel. The eﬀective charge of neutron is ¯e = −eZ/A, the emitted γ-ray wave number kγ = ϵγ/ℏc, ϵγ = E −ϵf the γ-ray energy, ϵf the single particle energy of the ﬁnal state calculated by the Woods-Saxon potential, and Q fi = T fiBfiAfi the tran- sition matrix [13], where Bfi is the parameter concerning the spectroscopic factor. The radial integral part, Tfi, is given by [13, 16] The factor v−1/2 = √m/ℏk is coming from the assumption of a unit-ﬂux incoming wave in the entrance channel. The eﬀective charge of neutron is ¯e = −eZ/A, the emitted γ-ray wave number kγ = ϵγ/ℏc, ϵγ = E −ϵf the γ-ray energy, ϵf the single particle energy of the ﬁnal state calculated by the Woods-Saxon potential, and Q fi = T fiBfiAfi the tran- sition matrix [13], where Bfi is the parameter concerning the spectroscopic factor. The radial integral part, Tfi, is given by [13, 16] In addition to neutron direct capture reaction, there is a semi-direct process coming out of E = 5 ∼10 MeV. But we do not discuss it because our interest in this work is less than E = 1 MeV. 2 Calculation where the scattering wave function calculated by a neutron-nucleus optical potential is In neutron resonance region for the nuclei studied in this work, the exit channel is classiﬁed only to the elastic scat- tering and the capture reaction, and other channels do not take part in. We also assume that the target nucleus is spherical. The entrance and the capture channels are spec- iﬁed by i = (Jπ, ji, li) and f = (Jπ f , nf , jf , lf ), respectively. Here, li is the relative orbital angular momentum between the projectile and the target and ji = li ± 1/2; ⃗J = ⃗S + ⃗li is the total spin of the system, ⃗S = ⃗s + ⃗sn the total spin, and s and sn the spins of target and projectile, respectively; n f is the principle quantum number, l f the orbital angular momentum, jf = l f ± 1/2 the total angular momentum of the bound state, and J f = J ± 1. χ( jili; r) = √ 4π √ 2li + 1ili i 2 ( I ji li (r) −U ji li O ji li (r) ) −−−−→ r→∞ √ 4π √ 2li + 1ili i 2 ( ei(kr−lπ 2 ) −U ji li e−i(kr−lπ 2 )) (6) (6) and u(nf jf lf ; r) is the radial part of the single particle wave function of the ﬁnal state. We here deﬁne the scattering matrix element U ji li = e−2iϕi. The angular momentum part, Afi is written as A fi = ˆJ f ˆjf ˆjiˆli √ 3 4π⟨li010\|lf 0⟩ × { ji s J Jf 1 jf } { li ji 1/2 jf l f 1 } . (7) The cross section of neutron capture from entrance channel i with spin-parity Jπ to capture channel f can be described by the R-matrix theory, which is given by [1, 2, 9, 10, 14, 15] (7) (7) σi→f (E) = π k2 gJ ∑ λ ie−iϕiΓ1/2 λniΓ1/2 λγ f (Eλ −E) −iΓλ/2 2 , (1) (1) So far, we considered the compound process and the direct process separately. 1 Introduction The direct process of those nuclei is strong enough that it aﬀects the evolution of stars and s-process. Besides light nuclei and magic nuclei, the direct process becomes signiﬁcant in neutron-rich nuclei as well because the num- ber of resonances available to form the compound state is expected to be small due to the small neutron threshold energy. For this reason, the direct process in neutron-rich nuclei has been actively investigated [5–8]. The neutron capture at low energies can be divided into two parts, that is the compound process and the direct pro- cess. Both of the processes emit γ-rays with the same en- ergy if the initial and ﬁnal states are identical. However, the compound process stops by a compound state before γ-emission one hand, the direct process instantly goes to the ﬁnal state by the electronic dipole radiation of incident neutron on the other hand. The compound process has a characteristic resonance structure in the cross section (see Fig. 2 , for instance). The strong peaks emerge when the The total neutron capture cross section is described in general by summing the results of the compound and di- rect processes. However, it was discussed [9, 10] that there is an interference eﬀect between these processes, similar to the potential scattering and compound one in the elas- tic channel. Such an interference eﬀect was investigated DOI: 10.1051/epjconf/201716300037 EPJ Web of Conferences 163, 00037 (2017) EPJ Web of Conferences 163, 00037 (2017) FUSION17 deﬁned by k = √ 2mE/ℏ, where E and m = A/(A + 1)mN are the incident neutron energy and the reduced mass, re- spectively. in proton capture reaction [11], however, it has not been clariﬁed if the interference really exists in the neutron cap- ture reaction. The evaluated neutron nucleus reaction cross section library, JENDL [12], therefore did not consider the eﬀect. However, several recent experiment works have shown unique data distributions in the cross sections of 9Be(γ, n) and 16O(n, γ)17O that one cannot reproduce only by the compound and direct processes. Mengoni and Ot- suka showed [13] that the cross sections can be reproduced theoretically if they take into account the interference ef- fect. If each resonance is isolated from others, Eq. (1) reads σBW,i→f (E) = π k2 gJ ∑ λ ΓλniΓλγ f (Eλ −E)2 + (Γλ/2)2 . (2) (2) Equation (2) is the so-called the multilevel Breit-Wigner formula. 1 Introduction This paper is organized as follows. In Sec. 2, we de- scribe the formalism to calculate the neutron capture cross section brieﬂy. Sec. 3 gives our result and Sec. 4 summa- rizes this work and gives the future perspectives. T fi = ∫∞ 0 u(n f jf lf ; r) rχ(jili; r) dr, (5) (5) 3 Result In order to take into account the non-local eﬀect of the optical potential on the scatter- ing wave function, the Perey and Buck method [18] with the non-local parameter β = 0.85 fm is adopted. Because 82Ge and 134Sn are the short half-life nuclei, the information about the resonance parameters evaluated from experiments is not available. It is not a simple task to calculate those parameters by a theoretical method. There- fore, we adopted, in this work, Γλni, Γλγ and Eλ given in TENDL-2015 [19], in which their resonance parameters are calculated on the basis of statistical properties regard- ing the nuclear excitations. In case of 13C, we used a newly evaluated resonance parameter of JENDL [20]. In the bottom panel of Fig. 1, the neutron capture cross section from E = 0.01 to 1 MeV is shown. The dotted line is the result of only the compound process. It is found that the direct process is essential to explain the experimental data [22] around 10−2 to 10−1 MeV. However, the interfer- ence eﬀect around the resonance at E = 152.4 keV (J = 2) is not strong as studied in 16O [16]. If resonance state is J 0, the ﬁnal state has two pos- sibilities in case of E1 radiation, that is J ± 1. In this case, we assume the equality in the γ decay widths, namely Γλγ f = Γλγ/2. Note that the interference eﬀect between the compound process and direct process occurs when the entrance and exit channels of the two processes are exactly identical. Figure 2 shows the neutron capture cross sections of 134Sn and 82Ge. We can see that the interference eﬀect gives a signiﬁcant change at the ﬁrst resonance appearing at E = 102.1 keV for 134Sn. The changes by the interfer- ence eﬀect can be also observed in the other resonances distributing from E = 0.5 to 1.0 MeV although the modi- ﬁcations are not as strong as the ﬁrst one. As seen in 13C, the interference eﬀect increases the cross section from low energy because the s-wave neutron capture into the 3p3/2 and 3p1/2 bound states is possible, and the compound and direct processes have a comparable cross section. In case of 82Ge, because of the multilevel resonances composed of J = 1/2 and 3/2 above E = 100 keV, the interference eﬀect gives complicated changes. 3 Result The ﬁrst and second terms are indentical to the Breit- Wigner formula and the direct capture cross section, re- spectively, and the third term is nothing but the one from the interference eﬀect. Figure 1 shows the neutron capture cross sections of 13C. The resultant 14C nucleus has the excited states of 2+ and 1−below the neutron threshold energy, and the 0+ ground state. Since the target has the initial spin of Jπ = 1/2−, the cross section is obtained by summing the results of total angular momentum, J = 0 to 3 based on the assump- tion that only E1 transition occurs. The top panel shows the cross section from E = 10−8 to 1 MeV. The solid and dashed lines are the results of compound process + direct process + interference eﬀect (Com+Dir+Int) and com- pound process + direct process (Com+Dir), respectively. The Woods-Saxon potential parameter V is determined so as to reproduce the most recent experimental data of the thermal neutron capture cross section, σγ(th) = 1.496 b [21] and at E = 64 keV [22] in case of Com+Dir+Int, as mentioned in the previous section. The interference eﬀect plays a signiﬁcant role from low energy region. This is because the s-wave neutron capture into the 1p1/2 bound state is concerned both with the compound and direct pro- cesses, and those cross sections are comparable. As inci- dent neutron energy increases, the contribution from the s-wave neutron becomes weak, and the p-wave neutron comes to play a role from E ∼0.01 MeV instead. Single particle wave functions and levels are calcu- lated by the Woods-Saxon potential, in which the param- eters are Rn = 1.236A1/3 fm, the diﬀuseness a = 0.62 fm, the potential depth V = −50 MeV, and the LS poten- tial VLS = 19.4/r d f(r) dr ⃗l · ⃗s MeV. In case of 13C, we ad- just V for each resonance to reproduce the neutron cap- ture cross section at thermal energy and E = 64 keV, that is V1p3/2 = −38.6, V2s1/2 = −51.4, and V1d5/2 = −48.0 MeV, retaining the other parameters. The scattering neu- tron wave function is calculated by the optical model with the Kunieda potential [17]. 2 Calculation The neutron capture cross sec- tion taking into account both processes simultaneously is given by [9, 10] where Γλ = Γλni + Γλγ f is the total decay width for reso- nance state λ, Γλni the neutron width, Γλγ f the γ width, and Eλ the resonance energy; gJ = (2J +1)/(2s+1)(2sn +1) is the statistical factor. The hard-sphere phase shift in chan- nel i is deﬁned by ϕi. Incident neutron wave number is σi→f (E) = π k2 gJ ∑ λ ie−iϕiΓ1/2 λniΓ1/2 λγ f (Eλ −E) −iΓλ/2 + Udir i→f 2 . (8) (8) 2 DOI: 10.1051/epjconf/201716300037 EPJ Web of Conferences 163, 00037 (2017) EPJ Web of Conferences 163, 00037 (2017) FUSION17 If each resonance with the same capture state is isolated, Eq. (8) becomes as we have done in Eq. (2) If each resonance with the same capture state is isolated, Eq. (8) becomes as we have done in Eq. (2) . discuss the inverse interference eﬀect in this work, how- ever it should be kept in mind that we may have an in- terference eﬀect on neutron capture cross sections work- ing oppositely to what we will show in the next section. One of the solutions to ﬁx the interference eﬀect would be to measure neutron capture cross section just below and above resonance as done for 16O [13]. discuss the inverse interference eﬀect in this work, how- ever it should be kept in mind that we may have an in- terference eﬀect on neutron capture cross sections work- ing oppositely to what we will show in the next section. σi→f (E) ≃σBW,i→f + σdir,i→f + π k2 gJ ∑ λ Γ1/2 λn Γ1/2 λγµ2Re ( −ieiϕiUdir(Eλ −E −iΓλ/2) ) (Eλ −E)2 + (Γλ/2)2 (9) . One of the solutions to ﬁx the interference eﬀect would be to measure neutron capture cross section just below and above resonance as done for 16O [13]. (9) 3 Result In order to calculate the interference eﬀect given in Eq. (9), we are confronted by the sign problem in the term proportional to Re(−ieϕiUdir(Eλ−E−iΓλ/2)). In this work, the following prescription is adopted; the interference ef- fect on the neutron capture cross section in the ﬁrst res- onance of 13C (J = 2) and 134Sn (J = 1/2) works con- structively below the resonance and destructively above it as found in the experimental data of 16O(n, γ) cross sec- tion [13]. Then, we apply the same sign to the interference term of the other resonances. In case of 82Ge, the same sign as 13C is adopted because the neutron capture reac- tion of 82Ge is dominated by the p-wave neutron, which causes the interference eﬀect to the ﬁrst resonance of 13C. In order to evaluate the eﬀect of the interference eﬀect in an astrophysical condition, we calculate the Maxwellian averaged cross section at kT = 30 and 300 keV. The re- sults comparing Com+Dir+Int and Com+Dir at kT = 30 keV are shown in Table 1. By introducing the interference eﬀect, the cross sections are increased for nuclei studied The sign of interference eﬀect mentioned above is en- tirely arbitrary. As is obvious, it is not clear whether the interference eﬀect works constructively below resonance and destructively above resonance as 16O(n, γ). We do not 3 EPJ Web of Conferences 163, 00037 (2017) EPJ Web of Conferences 163, 00037 (2017) FUSION17 DOI: 10.1051/epjconf/201716300037 10−8 10−6 10−4 10−2 100 10−3 10−2 10−1 100 101 Com+Dir exp. Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) 10−2 10−1 100 10−5 10−4 10−3 10−2 10−1 100 101 Com+Dir exp. Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) Com Figure 1. Neutron capture cross section for 13C from E = 10−8 to 1 MeV (top) and E = 0.01 to 1 MeV (bottom). The solid and dashed lines are the results of compound process + direct process + interference eﬀect (Com+Dir+Int) and compound+direct pro- cess (Com+Dir), respectively. Note that the resonance parameter of 13C is taken from Ref. [20]. The experimental data are taken from Ref. [21–23]. 10−2 10−1 100 10−2 10−1 100 101 Com+Dir Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) 134Sn(n, ) γ 10−2 10−1 100 10−1 100 101 Com+Dir Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) 82Ge(n, ) γ Figure 2. 3 Result The results of compound process + direct process + interference eﬀect (Com+Dir+Int) and compound process + direct process (Com+Dir) are listed. The variation is calculated by (σ(Com + Dir + Int)/σ(Com + Dir) −1) × 100. 13C 82Ge 134Sn Com+Dir+Int 0.0776 0.6367 0.2566 Com+Dir 0.0780 0.6115 0.2591 Variation (%) -0.5 +4.1 -1.0 Table 2. Same as Table 1 but at kT = 300 keV. 13C 82Ge 134Sn Com+Dir+Int 0.0700 0.3574 0.3511 Com+Dir 0.0692 0.3412 0.2690 Variation (%) +1.1 +4.7 +30 in this work. In particular, the neutron capture cross sec- tion for 134Sn changes considerably as compared to the other two nuclei. The Maxwellian averaged cross section at kT = 300 keV is shown in Table 2. Contrary to the results obtained for kT = 30 keV, the interference eﬀect decreases the neutron capture cross sections of 13C and 134Sn. On the other hand, the variation of 82Ge by includ- ing the interference eﬀect is not changed signiﬁcantly from that for kT = 30 keV. The diﬀerence of the interference eﬀect at kT = 30 and 300 keV can be understood by the followings; Because the Maxwell-Boltzmann distribution has an arched curve with the peak at E = kT, the cross section is increased by the interference eﬀect if the temperature is suﬃciently smaller than the ﬁrst resonance. However, if the Maxwell- Boltzmann distribution shifts to higher energy as the tem- perature increases, the cross section is aﬀected not only by the constructive part but also by the destructive part of the interference eﬀect. This case is realized in 13C and 134Sn. In case of 82Ge, because there are a lot of resonances from E = 0.01 to 1 MeV, the interpretation of the diﬀerence be- tween kT = 30 and 300 keV is not simple. However, the reduction of the capture cross section above E = 700 keV, as seen in Fig. 2, results in the smaller Maxwellian aver- aged cross section at kT = 300 keV than that at kT = 30 keV. 3 Result Neutron capture cross sections of 134Sn (top) and 82Ge (bottom) from E = 0.01 to 1 MeV. The results of compound process + direct process + interference eﬀect (Com+Dir+Int), compound process + direct process (Com+Dir) are shown by the solid and dashed lines, respectively. The resonance parameters of 134Sn and 82Ge are taken from TENDL [19]. 13C 82G 134S 10−8 10−6 10−4 10−2 100 10−3 10−2 10−1 100 101 Com+Dir exp. Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) 10−2 10−1 100 10−2 10−1 100 101 Com+Dir Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) 134Sn(n, ) γ 10−2 10−1 100 10−1 100 101 Com+Dir Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) 82Ge(n, ) γ Incident Neutron Energy (MeV) 10−2 10−1 100 10−1 100 101 Com+Dir Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) 82Ge(n, ) γ 10−2 10−1 100 10−5 10−4 10−3 10−2 10−1 100 101 Com+Dir exp. Incident Neutron Energy (MeV) Com+Dir+Int σγ (mb) Com σγ (mb) 10 Incident Neutron Energy (MeV) Figure 2. Neutron capture cross sections of 134Sn (top) and 82Ge (bottom) from E = 0.01 to 1 MeV. The results of compound process + direct process + interference eﬀect (Com+Dir+Int), compound process + direct process (Com+Dir) are shown by the solid and dashed lines, respectively. The resonance parameters of 134Sn and 82Ge are taken from TENDL [19]. Figure 1. Neutron capture cross section for 13C from E = 10−8 to 1 MeV (top) and E = 0.01 to 1 MeV (bottom). The solid and dashed lines are the results of compound process + direct process + interference eﬀect (Com+Dir+Int) and compound+direct pro- cess (Com+Dir), respectively. Note that the resonance parameter of 13C is taken from Ref. [20]. The experimental data are taken from Ref. [21–23]. Figure 2. Neutron capture cross sections of 134Sn (top) and 82Ge (bottom) from E = 0.01 to 1 MeV. The results of compound process + direct process + interference eﬀect (Com+Dir+Int), compound process + direct process (Com+Dir) are shown by the solid and dashed lines, respectively. The resonance parameters of 134Sn and 82Ge are taken from TENDL [19]. 13C 82Ge 134Sn Com+Dir+Int 0.0700 0.3574 0.3511 Com+Dir 0.0692 0.3412 0.2690 Variation (%) +1.1 +4.7 +30 Table 1. The Maxwellian averaged cross sections at kT = 30 keV of 13C, 82Ge, and 134Sn in unit of mb. 4 Summary We have estimated the interference eﬀect of the neutron capture cross sections on the neutron-rich nuclei 82Ge and 134Sn, and light nucleus 13C. The neutron capture cross section of the compound process was calculated by the resonance parameters and that of the direct process was 4 EPJ Web of Conferences 163, 00037 (2017) FUSION17 DOI: 10.1051/epjconf/201716300037 [2] E. P. Wigner and L. Eisenbud, Phys. Rev. 72, 29 (1947). calculated by the potential model. The phase problem in the interference eﬀect was chosen so that the interference eﬀect became constructive below the resonance and de- structive above it. [3] Y. Nagai, et al., Astrophys. J. 372, 683 (1991). [4] M. Igashira et al., Astrophys. J. 441, L89 (1995). In case of 13C, it was found that the interference eﬀect increased the neutron capture cross section from E = 10−8 to 10−2 MeV signiﬁcantly. However, its eﬀect was rela- tively small around the ﬁrst resonance. In case of 134Sn and 82Ge, the interference eﬀect was clearly observed around the resonances. [5] G. J. Matthews, A. Mengoni, F.-K. Thielemann, and W. A. Fowler, Astrophys. J. 270, 740 (1983). [6] L. Bonneau, T. Kawano, T. Watanabe, and S. Chiba, Phys. Rev. C 75, 054618 (2007). [7] T. Rauscher, Nucl. Phys. A834, 635 (2010). [8] S. Chiba, et al., Phys. Rev. C 77, 015809 (2008). The Maxwellian averaged cross sections at kT = 30 and 300 keV were calculated. The changes of the neutron capture cross section by the interference eﬀect were sen- sitive in case of kT = 30 keV for nuclei studied in this work. When the temperature was set to be kT = 300 keV, the interference eﬀect became weak because of the con- structive and destructive contribution of the interference eﬀect caused by neighboring resonances. [9] J. E. Lynn, The theory of nuclear resonance reac- tions, Oxford : Clarendon Press, (1968). [10] I. Lovas, Sov. Phys. JETP, 14, 840 (1962). [11] C. Rolfs and R. E. Azuma, Nucl. Phys. A227, 291 (1974). [12] K. Shibata et al., J. Nucl. Sci. Technol. 48, 1 (2011). [13] A. Mengoni and T. Otsuka, AIP Conference Proceed- ings (New York, 2000) 529, 119 (2000). This work suggests that the interference eﬀect may have a large impact on neutron-rich nuclei as well as other light nuclei. 4 Summary However, in order to put forward the in- terference eﬀect in the neutron capture reactions practi- cally, we must solve the problems about the use of arti- ﬁcial resonance parameters, the phase problem, and the partial γ-decay width. Thus, new experimental measur- ing cross section and γ-ray energy and its strength around resonances is required and it will provide a further under- standing of the interference eﬀect. [14] P. Descouvemont and D. Baye, Rep. Prog. Phys. 73, 036301 (2010). ( ) [15] A. M. Lane and J. E. Lynn, Nucl. Phys. 17, 563 (1960). [16] A. Mengoni, T. Otsuka, M. Ishihara, Phys. Rev. C 52, R2334 (1995). [17] S. Kunieda, et al., J. Nucl. Sci. Techno. 44, 838 (2007). [18] G. Perey and B. Buck, Nucl. Phys. 32, 353 (1962). [19] A. J. Koning and D. Rochman, Nuclear Data Sheets 113, 2841 (2012). Acknowledgment [20] N. Iwamoto, private communication. This work is supported by YUGOKENKYU project at Japan Atomic Energy Agency. [21] R.B. Firestone, Zs. Revay, Phys. Rev. C 93, 054306 (2016). [22] T. Shima, T. Kii, T. Kikuchi, and F. Okazaki, JAERI Conference Proceedings, 4, No.97, 131 (1996). [1] E. P. Wigner, Phys. Rev. 70, 15 (1946); Phys. Rev. 70, 606 (1946). References [23] O. Schwerer, editor. EXFOR formats description for users (EXFOR basics) (IAEA-NDS-206). Vienna: International Atomic Energy Agency (2008). 5 5
https://openalex.org/W2400149985	https://www.jmir.org/2016/5/e111/PDF	English	null	Preexposure Prophylaxis Modality Preferences Among Men Who Have Sex With Men and Use Social Media in the United States	JMIR. Journal of medical internet research/Journal of medical internet research	2,016	cc-by	9,017	Abstract Background: Preexposure prophylaxis (PrEP) is available as a daily pill for preventing infection with the human immunodeficiency virus (HIV). Innovative methods of administering PrEP systemically or topically are being discussed and developed. ive of our study was to assess attitudes toward different experimental modalities of PrEP administration. j j y p Methods: From April to July 2015, we recruited 1106 HIV-negative men who have sex with men through online social media advertisements and surveyed them about their likelihood of using different PrEP modalities. Participants responded to 5-point Likert-scale items indicating how likely they were to use each of the following PrEP modalities: a daily oral pill, on-demand pills, periodic injection, penile gel (either before or after intercourse), rectal gel (before/after), and rectal suppository (before/after). We used Wilcoxon signed rank tests to determine whether the stated likelihood of using any modality differed from daily oral PrEP. Related items were combined to assess differences in likelihood of use based on tissue or time of administration. Participants also ranked their interest in using each modality, and we used the modified Borda count method to determine consensual rankings. Results: Most participants indicated they would be somewhat likely or very likely to use PrEP as an on-demand pill (685/1105, 61.99%), daily oral pill (528/1036, 50.97%), injection (575/1091, 52.70%), or penile gel (438/755, 58.01% before intercourse; 408/751, 54.33% after). The stated likelihoods of using on-demand pills (median score 4) and of using a penile gel before intercourse (median 4) were both higher than that of using a daily oral pill (median 4, P<.001 and P=.001, respectively). Compared with a daily oral pill, participants reported a significantly lower likelihood of using any of the 4 rectal modalities (Wilcoxon signed rank test, all P<.001). On 10-point Likert scales created by combining application methods, the reported likelihood of using a penile gel (median 7) was higher than that of using a rectal gel (median 6, P<.001), which was higher than the likelihood of using a rectal suppository (median 6, P<.001). The modified Borda count ranked on-demand pills as the most preferred modality. There was no difference in likelihood of use of PrEP (gel or suppository) before or after intercourse. Conclusions: Participants typically prefer systemic PrEP and are less likely to use a modality that is administered rectally. Although most of these modalities are seen as favorable or neutral, attitudes may change as information about efficacy and application becomes available. Preexposure Prophylaxis Modality Preferences Among Men Who Have Sex With Men and Use Social Media in the United States Eric William Hall1, MPH; Walid Heneine2, PhD; Travis Sanchez1, MPH, DVM; Robert Craig Sineath1, MPH; Patrick Sullivan1, DVM, PhD 1Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, United States 2National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atl Corresponding Author: Eric William Hall, MPH Department of Epidemiology Rollins School of Public Health Emory Univeresity GCR 432 1518 Clifton Road Atlanta, GA, 30322 United States Phone: 1 4047278965 Fax: 1 4047128392 Email: eric.w.hall@emory.edu Abstract Further data on modality preference across risk groups will better inform PrEP development. (J Med Internet Res 2016;18(5):e111) doi: 10.2196/jmir.5713 JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Hall et al Original Paper http://www.jmir.org/2016/5/e111/ J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 1 (page number not for citation purposes) preexposure prophylaxis; PrEP; men who have sex with men; MSM; HIV prevention; Facebook; survey Recruitment We collected data through a study that primarily explored alternative methods for delivering consent information and maximizing retention in online surveys [27] (funded by NICHD Research Grant 1R21-HD074502-01A1). Participants were recruited through targeted advertisements on a social media website (Facebook) from April 2015 to July 2015. Recruitment advertisements appeared to users in the United States who indicated on their Facebook profile that they are male, over 18 years of age, and interested in men. People who clicked on the advertisements were directed to an online consent module and a short screener to determine eligibility. To be eligible for the survey, users had to be male, between 18 and 34 years old, and not report having sex only with women in the past year. Men who reported never having oral or anal sex with a man were removed from the analysis dataset. Results from these PrEP efficacy studies show varying degrees of HIV risk reduction that ranges from 0% to 75%. However, this wide range is most commonly attributed to varying levels of adherence, because PrEP efficacy is much higher among participants who demonstrated consistent use [10-14]. For example, when blood samples were analyzed in the iPrEx study, HIV incidence reduction was 92% among participants who had the drug detectable in their blood and 99% among participants who had drug levels corresponding to daily use (both compared with the placebo group) [15,16]. Eligible men were given an online survey that collected demographic information such as age, education, race or ethnicity, zip code, and self-identified sexual orientation. The survey also collected information about sexual history and current sexual practices, history of HIV testing, and relationship status. All study materials and procedures were approved by the Emory University Institutional Review Board. In 2014, the US Centers for Disease Control and Prevention and the World Health Organization released guidelines that recommend the use of daily oral PrEP in populations with an elevated risk of HIV infection [17,18]. However, barriers such as cost, the burden of taking a daily pill, and concerns about potential health effects (both long-term and short-term effects) have led to PrEP being underused by eligible people [13,19-21]. Participants who reported a negative or unknown HIV status were asked about their knowledge of PrEP and history of use. (J Med Internet Res 2016;18(5):e111) doi: 10.2196/jmir.5713 preexposure prophylaxis; PrEP; men who have sex with men; MSM; HIV prevention; Facebook; survey http://www.jmir.org/2016/5/e111/ J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 1 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Hall et al Hall et al cervicovaginal fluid, which is also likely an indication of adherence [23]. A recent study among MSM (iPERGAY) investigated the efficacy of intermittent TDF/FTC pills taken just before and after sexual encounters, but the study was stopped early because initial analysis found comparable protection against HIV infection between these on-demand PrEP regimens and daily PrEP [24]. Introduction Although improvements in treatment have extended the life expectancy of people infected with the human immunodeficiency virus (HIV), there are still a troubling number of new HIV infections each year. In particular, HIV incidence rates among men who have sex with men (MSM) are increasing in North America and several other regions of the world [1,2]. In 2014, 70% of all new infections in the United States occurred among MSM [3]. To reduce the number of new infections, prevention strategies targeted toward specific risk groups are needed. Researchers are investigating the delivery of PrEP as oral pills that are used intermittently (ie, less than a daily basis), topical gels, vaginal rings, and long-lasting injections [21,25,26]. Because high levels of PrEP efficacy are dependent on adherence, there is obvious interest in developing administration modalities that target groups are willing to use. To help guide these research efforts, we sought to assess attitudes among MSM toward a variety of potential modalities of PrEP administration. A recent and exciting strategy, HIV preexposure prophylaxis (PrEP), involves using antiretroviral medication to reduce the risk of HIV infection among HIV-negative individuals. In 2010, this concept was first demonstrated in humans with publication of results from the iPrEx study, a randomized controlled trial that tested the efficacy of tenofovir disoproxil fumarate (TDF) in combination with emtricitabine (FTC) among MSM [4]. The iPrEx trial demonstrated a 44% (95% CI 15%-63%) reduction in HIV incidence among men who were taking TDF/FTC as a daily oral pill compared with a placebo group. Since then, other randomized controlled trials have indicated that a daily PrEP pill can reduce risk of HIV transmission in HIV-discordant couples [5], sexually active heterosexual men and women [6], and intravenous drug users [7]. However, 2 other trials showed no protective effect in heterosexual women in high-risk areas of Africa, but both of these studies had problems with adherence [8,9]. J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 2 (page number not for citation purposes) http://www.jmir.org/2016/5/e111/ Recruitment xis modalities presenteda to online survey respondents aged 18–34 years, by type of anal sex with male partner(s) in the July 2015 Rectal suppository 3 hours after receptive intercourse bParticipants who indicated they had both insertive and receptive anal sex in the past year were presented all modalities. Modalities presented to participants who said they only had insertive anal sex, receptive anal sex, or no anal sex in the past year are indicated by an “X”. Depending on their response, participants were then presented with a complete list (for each sexual behavior group) and asked to rank the modalities from most likely to least likely to use. c bParticipants who indicated they had both insertive and receptive anal sex in the past year were presented all modalities. Modalities presented to participants who said they only had insertive anal sex, receptive anal sex, or no anal sex in the past year are indicated by an “X”. Depending on their response, participants were then presented with a complete list (for each sexual behavior group) and asked to rank the modalities from most likely to least likely to use. cIncludes 2 pills within 24 hours before sex and 2 separate 1-pill doses within 2 days after sex. unlikely”). Responses of “neither likely or unlikely” were set to missing. We used logistic regression to determine unadjusted odds ratios (ORs) and corresponding confidence intervals for demographic variables of interest. Participants were also asked to rank their interest in using each of the different potential methods of PrEP administration. The number of modalities that each participant could rank depended on the type of sex he indicated having in the past 12 months. If a respondent said he had both insertive and receptive anal sex in the past year, he could rank all 9 potential modalities. However, if a respondent indicated he only had receptive anal sex in the past 12 months, he was prompted to rank only 7 potential modalities (4 that are applied rectally, 2 applied orally, and an injection). We used the modified Borda count method to determine the order of preference for the different PrEP modalities [29]. Each modality was assigned a number of points that corresponded to the position in which it was ranked by the participant. The number of points given to a participant’s first choice was equal to the number of modalities he actually ranked. Recruitment We then summed points for each modality to create a collective ranking. Since the number of options presented to an individual depended on the type of sex he reported in the past 12 months (eg, a participant who only reported insertive anal sex was not presented with modalities that are administered rectally), we stratified cumulative rankings by reported sexual practices. Statistical Analyses We did all analyses using SAS v9.4 (SAS Institute Inc). Participants who did not respond to any of the PrEP modality Likert-scale items (n=318) were removed from the analysis dataset. We summarized the likelihood of using each PrEP modality by finding the mean, median, and mode of the 5-point Likert-scale item response. Since these items are ordinal and not interval data, we used nonparametric tests for statistical inferences. We used Wilcoxon signed rank tests to determine whether the reported likelihood of using each of the 8 experimental PrEP modalities differed from the likelihood of using PrEP as a daily oral pill. Each test was considered statistically significant at alpha=.05. http://www.jmir.org/2016/5/e111/ J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 3 (page number not for citation purposes) Recruitment Participants who had not previously used PrEP answered 5-point Likert-scale items that asked how likely they were to use different PrEP modalities to reduce the risk of getting HIV. They were asked about 9 Likert-scale items, 1 for each of the modalities listed in Table 1. Participants were only asked about modalities that involved penile application if they reported having insertive anal sex in the past year. Likewise, participants were only asked about the rectal modalities if they reported having receptive anal sex in the past year. We collected responses to each Likert-scale item in the following format: 1=very unlikely, 2=somewhat unlikely, 3=neither likely or unlikely, 4=somewhat likely, 5=very likely. Although a daily oral TDF/FTC pill is the only approved and recommended form of PrEP, there is growing interest in developing new methods for administering antiretroviral drugs as prevention. Topical applications of PrEP have been studied in 2 clinical trials of high-risk women who used a TDF-based vaginal gel either before and after sex [22] or on a daily basis [8]. Although the results from these trials present conflicting conclusions, further analysis indicates that the efficacy of vaginal gel depends on the concentration of tenofovir in the XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Hall et al Table 1. Preexposure prophylaxis modalities presenteda to online survey respondents aged 18–34 years, by type of anal sex with male partner(s) in the past year, United States, April–July 2015. No anal sex Receptive only Insertive only Modalities X X Xb Daily oral pill X X X On-demand pillsc X X X Injection every 1–3 months X Penis gel before insertive intercourse X Penis gel after insertive intercourse X Rectal gel before receptive intercourse X Rectal gel after receptive intercourse X Rectal suppository 30 minutes before receptive intercourse X Rectal suppository 3 hours after receptive intercourse aThe survey included individual Likert-scale items asking the likelihood of using each modality. bParticipants who indicated they had both insertive and receptive anal sex in the past year were presented all modalities. Modalities presented to participants who said they only had insertive anal sex, receptive anal sex, or no anal sex in the past year are indicated by an “X”. Depending on their response, participants were then presented with a complete list (for each sexual behavior group) and asked to rank the modalities from most likely to least likely to use. Results In the previous 12 months, 599 (54.16%) participants reported having both insertive and receptive anal sex with a male partner, 202 (18.26%) reported participants identified as homosexual (965/1106, 87.25%) and reported having had anal sex with a male partner in the past month (964/1106 87.16%). In the previous 12 months, 599 (54.16%) participants reported having both insertive and receptive anal sex with a male partner, 202 (18.26%) reported p p p g receptive anal sex with a male partner, 202 (18.26% Table 2. Demographic and sexual behavior characteristics of about preexposure prophylaxis (PrEP) for HIV infection, Unit Characteristics Age (years) a 18–24 25–29 30–34 Race/ethnicity White, non-Hispanic Black, non-Hispanic Hispanic Other US census region Midwest Northeast South West Unknown Highest level of education High school or less Some college, associate degree, or technical degree Bachelor degree Any graduate or professional school Unknown Sexual identity Homosexual Bisexual Otherb Type of anal sex with male partner(s) in past 12 months Insertive only Receptive only Both insertive and receptive No anal sex Previously heard of PrEP aMean 25.2 years, median 25 years, range 18–34 years. bThere were 5 participants who indicated heterosexual, 15 ind Figure 1 displays the stated likelihood of using e modality to reduce the risk of contracting HIV. Ove Table 2. Demographic and sexual behavior characteristics of 1106 men who have sex with men, aged 18–34 years, participating in an online survey about preexposure prophylaxis (PrEP) for HIV infection, United States, April 2015–July 2015. (%) n Characteristics Age (years) a (49.01) 542 18–24 (30.20) 334 25–29 (20.80) 230 30–34 Race/ethnicity (72.33) 800 White, non-Hispanic (4.61) 51 Black, non-Hispanic (16.37) 181 Hispanic (6.69) 74 Other US census region (19.62) 217 Midwest (22.69) 251 Northeast (35.44) 392 South (20.52) 227 West (1.72) 19 Unknown Highest level of education (7.23) 80 High school or less (27.67) 306 Some college, associate degree, or technical degree (34.72) 384 Bachelor degree (30.29) 335 Any graduate or professional school (0.09) 1 Unknown Sexual identity (87.25) 965 Homosexual (9.58) 106 Bisexual (3.16) 35 Otherb Type of anal sex with male partner(s) in past 12 months (18.26) 202 Insertive only (14.74) 163 Receptive only (54.16) 599 Both insertive and receptive (12.48) 138 No anal sex (74.50) 824 Previously heard of PrEP aMean 25.2 years, median 25 years, range 18–34 years. Results There were 3990 participants who started the online survey and answered eligibility questions. Of 1921 men who met the eligibility requirements for the survey, 4 reported having tested positive for HIV and 493 reported never having oral or anal sex with a man. We further limited the final analysis dataset to the 1106 participants who answered at least one of the questions related to PrEP. Individual Likert-scale items that asked about topical application sites were summed to create separate Likert scales based on application method and time of application. This resulted in three 10-point Likert scales based on method (penile gel, rectal gel, and rectal suppository) and two 15-point Likert scales based on time of application (before intercourse, after intercourse) [28]. Table 2 summarized the demographic characteristics of the 1106 participants included in the analysis. Almost half of the participants (542/1106, 49.01%) were between 18 and 24 years old and the mean age was 25.2 years. The highest proportion (800/1106, 72.33%) of participants were white, and most had received some level of education past secondary school (1025/1106, 92.68%). The highest proportion of respondents lived in the south (392/1106, 35.44%) but there were at least 200 respondents from all 4 US census regions. The majority of To assess demographic associations with likelihood of using each modality, we dichotomized individual Likert-scale item responses so that “somewhat likely” or “very likely” indicated likelihood of use (versus “somewhat unlikely” or “very XSL•FO RenderX Hall et al Hall et al JOURNAL OF MEDICAL INTERNET RESEARCH having only insertive sex, and 163 (14.74%) reported having only receptive sex. There were 138 (12.48%) participants who did not have anal sex in the previous 12 months. The majority of participants (824, 74.50%) had previously heard of people using PrEP to reduce the risk of getting HIV. having only insertive sex, and 163 (14.74%) reported having only receptive sex. There were 138 (12.48%) participants who did not have anal sex in the previous 12 months. The majority of participants (824, 74.50%) had previously heard of people using PrEP to reduce the risk of getting HIV. participants identified as homosexual (965/1106, 87.25%) and reported having had anal sex with a male partner in the past month (964/1106 87.16%). http://www.jmir.org/2016/5/e111/ J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 4 (page number not for citation purposes) y , y , g y bThere were 5 participants who indicated heterosexual, 15 indicated unsure, 14 indicated other, and 1 who did not indicate sexual identity. J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 4 Figure 1. Stated likelihood of using different preexposure prophylaxis modalities among 1106 men who have sex with men, aged 18-34 years, participating in an online survey, United States, April–July 2015. J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 5 (page number not for citation purposes) Results P-valuec Mode Median (IQRb) Mean (SD) n Individual Likert-scale items for each modalitya Reference 4 4 (2) 3.31 (1.4) 1036 Daily oral pill <.001 5 4 (2) 3.63 (1.4) 1105 On-demand pillsd .02 5 4 (3) 3.27 (1.5) 1091 Injectione .001 5 4 (3) 3.45 (1.4) 755 Penile gel before intercourse .28 5 4 (3) 3.31 (1.5) 751 Penile gel after intercourse <.001 4 3 (2) 3.06 (1.5) 791 Rectal gel before intercourse <.001 1 3 (2) 3.00 (1.5) 794 Rectal gel after intercourse <.001 1 2 (3) 2.58 (1.4) 792 Rectal suppository before intercourse <.001 1 3 (3) 2.90 (1.5) 791 Rectal suppository after intercourse aThe 5-point Likert-scale items where 1=very unlikely, 2=somewhat unlikely, 3=neither likely or unlikely, 4=somewhat likely, 5=very likely. bIQR: interquartile range. cP-values calculated using Wilcoxon signed rank tests with alpha=.05. dConsisting of 2 pills 24 hours before sex and 2 separate 1-pill doses after. eEvery 1–3 months. of using different preexposure prophylaxis modalities for HIV infection among 1106 men who have sex with men, aged n an online survey, United States, April–July 2015. likelihood of using the following modalities: daily oral pills (OR 3.10, 95% CI 1.35–7.13), penis gel before intercourse (OR 6.59, 95% CI 1.54–28.24), penis gel after intercourse (OR 2.82, 95% CI 1.05–7.59), rectal gel before intercourse (OR 4.28, 95% CI 1.73–10.61), rectal gel after intercourse (OR 2.52, 95% CI 1.14–5.58) and a suppository before intercourse (OR 2.69, 95% CI 1.26–5.77). On 10-point Likert scales created by combining modalities by topical application methods, the reported likelihood of using a penile gel (median 7) was higher than that of using a rectal gel (median 6, P<.001; Table 4). However, the likelihood of using a rectal gel was higher than that of using a rectal suppository (median 6, P<.001). There was no statistically significant difference in the reported likelihood of using a topical PrEP modality (gel or suppository) before or after intercourse. Figure 2 shows the modified Borda count rankings, stratified by type of anal sex in the past 12 months. On-demand pills were the top-ranked modality for each sexual behavior group. In general, modalities administered orally were ranked highest and modalities administered rectally were ranked lowest. When individual Likert-scale item responses were dichotomized, stated likelihood of using daily oral pills differed by race/ethnicity, age category, and highest level of education (Multimedia Appendix 1). Results bThere were 5 participants who indicated heterosexual, 15 indicated unsure, 14 indicated other, and 1 who did not indicate sexual identity. Figure 1 displays the stated likelihood of using each PrEP modality to reduce the risk of contracting HIV. Overall, over half of participants stated they would be somewhat likely or very likely to use on-demand pills (685/1105, 61.99%), penile gel (438/755, 58.01% before intercourse and 408/751, 54.33% after intercourse), a periodic injection (575/1091, 52.70%), or a daily oral pill (528/1036, 50.97%). The majority (437/792, 55.18%) of respondents indicated they would be very unlikely Receptive only aMean 25.2 years, median 25 years, range 18–34 years. bThere were 5 participants who indicated heterosexual, 15 indicate bThere were 5 participants who indicated heterosexual, 15 indicate gel (438/755, 58.01% before intercourse and 408/751, 54.33% after intercourse), a periodic injection (575/1091, 52.70%), or a daily oral pill (528/1036, 50.97%). The majority (437/792, 55.18%) of respondents indicated they would be very unlikely Figure 1 displays the stated likelihood of using each PrEP modality to reduce the risk of contracting HIV. Overall, over half of participants stated they would be somewhat likely or very likely to use on-demand pills (685/1105, 61.99%), penile http://www.jmir.org/2016/5/e111/ XSL•FO RenderX XSL•FO RenderX Hall et al Hall et al JOURNAL OF MEDICAL INTERNET RESEARCH Table 3). Compared to the currently available daily pills (median 4), respondents reported a higher likelihood of using on-demand pills (median 4, P<.001) or a penile gel before intercourse (median 4, P=.001). However, participants reported a significantly lower likelihood of using each of the rectal modalities (all P<.001) compared with a daily oral pill. or somewhat unlikely to use a rectal suppository before intercourse. When the responses were analyzed on a 5-point scale, the mean responses ranged from 2.58 (rectal suppository before intercourse, median 2) to 3.63 (on-demand pills, median 4; Figure 1. Stated likelihood of using different preexposure prophylaxis modalities among 1106 men who have sex with men, aged 18-34 years, participating in an online survey, United States, April–July 2015. J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 5 (page number not for citation purposes) http://www.jmir.org/2016/5/e111/ http://www.jmir.org/2016/5/e111/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Hall et al Table 3. Stated likelihood of using different preexposure prophylaxis modalities for HIV infection among 1106 men who have sex with men, aged 18–34 years, participating in an online survey, United States, April–July 2015. http://www.jmir.org/2016/5/e111/ J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 6 (page number not for citation purposes) Results Compared with white participants, black participants had higher odds of reporting a favorable sing different preexposure prophylaxis topical modalities, by method and time of application, among 1106 men who years, participating in an online survey, United States, April–July 2015. Table 4. Stated likelihood of using different preexposure prophylaxis topical modalities, by method and time of application, among 1106 men who have sex with men, aged 18–34 years, participating in an online survey, United States, April–July 2015. ble 4. Stated likelihood of using different preexposure prophylaxis topical modalities, by method and time of application, ve sex with men, aged 18–34 years, participating in an online survey, United States, April–July 2015. Table 4. Stated likelihood of using different preexposure prophylaxis topical modalities, by method and time of application, among 1106 men who have sex with men, aged 18–34 years, participating in an online survey, United States, April–July 2015. P-valuec Mode Median (IQRb) Mean (SD) n Combined Likert scalesa Application method d <.001 10 7 (4) 6.76 (2.7) 750 Penile gel, anytime Reference 2 6 (4) 6.06 (2.9) 790 Rectal gel, anytime <.001 2 6 (6) 5.48 (2.8) 790 Rectal suppository, anytime Time of application e Reference 3 9 (6) 8.87 (3.8) 585 Before intercourse .14 3 9 (6) 9.02 (4.0) 584 After intercourse aCreated from original 5-point Likert-scale items where 1=very unlikely, 2=somewhat unlikely, 3=neither likely or unlikely, 4=somewhat likely, 5=very likely. b bIQR: interquartile range. Q q g cP-values calculated using Wilcoxon signed rank tests with alpha=.05. Q q g cP-values calculated using Wilcoxon signed rank tests with alpha=.05. dBased on a 10-point scale created by adding the two 5-point Likert-scale items for each application method. ased on a 15-point scale created by adding the three 5-point Likert-scale items for each time of application. J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 6 http://www.jmir.org/2016/5/e111/ (page number not for citation purposes) O http://www.jmir.org/2016/5/e111/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Hall et al Figure 2. Modified Borda count ranking of different preexposure prophylaxis modalities, stratified by type of anal sex with a male partner in the past year, among 1106 men who have sex with men, aged 18–34 years, participating in an online survey, United States, April-July 2015. Respondents were presented with a different number of modalities to rank, depending on the type of anal sex they reported in the past 12 months. results [4]. While our analysis did not investigate reasons against taking PrEP, previous research has indicated that concerns about health (both long-term consequences and immediate side effects), unknown efficacy, possibility of developing drug resistance, cost, and risk perception can all be barriers to use [13,19,20]. Discussion http://www.jmir.org/2016/5/e111/ J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 7 (page number not for citation purposes) Discussion However, there are some noticeable differences in the likelihoods of using each modality. However, there are some noticeable differences in the likelihoods of using each modality. The preference for intermittent oral PrEP is evident across this analysis. The highest proportion of participants indicated they would use on-demand pills, which was also the top consensus rank for each of the 4 modified Borda count groups. This preference is particularly of interest when viewed with the growing evidence base demonstrating the efficacy of intermittent (ie, less than daily) oral PrEP [24]. Community surveys have shown that the majority of condomless anal intercourse events appear to be anticipated in advance or infrequent enough to make event-driven or time-driven PrEP regimens feasible [34-36]. Furthermore, Parsons et al recently found that MSM overestimate the likelihood of having sex and are much better at predicting when they would not have sex [37]. This has implications on counseling related to intermittent PrEP use. Parsons et al concluded that counseling messages should encourage individuals to skip a daily dose only when they are sure there is no chance they will have sex the following day. Several forms of slow-acting injectable PrEP are being studied [26], and our study indicates they may have similar acceptability to daily oral pills. For most of the modalities (daily oral pills, both penis gels, both rectal gels, and suppository before intercourse), our study indicated that black participants were more likely than white participants to use them. Since there is not a reference population that can be used as a comparison to assess representativeness, there have not been any other studies that characterized sampling biases using online convenience samples. However, this approach is one of the most common in the field, is consistent with the body of published literature, and allows researchers to quickly collect behavior information on a large number of MSM [40]. Our advertisements targeted young-adult MSM in the United States, which limits the ability to generalize these results to other age groups, regions, or populations affected by the HIV epidemic. Likelihood and acceptability of use is likely to vary depending on perceived risk and cultural acceptability. It is important to note that attitudes toward topical PrEP modalities differ by administration site. Limitations Several limitations need to be considered when interpreting these results. First, because Likert-scale items are not interval in nature, only the direction of preference can be determined. The magnitude of preference cannot be adequately determined or compared (ie, we cannot say “how much more likely” somebody is to use a single modality over another). Second, we asked participants to state their likelihood of using different modalities without any specifications of cost, efficacy, or Conclusion Previous research has shown that PrEP has the potential to reduce risk of HIV transmission, but adherence is essential to ensure efficacy. In order to overcome the many barriers to PrEP uptake, we need to develop modalities that are feasible and likely to be used. Among this specific population of MSM in the United States, intermittent systemic approaches seem to be preferred. For topical methods, those that involve application on the penis are preferred over rectal application, and gels are preferred over suppositories. However, further analysis is needed to determine why people would not use particular modalities. Other studies similar to this one need to be carried out for other groups that will be targeted for the newer PrEP modalities. While there may not be a single PrEP modality that is used by everybody, our study and future ones like it can help determine which technology is most likely to be adopted by specific communities. Discussion Although gels applied to the penis were generally viewed as acceptable, the 4 rectal modalities were the only Likert-scale items in which more than half of participants reported that they were unlikely to use them. While the combined Likert scales indicated that rectal gels are seen more favorably than rectal suppositories, both were less likely to be used than penile gels. Most research conducted on PrEP in a gel form has focused on vaginal gels for women in Africa [8,22], but there is an ongoing phase 2 trial on rectal gel microbicides in MSM (MTN-017) [38]. Phase 1 research indicated that 75% to 100% of recipients found the experimental gels to be acceptable [39]. Acknowledgments This work was funded by Eunice Kennedy Shriver National Institute of Child and Health and Human Development (NICHD) Research Grant 1R21-HD074502-01A1. The findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Discussion Our findings indicate that 51% of MSM would be likely to take PrEP as a daily oral pill, which is consistent with the range seen in previous studies in which between 46.1% and 78.5% of MSM said they would be willing to use daily oral PrEP [19,30-33]. The varying range of acceptability is likely a result of some studies stipulating specific scenarios in which PrEP is either offered free of charge, does not cause side effects, or is 80% effective against preventing HIV infection. Some of this variation in acceptability could be related to when the survey was conducted in relation to the public release of the iPrEx These results provide comparative insights on how likely MSM in our sample were to use a variety of hypothetical PrEP modalities. Overall, attitudes toward using PrEP to reduce the risk of contracting HIV were generally neutral or favorable, with a reported likelihood of use ranging from 32% (rectal suppository before intercourse) to 62% (on-demand pills). J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 7 (page number not for citation purposes) J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 7 (page number not for citation purposes) XSL•FO RenderX Hall et al Hall et al JOURNAL OF MEDICAL INTERNET RESEARCH possible side effects. As this information (specific to each modality) becomes available, we would expect the reported likelihood of use to change. Furthermore, we asked participants about different modalities of administration based on the type of sex they reported having in the past year. As a result, the attitudes toward any of the modalities administered rectally represent only those of the participants who recently received anal sex (and vice versa for penile application and insertive anal sex). There is potential for selection bias in the analysis dataset. We excluded 318 respondents from the analysis because they did not respond to any of the PrEP modality Likert-scale items. Those excluded respondents were more likely to be African American (8.2% vs 4.6%, P=.009) and more likely to report not having anal sex with a male in the past year (27.8% vs 12.5%, P≤.001), compared with our sample of 1106 participants. Third, our recruitment methods targeted social media users and our sample may not be representative of the general community of MSM. 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A phase 1 randomized, open label, rectal safety, acceptability, pharmacokinetic, and pharmacodynamic study of three formulations of tenofovir 1% gl (the CHARM-01 Study). PLoS One 2015;10(5):e0125363 [FREE Full text] [doi: 10.1371/journal.pone.0125363] [Medline: 25942472] 40 S h T S i h A D D Di E L k A D l i W b b d HIV b h i l ill il j Abbreviations Edited by G Eysenbach; submitted 01.03.16; peer-reviewed by J Topolovec-Vranic, S LeGrand, C Grov; comments to author 24.03.16; revised version received 29.03.16; accepted 31.03.16; published 19.05.16 Please cite as: Hall EW, Heneine W, Sanchez T, Sineath RC, Sullivan P Preexposure Prophylaxis Modality Preferences Among Men Who Have Sex With Men and Use Social Media in the United States J Med Internet Res 2016;18(5):e111 URL: http://www.jmir.org/2016/5/e111/ doi: 10.2196/jmir.5713 PMID: 27199100 ©Eric William Hall, Walid Heneine, Travis Sanchez, Robert Craig Sineath, Patrick Sullivan. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 19.05.2016. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be included. J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 11 (page number not for citation purposes) J Med Internet Res 2016 \| vol. 18 \| iss. 5 \| e111 \| p. 11 (page number not for citation purposes) http://www.jmir.org/2016/5/e111/ XSL•FO RenderX
W4213440708.txt	https://www.mdpi.com/1996-1073/15/5/1623/pdf?version=1645675809	en		A Mathematical Model of Electrical Arc Furnaces for Analysis of Electrical Mode Parameters and Synthesis of Controlling Influences	Energies	2,022	cc-by	8,463	energies Article A Mathematical Model of Electrical Arc Furnaces for Analysis of Electrical Mode Parameters and Synthesis of Controlling Influences Andriy Lozynskyy 1 , Jacek Kozyra 1, , Zbigniew Łukasik 1 , Aldona Kuśmińska-Fijałkowska 1 , Andriy Kutsyk 2,3 , Yaroslav Paranchuk 2 and Lidiia Kasha 2 1 2 3 Citation: Lozynskyy, A.; Kozyra, J.; Łukasik, Z.; Kuśmińska-Fijałkowska, A.; Kutsyk, A.; Paranchuk, Y.; Kasha, L. A Mathematical Model of Faculty of Transport, Electrical Engineering and Computer Science, University of Technology and Humanities in Radom, 26-600 Radom, Poland; a.lozynskyy@uthrad.pl (A.L.); z.lukasik@uthrad.pl (Z.Ł.); a.kusminska@uthrad.pl (A.K.-F.) Institute of Power Engineering and Control System, Lviv Polytechnic National University, 79-013 Lviv, Ukraine; andrii.s.kutsyk@lpnu.ua (A.K.); yaroslav.s.paranchuk@lpnu.ua (Y.P.); lidiia.v.kasha@lpnu.ua (L.K.) Faculty of Electrical and Computer Engineering, Rzeszow University of Technology, 35-959 Rzeszow, Poland; a.kutsyk@prz.edu.pl Correspondence: j.kozyra@uthrad.pl Abstract: The synthesis of a real-time model of the electric mode of an arc steelmaking furnace is shown. This model is based on the method of medium-voltage and can be used to analyze the impact of the arc steelmaking furnace on the supply network at different stages of the melting process at different control systems of the electromechanical electrode movement system, and to create model-predictive control systems of electrode movement, when control using hardware in the loop technology. To implement the chosen method, the dynamic arc resistance was used. The last was obtained on the basis of the averaged experimentally taken volt-ampere characteristics of the arc. In paper was proposed its approximation as a current function and arc length. The simulation results obtained using the created model for different operation modes of the arc steelmaking chipboard for a system with a traditional regulator of the electrode movement system are demonstrated. Electrical Arc Furnaces for Analysis of Electrical Mode Parameters and Synthesis of Controlling Influences. Keywords: arc steelmaking furnace; mathematical model; dynamic volt-ampere characteristic of the arc; hardware in the loop; control of electrode movement Energies 2022, 15, 1623. https:// doi.org/10.3390/en15051623 Academic Editor: Andrea Mariscotti 1. Introduction Received: 23 January 2022 Accepted: 17 February 2022 Published: 22 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Modern high-tech systems, which are increasingly used in industry, place increasing demands on the quality of electricity. As a rule, the basic requirements for the quality of electricity in modern production are put forward to ensure the appropriate parameters of the supply voltage and harmonic composition of the current. Violation of electricity quality parameters can cause significant losses to the manufacturer both due to violation of the technological mode of production, and due to equipment failure or forced downtime. The main threats in terms of electricity quality are long-term and short-term voltage fluctuations, the appearance of harmonic current components due to the connection of nonlinear, abruptly changing and asymmetric loads. An example of such a load is an arc steelmaking furnace [1–3]. It should also be noted that steel arc furnaces are a source of rapid voltage fluctuations [4,5], which occur due to the asymmetry of currents and voltages. Today, the melting of scrap metal in arc steel furnaces provides about 1/3 of annual world steel production. A typical arc steelmaking furnace (EAF) during its operation consumes approximately 450 kWh of electrical energy for smelting one ton of steel [6]. The principle of melting scrap in chipboard is to convert electrical energy into thermal energy, which is dissipated from electric arcs and melts the materials loaded into the furnace. Arcs burn between graphite electrodes and conductive scrap (3-phase system without neutral) and Energies 2022, 15, 1623. https://doi.org/10.3390/en15051623 https://www.mdpi.com/journal/energies Energies 2022, 15, 1623 2 of 19 are characterized by low voltage (400–1000 V) and high currents (40–60 kA) for each phase. The temperature in the arc column can reach 8500 K, which provides the ability to quickly and efficiently melt scrap. In alternating current furnaces, the power supply system consists of a power transformer with variable taps and, sometimes, a series reactor connected to the high voltage side to increase the reactance of the system. The electric arc length is regulated by the electrodes movement system, which provides to set a given power into the furnace, which is determined by the melting mode. In the melting process, the harmonic distortions of voltage and current signals change from larger values of the harmonic distortion factor (THD) in the initial stages of melting to smaller in the stages of purification and heating [7,8]. Random change of the arc length during the melting process leads to a significant change in the parameters of the electric mode and the corresponding reaction on the part of the electrode movement control system. It is necessary to deal with systems by different tempo both in the system of electrode movement and taking into account the interaction between the electrode movement system and electromagnetic processes in the furnace space. This, in turn, will require the use of special approaches when conducting research using the created mathematical model of EAF. Thus, the model for studying the influence of EAF on the power supply network should contain a model of the electric circuit taking into account the nonlinearity of the volt-ampere characteristic of the arc, the model of the control system of EAF electrode movement and the model of random arc length processes [9–13]. Depending on the class of tasks, researchers often use simplified models of individual subsystems. In particular, in the synthesis of EAF electrodes movement control systems, the model of the power supply system is ignored as a whole, using only Volt-Ampere characteristics at different arc lengths [14–16]. On the other hand, when studying the influence of EAF on the electrical network, the processes in the EAF electrode movement are not taken into account, using Volt-Ampere characteristics of arcs at given values of arc length or given laws of its change [1–3,8,17–19]. In both cases, the Volt-Ampere characteristics of the arc are used, and therefore it is not surprising, that much of the work is devoted to the modeling of the electric arc [7,9,20–22]. Thus, a significant part of EAF models used in the control systems studing, does not take into account nonlinearities and changes in parameters. They also are based on the use of static Volt-Ampere characteristics. The above works did not address the task of ensuring the functioning of models in real time. At the same time, the creation of such models in combination with the technology of “hardware-in-the-loop” significantly expands the possibilities of research and synthesis of modern control algorithms. The purpose of this article is to develop such a EAF model. A feature of EAF is the presence of different tempo subsystems and a significant range of changes in its parameters in the melting modes (in particular, due to changes in the dynamic resistance of the arc). Reducing the numerical integration step to ensure the required modeling accuracy significantly slows down the calculation and hinders the operation of the model in real time. This complicates the task of choosing the method of numerical integration for real-time models, as noted in [23–25]. In particular, in [25] it was noted about the difficulties in applying the widely used trapezoidal rule and backward Euler method for this class of problems. In particular, the application of the backward Euler method requires a significant reduction in the step of numerical integration and thus an increase in computing power and speed of calculation. Instead, the use of the trapezoid method, although it allows to increase the step of numerical integration, however, can lead to numerical oscillations and violations of the stability of the calculation. Therefore, it is important to apply new approaches to real-time modeling of EAF systems. One of such approaches, proposed in [23], will be used in this paper to create a comprehensive model for the analysis of the electrical operation mode of EAF. A feature of this approach is the application of a new method of algebraization of differential equations—the method of average voltages at the step of numerical integration (average voltage values in integration step AVIS). The advantage of this method is the increased numerical stability of the calculation, as well as the possibility of calculation with an increased step of numerical Energies 2022, 15, 1623 3 of 19 integration and maintaining the required accuracy of the calculation, which is justified in the works [23,24]. This approach has proven itself in the synthesis of real-time models of various electromechanical systems [24–26] and has been used to set up excitation systems for synchronous generators [27,28], including at the South Ukrainian NPP. The application of this approach in the EAF model will require the creation of a slightly different from the existing model of the arc space. Synthesis a real-time EAF model, which can be used in both—model predictive control systems and to establish control systems for the movement of EAF electrodes and analysis of the impact of EAF on the power supply network in different control systems is an urgent problem to solve. The article is organized as follows: Section 2 presents, for the first time, a mathematical model of an electric branch with an arc, created using the method of average voltages at the step of numerical integration. Section 3 describes a mathematical model of arc space, in which new methods are used to approximate the volt-ampere characteristics of the arc and determine the dynamic resistance of the arc as a parameter of the developed model. Section 4 describes the mathematical models of the of the power supply system of the EAF elements, namely: short network with furnace space (arc), furnace transformer and power supply network with choke, created using the AVIS method. The peculiarity of these models is their representation in the form of multipoles. Section 5 presents the calculation scheme of the EAF power supply system formed by a combination of the previously described multipole elements and briefly describes the calculation algorithm (a detailed description of the algorithm for mathematical modeling of electrical systems using AVIS and multipole theory is given in [24]). Section 6 for the first time describes a mathematical model of the chipboard electrode movement system created using the AVIS method. Section 7 presents the results of studies of established symmetric and asymmetric modes in the EAF’s power supply system, as well as transient modes in EAF when the arc length is changing and during melting. The adequacy of the model was confirmed by comparison with the simulation results obtained by other authors using other approaches and the results of the experiment. 2. Mathematical Model of an Electric Branch with an Electrical Arc In accordance with the main provisions of the method of average voltages in numerical integration step [23], the electric branch with inductance, active resistance and arc (Figure 1) is described by the following equation: 1 ∆t t0Z+∆t (u − u R − u L − u arc (i ))dt = 0, (1) t0 where uR , uL , uarc —instantaneous values of voltage on active resistance, voltage on inductance, arc voltage, respectively; u—voltage applied to the branch; t0 —is the start time of integration step, ∆t—is the numerical integration step. Figure 1. Circuit of an electric branch with inductance, active resistance and arc. The instantaneous values of voltages on the active resistance and on the arc are represented by the formulas: u R = u R0 + ∆u R , u arc = u arc0 + ∆u arc , (2) Energies 2022, 15, 1623 4 of 19 where uR0 , uarc0 —voltage values at the start of the step; ∆uR , ∆uarc —voltage gains at the step of numerical integration. The voltage increments at the step of numerical integration are determined using the Taylor series: ∆u R = (k) m d(k) u arc0 (t − t0 )k d(k) u R0 (t − t0 )k · , ∆u = · arc ∑ (k) (k) k! k! k =1 dt k =1 dt m ∑ (3) (k) where d (uk)R0 , d u(karc0 —k-th voltage derivatives on the active resistance and on the arc at the dt dt ) beginning of the step; m—is the number of derivatives taken into account (method order). Taking into account the dependences between voltage and current for the active resistance of the circuit R and for the arc, respectively: u R (t) = Ri (t), u arc (t) = f (i ), we obtain expressions for increments (4) and (5): ∆u R = m ∑ k =1 R d ( k ) i0 ( t − t0 ) k · k! dt(k) (4) d(k) u arc0 d(k) i0 (t − t0 )k ∑ di(k) dt(k) · k! k =1 (5) di0 du arc0 di0 di0 ∆t, ∆u arc = ∆t = Rd0 ∆t dt di dt dt (6) ∆u arc = m For the 1st order method: ∆u R = R where Rd = dudiarc —dynamic arc resistance (determined on the basis of instantaneous values of arc voltage uarc and current (i); Rd0 —dynamic arc resistance at the beginning of the step. Note that the dynamic resistance of the arc depends on the current of the arc and the length of the arc and is determined at each step. Taking into account (2), (6) and the equation for the voltage on the inductance (the di (t) value of the inductance L is constant) u L (t) = L dt from Equation (1) we obtain for the method of the 1st order: R + Rd0 L U − u R0 − u arc0 − + (7) ( i1 − i0 ) = 0 2 ∆t where i0 —current of circuit at the beginning of the integration step, i1 —is the current at the t0R +∆t 1 udt—the average value of the voltage applied to end of the integration step, U = ∆t t0 the circuit at the numerical integration step. From Equation (7) we obtain the expression for determining the current of the branch at the end of the step: i1 = i0 + R + Rd0 L + 2 ∆t −1 (U − Ri0 − u arc0 ). (8) The effective value of the arc voltage at each step of integration is calculated by the following formula: u arc1 = u arc0 + Rd ∆i, (9) where ∆i = i1 − i0 —the increase in arc current on the step. For the method of average voltages integration step of the 2nd order, the equations of voltage increments at the active resistance and at the arc will look like: ∆u R = R di0 d2 i0 ∆t2 ∆t + R 2 , dt dt 2 (10) Energies 2022, 15, 1623 5 of 19 du arc0 di0 d2 u arc0 d2 i0 ∆t2 di dRd d2 i0 ∆t2 ∆t + = Rd0 0 ∆t + , (11) 2 2 di dt dt di dt2 2 di dt 2 In this case, from Equations (1), (2), (10) and (11) we obtain: R + Rd0 ∆t L dRd0 di0 R + Rd0 L U − Ri0 − u arc0 + + ) − + i0 − ( R + i = 0, (12) 3 ∆t 6 di dt 3 ∆t 1 ∆u arc = The expression for determining the circuit current at the end of the integration step will take the form: R + Rd0 L −1 ∆t dRd0 di0 i1 = i0 + + ) , (13) (U − Ri0 − u arc0 ) − ( R + 3 ∆t 6 di dt 3. Mathematical Model of Arc Space of Electrical Arc Furnace According to the chosen approach to EAF modeling, the description of the arc space does not use the arc model in the form of a volt-ampere characteristic, but rather the change of the dynamic arc resistance, which depends on both the current and the arc length. We demonstrate the application of this approach on the example of EAF-200. Experimentally obtained averaged dynamic Volt-Ampere characteristics for such a furnace are shown in Figure 2. Figure 2. Dynamic Volt-Ampere characteristics of EAF. The dynamic Volt-Ampere characteristic of the arc has different curves when the current decreases and increases, and is also asymmetric. Then, obtained on the basis of experimental dynamic Volt-Ampere characteristics of the arc, the dependences of the dynamic resistance of the arc Rd ( Iarc , larc ) for different lines will have the form shown in Figure 3. The kind of the Volt-Ampere characteristic, which corresponds to the current drop, for a single arc length can be successfully approximated as: Uarc = A0 · th( β · Iarc ) i f larc = const, where A0 and β approximation coefficients. (14) Energies 2022, 15, 1623 6 of 19 Figure 3. Dependences of dynamic resistance change of Rd (Iarc , larc ) for different curves of dynamic Volt-Ampere characteristic: (a)—for current increasing, (b)—for current decreasing. Then the equation for determining the dynamic resistance Rd will look like: Rd ( Iarc ,) = A0 · β · 1 − th( β · Iarc )2 , (15) The change in dynamic resistance Rd obtained on the basis of experimental volt-ampere characteristics and its approximation by the equation for Rd (Iarc ) when Iarc = 10 mm are given in Figure 4. Significant deviations observed in the regions of large currents are explained primarily by the shortcomings of the construction of the Volt-Ampere characteristic based on experimental data. Figure 4. Dependences of the change Rd , obtained on the basis of experimental data and when approximating the curves of the volt-ampere characteristic using (a) hyperbolic tangent functions; (b) modified Mexican hat wavelet. In the case of current increasing, the approximation formula is much more complex. Analysis of different models of the current-voltage characteristic curves approximation is given in [22]. We need the derivative of a rather complex model to approximate the arc volt-ampere characteristic to determine Rd . In our opinion, it is more appropriate to approximate the dependence Rd (Iarc ) for a given arc length. If we ignore the asymmetry, the obtained dependence of the change in dynamic resistance resembles the curve obtained when using the Mexican hat wavelet [29]. Energies 2022, 15, 1623 7 of 19 The equation for approximating the characteristic Rd (Iarc ) for a given arc length can be described as: ! 2 ! Iarc 2· a41 0.4337 Iarc · exp − a31 · Rd (Iarc ) = √ (16) · a11 · 1 − σ · a21 σ σ where a11 , a21 , a31 , a41 are required parameters. The change in dynamic resistance Rd obtained on the basis of experimental voltampere characteristics and its approximation using (16) for Iarc = 10 mm at a11 = 9.4, a21 = 0.38, a31 = 18, a41 = 2 and σ = 51, 000 are given in Figure 4b. To take into account the asymmetry of the Volt-Ampere characteristic of the arc, it is necessary to separately determine the coefficients a11 , a21 , a31 , a41 and σ at Iarc < 0 and Iarc ≥ 0. Approximating the obtained values of the coefficients at different arc lengths using polynomials of the appropriate order, we obtain the following equation to determine the dynamic resistance as a function of current and arc length: if Iarc · dIdtarc > 0 then: 2 ! 2· a41 ( larc ) ! 0.4337 Iarc Iarc Rd ( Iarc , larc ) = p · a11 ( larc ) · 1 − exp − a31 ( larc ) · σ ( larc ) · a21 ( larc ) σ ( larc ) σ ( larc ) if Iarc · dIarc dt < 0 then: Rd ( Iarc , larc ) = a0 ( larc ) · 1− Iarc th σ ( larc ) · 0.5 2 !! where: a11 ( larc ) = 0.9658 · larc − 0.1282; a21 ( larc ) = 2.0477 · 10−5 · larc 3 − 0.0005 · larc 2 + 0.0019 · larc + 0.3992; a31 ( larc ) = −0.0026 · larc 3 + 0.0939 · larc 2 − 0.3429 · larc + 14.0293; a41 ( larc ) = 2; σ ( larc ) = 0.0093 · larc 2 − 0.4513 · larc + 9.2986 · 10, 000; a0 ( larc ) = 0.0004 · larc 4 − 0.0124 · larc 3 + 0.1468 · larc 2 + 0.0081 · larc + 0.1418 /1000. The obtained dependences of the change of dynamic resistance Rd (Iarc , larc ) when applying the found approximation functions are given in Figure 5. Figure 5. Approximate dependences of dynamic arc resistance changing as a function of current and arc length: (a) for current increasing, (b) for current decreasing. Energies 2022, 15, 1623 8 of 19 Comparison of the obtained dependences of the dynamic resistance change with the ones shown in Figure 3 dependences allows us to state, that the dependences obtained on the basis approximation functions application of reproduce the nature of the change in dynamic resistance with a change in current and arc length. 4. Mathematical Models of Power System Elements of Electrical Arc Furnace According to the approach described in [23,24], the calculation scheme of a three-phase short network with a furnace space (arc) is presented in the form of a six-pole (Figure 6). Figure 6. Calculation scheme of a short network with a furnace space as a six-pole. Such a multipole is described by a vector Equation (17): t0Z+∆t → → 1 i SL + GSL ∆t → v SL dt + C SL = 0 (17) t0 which connects the currents of the outer branches of the multipole →i SL = iSLA1 , iSLB1 , iSLC1 , iSLA2 , iSLB2 , iSLC2 T with the average values of the potentials of the external poles at the step of numerical ! integration 1 ∆t t 0R +∆t → → v SL dt, v SL = t0 → v SL1 v SL2 → → , v SL1 = (vSLA1 , vSLB1 , vSLC1 ) T , v SL2 = (vSLA2 , vSLB2 , vSLC2 ) T . → Equation (17) is obtained from Equation (8), taking into account that the vector of the average values of the applied voltage at the integration step is equal to: → U SL 1 = ∆t t0Z+∆t → t0 1 v SL1 dt − ∆t t0Z+∆t → v SL2 dt. t0 The coefficients of Equation (17) are defined as a matrix:  GSL     =    1 R∗− SLA 0 0 1 − R∗− LA 0 0  → C SL     =    1 0 0 − R∗− 0 LA −1 1 R∗ LB 0 0 − R∗− LB −1 0 R∗ LC 0 0 1 0 0 R∗− 0 LA −1 1 − R∗ LB 0 0 R∗− LB −1 0 − R∗ LC 0 0 1 − R ∗− R i + u + iSLA0 ( ) SLA SLA0 arcA0 SLA 1 − R ∗− R i + u + i ( ) arcB0 SLB SLB0 SLB0 SLB 1 − R ∗− R i + u + i ( ) SLC SLC0 arcC0 SLC0 SLC 1 R ∗− R i − u − i ( ) SLA SLA0 arcA0 SLA0 SLA 1 R ∗− SLB ( RSLB iSLB0 − u arcB0 ) − iSLB0 1 R ∗− SLC ( RSLC iSLC0 − u arcC0 ) − iSLC0 0 0 1 − R∗− LC 0 0 −1 R ∗  LC                  R∂C0 R∂A0 R∂B0 SLA SLB SLC where R∗SLA = RSLA + + L∆t , R∗SLB = RSLB + + L∆t , R∗SLC = RSLC + + L∆t —step phases 2 2 2 resistances, iSLA = −iSLA1 = iSLA2 , iSLB = −iSLB1 = iSLB2, iSLC = −iSLC1 = iSLC2—phase currents. Energies 2022, 15, 1623 9 of 19 The three-phase transformer is represented by a 12-pole (see Figure 7), the outer circuits of which are the winding outputs Figure 7. Calculation scheme of a three-phase transformer. Using an approach similar to the network model with an arc had need applied, we obtain the vector equation of a three-phase transformer: → i TR + GTR 1 ∆t t0Z+∆t → → v TR dt + C TR = 0 (18) t0 T where →i TR = −→i tr , →i tr – vector of external branches currents; →i tr = iTA , iTB , iTC , iTa , iTb , iTc T —vector of primary and secondary transformer windings currents; →v TR = vTA1 , vTB1 , vTC1, vTa1 , vTb1, vTc1 , vTA2, vTB2 , vTC2, vTa2 , vTb2 , vTc2 T — vector of external poles potentials. The coefficients of Equation (18) are defined as follows:  →  → −1 − R ∗ R i + i 1 1 tr0  TR tr0 ^ → TR  R∗ − −R∗ − TR TR , C   → GT = TR =  −1 −1 →  −R∗ TR R∗ TR 1 R ∗− R i − i tr0 TR tr0 TR LTR where R∗TR = RTR 2 + ∆t —matrix of step resistances of the transformer; RTR = diag RTA , RTB , RTC , RTa , RTb , RTc — matrix of windings active resistances; LTR —matrix of self- and mutual- inductances of windings:   L AA L AB L AC L Aa L Ab L Ac  L BA L BB L BC L Ba L Bb L Bc     LCA LCB LCC LCa LCb LCc    LTR =    L aA L aB L aC L aa L ab L ac   L LbB LbC Lba Lbb Lbc  bA LcA LcB LcC Lca Lcb Lcc A three-phase network with a choke is represented by a six-pole (Figure 8): Similarly, applying of AVIS method into the electrical circuit of a three-phase network with a choke, we obtain the Equation (19): i N A1 = i N A0 + i NB1 = i NB0 + i NC1 = i NC0 + RN A 2 R NB 2 R NC 2 + + + LN A ∆t L NB ∆t L NC ∆t −1 −1 −1 (UN A + EN A − R N A i N A0 ) (UNB + ENB − R N A i NB0 ) (UNC + ENC − R N A i NC0 ) (19) Energies 2022, 15, 1623 10 of 19 where RNA , RNB , RNC —resistance of phases; LNA , LNB , LNC inductance of phases t0R +∆t t0R +∆t t0R +∆t 1 1 1 in choke; UN A = ∆t u N A dt, UNB = ∆t u NB dt, UNC = ∆t u NC dt—average t0 t0 of a branch voltages at the integration step; EN A = ENC = 1 ∆t t0R +∆t 1 ∆t t0R +∆t t0 e N A dt, ENB = t0 1 ∆t t0R +∆t e NB dt, t0 e NC dt—average values of EMF in integration step. t0 Figure 8. Calculation scheme of a three-phase network with a choke. Based on the system of Equation (19), the vector equation of a three-phase network with a choke as a six-pole can be written as: → 1 i N + GN ∆t t0Z+∆t → → v N dt + C N = 0 (20) t0 → where i N = (i N A , i NB , i NC , −i N A , −i NB , −i NC ) T —vector of currents of outer brunches, → v N = (v N A1 , v NB1 , v NC1 , v N A2 , v NB2 , v NC2 ) T —vector of potentials of external poles:  GN     =    1 R∗− NA 0 0 1 − R∗− NA 0 0 where R∗ N A = R N2 A + network with choke. 1 0 0 − R∗− 0 NA −1 1 R∗ NB 0 0 − R∗− NB −1 0 R∗ NC 0 0 1 0 0 R∗− 0 NA 1 1 − R∗− 0 0 R∗− NB NB 1 0 − R∗− 0 0 NC  −1 − R ∗ N A ( R N A i N A0 ) + i N A0  − R ∗ −1 ( R i  NB NB0 ) + i NB0   NB   1 →  − R ∗− NC ( R NC i NC0 ) + i NC0  C SL =   1  R ∗−  N A ( R N A i N A0 ) − i N A0   1  R ∗−  R i − i ( ) NB NB0 NB0 NB 1 R ∗− R i − i ( ) NC NC0 NC0 NC LN A ∆t , R∗ NB = R NB 2 + L NB ∆t , R∗ NC = R NC 2 + 0 0 1 − R∗− NC 0 0 1 R∗− NC L NC ∆t —step      ,    resistances of 5. Simulation Model and Calculation Algorithm of Power Supply System of Electrical Arc Furnace The mathematical model of the arc power supply system is formed from the models of its separate multipole elements connected according to the calculation scheme shown in Figure 9. Energies 2022, 15, 1623 11 of 19 Figure 9. Calculation scheme of arc power supply system. The outer branches of the multipoles are interconnected at the nodes of the electrical circuit. The connection method is mathematically described by incidence matrices, which indicate with which independent nodes of the system the poles of the multipole element are connected. For schemes, which shown in Figure 9, the incidence matrices of elements  are following: for a three-phase network with a choke Π N         1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1    =    0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0      , Π TR =            —for transformer, ΠSL =       0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0         —for the short network with furnace space. According to the algorithm described in [24], the average in integration step valt0R +∆t → → → 1 ues of the potentials of the independent nodes in system V S = ∆t v S dt, v S = t0 (v1 , v2 , v3 , v4 , v5 , v6 )T we can find from Equation (21): 1 GS ∆t t0Z+∆t → → v S dt + C S = 0, (21) t0 matrices of coefficients which are determined on the basis of coefficients of Equations (17), (18) and (20) and incidence matrices as: → → → → T GS = Π N G N Π TN + Π TR GTR Π TTR + ΠSL GSL ΠSL , C S = Π N C N + Π TR C TR + ΠSL C SL . The system (21) is solved at each integration step with respect to the vector of averages in step values of potentials of the independent nodes in the system, then determine the averages value of the pole potentials of each element can be rewriten as: Energies 2022, 15, 1623 12 of 19 t0R +∆t → → → 1 v N dt = Π TN · V S = Π TN · for three-phase network with a choke V N = ∆t t0 ! ! t0R +∆t t0R +∆t t0R +∆t → → → → → 1 1 1 T T v S dt , for transformer V TR = ∆t v TR dt = Π TR · V S = Π TR · ∆t v S dt ∆t t0 t0 → and for the short network with furnace space V SL = ! t0R +∆t → 1 v S dt . ∆t 1 ∆t t0R +∆t t0 t0 → → T · V = ΠT · v SL dt = ΠSL S SL t0 After determining the average in the step values of the pole potentials of each element by Equations (17), (18) and (20), can be determined the vectors of the currents of the outer branches: t0R +∆t → → → 1 v N dt − C N , i N = −G N ∆t → 1 i TR = −GTR ∆t → 1 i SL = −GSL ∆t t0 t0R +∆t → → t0 t0R +∆t → → v TR dt − C TR , v SL dt − C SL . t0 The calculation algorithm, which was described above, starting from finding of coefficients of vector equations of elements-multipoles (17), (18), (20) and finishing with definition of vectors of currents of external branches is cyclic. 6. Mathematical Model of the Electrode Movement System The electric drive of the electrode movement system (see Figure 10) uses a DC motor for each phase. Its supply voltage is formed by a thyristor converter based on the mismatch signal ∆arc , which is defined by Equation (22): ∆ arc = aIarc − bUarc (22) where Iarc, Uarc—current values of arc current and voltage; a, b—proportionality factros (setpoint). Figure 10. Structural-functional diagram of the electrode movement system. The value of the electrode movement is defined as: le = K M t0Z+∆t ωdt t0 (23) Energies 2022, 15, 1623 13 of 19 where KM —the transmission coefficient of the mechanical system; J—the moment of inertia; ω—the angular speed of the motor. The length of the arc larc is determined by the sum of the displacement of the electrode le and the displacement of the charge in the melt process ldist , which are perturbations in the furnace space larc = le ± ldist . It should be noted that ldist changes randomly and the task of the automated control system (ACS) is to ensure that these perturbations work out. Mathematical description of the DC motor by the method of medium voltages in the integration step of the first order is described in [30] and has the form: i a1 = Ua − KΦ ω0 − R a i a0 + ! ! 2 ∆t KΦ Ra La + − i a0 × 2 ∆t 3J ω1 = ω0 + KΦ ∆t (i a0 + i a1 ) 2J K2 ∆t La Ra + − Φ 2 ∆t 6J ! −1 (24) (25) where ia0 , ia1 —the armature current of the motor at the beginning and end of the integration step; ω 0 , ω 1 —angular rotation speed at the beginning and end of the integration step; Ua —supply voltage; Ra —the active resistance of the armature winding; La —inductance of the armature winding; KΦ —design factor; J—moment of inertia. In the described model, the value of the static torque of the motor, which is small compared to the dynamic torque, is neglected. The magnitude of the electrodes movement is defined by the following equation: le1 = le0 + K M ∆t ( ω0 + ω1 ) 2 (26) 7. The Results of Process Investigation The results of calculation of steady symmetrical modes in the arc power supply system for different values of arc length: 2 mm (in Figure 11) and 20 mm (in Figure 12) are given in Figures 11 and 12. As shown by the results of studies, increasing the length of the arc leads to an increase in the dynamic resistance of the arc, an increase in arc voltage and a decrease in current in the supply circuit. Figure 11. Dynamic arc resistance (a), instantaneous values of arc voltages (b), instantaneous values of arc currents (c), instantaneous values of mains currents (d) in steady state for arc length 2 mm. Energies 2022, 15, 1623 14 of 19 Figure 12. Dynamic arc resistance (a), instantaneous values of arc voltages (b), instantaneous values of arc currents (c), instantaneous values of mains currents (d) in steady state for arc length 20 mm. Increasing the length of the arc also leads to a deterioration of the shape of the current in the arc supply circuit and the shape of the current (Figure 12c) consumed from the mains (Figure 12d). The results of calculation of the steady asymmetric mode in the power supply system of arcs for different values of arc length in phases: phase A—2 mm, phase B—8 mm, phase C—20 mm are shown in Figure 13. Figure 13. Dynamic arc resistance (a), instantaneous values of arc voltages (b), instantaneous values of arc currents (c), instantaneous values of mains currents (d) in steady asymmetric mode for arc length: phase A—2 mm, phase B—8 mm, phase C—20 mm. Energies 2022, 15, 1623 15 of 19 In Figure 14 are shown the results of the calculation of the transient mode of arc ignition and the output to the operating mode, which is determined by the ACS for the electrodes movement which described above (Figure 10). The electrode movement system provides the formation of the arc length for which the mismatch signal defined by expression (22) becomes equal to 0. Figure 14. Arc length (a), current value of arc current (b), current value of arc voltage (c), dynamic arc resistance (d) in transient electrode movement mode. The nature of the change in the obtained dependences of current and arc voltage, current and voltage of the power supply network corresponds to the experimentally removed dependences given in the publications of other authors, in particular [18]. In addition, the models created on the basis of the proposed approach can function in combination with physical objects, which createsmodel predictive control. The simulation results completely repeat the dependences obtained using the EAF model, in which the dynamic volt-ampere characteristics of the arc are tabulated and the 4th order Runge-Kutta method is used for calculations. However, the model proposed in this article has a higher (several times) calculation speed and numerical stability (the ability to perform calculations over long time intervals).The adequacy of the mentioned model of EAF with approximated volt-ampere characteristics of arcs was confirmed using the methods of statistical data analysis, in particular the method of one-way analysis of variance [31], which based on the following equation: k t= N−k · k−1 2 ∑ ni · ( x i − x ) i =1 k ni ∑ ∑ xij − xi i =1 j =1 (27) 2 Energies 2022, 15, 1623 16 of 19 k where N = ∑ ni —input number; k—number of samples; ni, i = 1, 2, . . . , k—number of i-th sample; xi = 1 ni i =1 ni · ∑ xij , i = 1, 2, . . . , k—average value of i-th sample; x = j =1 1 N k ni ∑ ∑ xij —summarized i =1 j =1 average value; M—factor of Barlett criteria [31]. ln 10 2 2 t = c ( N − k) · lgs − ∑ (ni − 1) · lgsi k c = 1 + 3·(k1−1) · ∑ n 1−1 − N1−k s2 where s2i = 1 n i −1 = 1 N −k i =1 i · ∑ (ni − 1) · s2i ni 2 · ∑ xij − xi —variance of the i-th sample j =1 These equations applying for data samples, which are shown in Figures 15 and 16. These dependencies correspond to the modes of the beginning of melting and the mode of completion of melting of wells. Figure 15. The dependences of the phase B current change for the melting start stage obtained experimentally (a) and with the help of a model with tabular volt-ampere characteristics and the application of the 4-th order Runge-Kutta method (b). Figure 16. The dependences of the phase B current change for the stage of melting completion obtained experimentally (a) and using a model with tabular volt-ampere characteristics and the application of the 4-th order Runge-Kutta method (b). Energies 2022, 15, 1623 17 of 19 To obtain the results of mathematical modeling on the model of EAF, perturbations were applied, which in their spectral characteristics correspond to the perturbations acting in a real furnace at this stage of melting. In Figure 15a the scale for I 10 mm = 25,000 A, for time—10 mm = 0.2 s; in Figure 16–scale for I-axes—10 mm = 20,000 A, for time—10 mm = 0.2 s. As can be seen from results in Table 1, the values of the relevant criteria obtained for different modes of operation of the EAF are less than the corresponding tabular values taken for 5%—the level of significance, which allowed us to conclude that the created system model adequately reflects the processes taking place in a real life project. Table 1. Statistical data analysis. Number of Samples Melting start Copletion of wells melting experiment 301 919 model Valid criterion value experiment 301 6001 model Valid criterion value Average Value Value of Criterion (14) Dispersion Value M Barlett Criterion 44,580 45,860 2.93 1.876 × 108 1648 × 108 1924 3.84 47,420 47,720 1.191 3.84 384 1037 × 108 1205 × 108 3079 384 It should be noted that the speed of the EAF model created using the method of medium voltage at the integration step and the use of dynamic arc resistance approximation is almost two orders of magnitude higher than the model with tabular volt-ampere characteristics and 4th order Runge-Kutta method, as well as the fact that the system retains numerical stability over long intervals of calculations. In further research authors plan to analyze the influence of different control systems (single-circuit—when implementing different laws of electrode movement control and double-circuit—with additional high-speed inductor control circuit) on the characteristics of the power supply network, and to synthesize new, effective laws of arc electrode movement control of EAF using fractional and intelligent controllers for single- and double-circuit control systems. 8. Conclusions Using the method of medium voltages at the step of numerical integration a mathematical model of the arc power system is developed. Such a model of the arc power system with electromechanical electrode movement system allows one to study transient and steady-state modes taking into account the nonlinearity of the Volt-Ampere characteristics of the arc and load asymmetry. The application of this method provides high model performance and numerical stability, which allows it to be used for real-time testing of electrode movement control systems using “hardware-in-the-loop” technology. The adequacy of the developed model is confirmed by comparison with the results of the experiment on real EAF. The proposed approximation of the dynamic arc resistance curve using a combination of Mexican hat wavelet and the derivative function th(x) provides a fairly accurate reproduction of the dynamic arc resistance obtained on the basis of experimental volt-ampere characteristics for different currents and arc lengths. Author Contributions: Conceptualization, A.L., J.K., A.K. and Z.Ł.; methodology, A.L., J.K., A.K., Z.Ł. and Y.P.; software, A.L., A.K. and J.K., validation, A.L., J.K., A.K., A.K.-F. and Y.P.; formal analysis, A.L., J.K., A.K., A.K.-F. and Y.P.; investigation, A.L., J.K., A.K., Y.P., Z.Ł. and L.K.; data curation, J.K., A.K.-F., Z.Ł. and L.K.; writing—original draft preparation, A.L. and A.K.; writing—review and editing, Y.P., J.K., A.K.-F., Z.Ł. and L.K.; visualization, A.L., J.K., A.K.-F. and L.K.; supervision, A.L. and J.K.; project administration, A.L., J.K., Z.Ł., A.K.-F. and A.K. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Energies 2022, 15, 1623 18 of 19 Institutional Review Board Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. 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https://openalex.org/W4286602952	https://nottingham-repository.worktribe.com/file/9098631/1/Effects%20of%20emotion%20and%20semantic%20relatedness	English	null	Effects of emotion and semantic relatedness on recognition memory: Behavioral and electrophysiological evidence	Psychophysiology	2,022	cc-by	12,124	Received: 13 January 2021 \| Revised: 17 March 2022 \| Accepted: 1 June 2022 Received: 13 January 2021 \| Revised: 17 March 2022 \| Accepted: 1 June 2022 DOI: 10.1111/psyp.14152 O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Effects of emotion and semantic relatedness on recognition memory: Behavioral and electrophysiological evidence Meng Han1 \| Bingcan Li1 \| Chunyan Guo1 \| Roni Tibon2,3 1Beijing Key Laboratory of Learning and Cognition, School of Psychology, Capital Normal University, Beijing, PR China Funding information Funding information British Academy Postdoctoral Fellowship, Grant/Award Number: SUAI/028 RG94188 and fp170046; Capacity Building for Sci-Tech Innovation - Fundamental Scientific Research Funds, Grant/Award Number: 025185305000/200; National Natural Science Foundation of China, Grant/ Award Number: 31671127 Abstract Some aspects of our memory are enhanced by emotion, whereas others can be unaffected or even hindered. Previous studies reported impaired associative memory of emotional content, an effect termed associative “emotional interfer- ence”. The current study used EEG and an associative recognition paradigm to investigate the cognitive and neural mechanisms associated with this effect. In two experiments, participants studied negative and neutral stimulus-pairs that were either semantically related or unrelated. In Experiment 1 emotions were relevant to the encoding task (valence judgment) whereas in Experiment 2 emo- tions were irrelevant (familiarity judgment). In a subsequent associative recog- nition test, EEG was recorded while participants discriminated between intact, rearranged, and new pairs. An associative emotional interference effect was ob- served in both experiments, but was attenuated for semantically related pairs in Experiment 1, where valence was relevant to the task. Moreover, a modulation of an early associative memory ERP component (300–550 ms) occurred for negative pairs when valence was task-relevant (Experiment 1), but for semantically related pairs when valence was irrelevant (Experiment 2). A later ERP component (550– 800 ms) showed a more general pattern, and was observed in all experimental conditions. These results suggest that both valence and semantic relations can act as an organizing principle that promotes associative binding. Their ability to contribute to successful retrieval depends on specific task demands. 2MRC Cognition & Brain Sciences Unit, University of Cambridge, Cambridge, UK 2MRC Cognition & Brain Sciences Unit, University of Cambridge, Cambridge, UK 3School of Psychology, University of Nottingham, Nottingham, UK associative recognition, EEG, emotion, ERPs, familiarity, old-new ERP effect, recollection, semantic relatedness, unitization, valence associative recognition, EEG, emotion, ERPs, familiarity, old-new ERP effect, recollection, semantic relatedness, unitization, valence Correspondence Correspondence Roni Tibon, School of Psychology, University of Nottingham, University Park, The University of Nottingham, Nottingham, NG7 2RD, UK. Email: roni tibon@nottinghmam ac uk Chunyan Guo, School of Psychology, Capital Normal University, No. 23 Baiduizijia, Fuwaidajie St. Haidian District, Beijing, 100048, PR China. Email: guocy@cnu.edu.cn 1 \| INTRODUCTION Previous studies investigating the effects of emotion on these ERP components yielded mixed results. Several studies used a context effect paradigm, in which neutral stimuli were encoded in the context of a neutral/nega- tive sentences (Maratos & Rugg, 2001) or scenes (Smith et al., 2004; Ventura-Bort et al., 2016), and were subse- quently discriminated from new stimuli via an old/new recognition judgment task. In one report (Ventura-Bort et al., 2016), the valence of the encoding context modu- lated the early and late old/new ERP effects. In another (Maratos & Rugg, 2001), a similar modulation was ob- served but only in one (out of two) of the experiments re- ported in the study. Conversely, in a third report (Smith et al., 2004), neither the early old/new component, nor the late one, were modulated by the valence of the con- text, but instead additional early ERP components were elicited for items encoded in emotional contexts. In ad- dition, several studies that employed a source-memory paradigm, in which participants are explicitly asked about the encoding context, reported increased modulation of the old/new ERP effects for emotional contexts (Minor & Herzmann, 2019; Newsome et al., 2012), whereas others reported an opposite pattern (Mao et al., 2015). Finally, using subjective measures of familiarity and recollection (via remember/know or confidence judgments), several studies reported stronger recollection for emotional vs. neutral stimuli, accompanied by larger ERP old/new ef- fects for the former (Schaefer et al., 2011; Ventura-Bort et al., 2016; Weymar et al., 2010, 2011). In the current study, we used an associative recognition task, together with the well-established distinction between familiar- ity and recollection, and the putative electrophysiologi- cal markers corresponding to these processes, to aid our understanding of the mechanisms that drive emotional effects on associative memory. In contrast to the facilitative effects of emotion on within-item binding, inter-item associative memory (i.e., remembering that two or more items were encoded to- gether) for emotional content is often hindered, an ef- fect termed associative “emotional interference” (Madan et al., 2012; Mao et al., 2015; Mather & Knight, 2008; Pierce & Kensinger, 2011; Rimmele et al., 2011). One method of accessing associative episodic memory is recognition, i.e., the judgment that currently presented items were previ- ously experienced together in a specific episodic context. In an associative recognition task, during an initial study phase, participants memorize pairs of stimuli. 1 \| INTRODUCTION Kensinger, 2009; MacKay et al., 2004; Mather & Sutherland, 2011). The term emotionally enhanced mem- ory (EEM) refers to the common finding that emotional events are remembered better than neutral ones; a phe- nomenon that had been demonstrated for memory of Convergent evidence indicate that emotions influence memory, but the ways in which they operate and man- ifest require further specification (Easterbrook, 1959; Psychophysiology. 2022;00:e14152. \| 1 of 16 https://doi.org/10.1111/psyp.14152 wileyonlinelibrary.com/journal/psyp This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Psychophysiology published by Wiley Periodicals LLC on behalf of Society for Psychophysiological Research. g p p y © 2022 The Authors. Psychophysiology published by Wiley Periodicals LLC on behalf of Society for Psychophysiological Research. \| 1 of 1 wileyonlinelibrary.com/journal/psyp HAN et al. 2 of 16 evidence, this distinction is supported by a large corpus of event-related potential (ERP) studies, revealing two distinct components which are differentially engaged in recognition judgments. The first component is considered to be the electrophysiological index of familiarity. It onsets ~300–500 ms post-stimuli presentation, elicits greater neg- ativity for new vs. old items, and is prominent over mid- frontal locations. The second component is considered to be an electrophysiological index of recollection. It onsets ~500–800 ms post-stimuli presentation, elicits greater pos- itivity to old vs. new stimuli, and is usually (though not always) prominent over parietal locations (reviewed by Curran, 2000; Diana et al., 2007; Rugg & Curran, 2007). emotional words, pictures, and stories (e.g., Bradley et al., 1992, with pictorial stimuli; Cahill & McGaugh, 1995, with stories; and LaBar & Phelps, 1998, with taboo words). According to one suggestion (Kensinger, 2009), this ad- vantage for emotional content, and negative content in particular, arises from increased engagement of sensory processes, leading to focused attention on intrinsic de- tails and to focal enhancement of the negative event. A similar account is the object-based framework (see also arousal-biased competition; Mather & Sutherland, 2011), which posits that emotional objects attract attention that enhances within-item binding of their constituent fea- tures (Mather, 2007). Accordingly, studies have shown that emotion facilitates memory for within-object mem- ory binding (Kensinger, 2009; Mather & Nesmith, 2008; Mather & Sutherland, 2011; Nashiro & Mather, 2011; Schmidt et al., 2011; Steinmetz et al., 2015). 2 \| EXPERIMENT 1 In experiment 1, participants memorized and retrieved picture-picture pairs while EEG was recorded. Half the pairs were of negative objects (e.g., gun, spider) and half of neutral objects (e.g., bunny, traffic light). In addition, half the pairs contained objects that were semantically related (e.g., bunny-carrot), whereas half contained objects with no pre-existing semantic relations (e.g., spider-bullet). During study, participants performed a valence judgment task, and at test they were asked to discriminate between intact, rearranged and new pairs. We predicted an overall reduction in associative mem- ory accuracy for negative vs. neutral pairs due to associative emotional interference. Importantly, possible moderation of this effect by semantic relatedness will crucially de- pend on the mnemonic processes promoted by semantic relations: either unitization or LOP. More specifically, if semantic relations promote unitization, then associative retrieval of semantically related pairs would benefit from within-item binding. In this case, we would expect an at- tenuation of the associative emotional interference effect for negative pairs that are semantically related (compared to unrelated). We therefore predicted that associative memory for negative-related pairs would be similar (if not comparable) to memory for neutral-related pairs. We further predicted that this attenuation will be accompa- nied by a modulation of the early ERP component—the electrophysiological marker of familiarity—for related but not for unrelated pairs. Thus, the early associative mem- ory ERP effect was expected to be greater for related pairs than for unrelated pairs. If, on the other hand, relatedness increases LOP, then we would not expect the associative emotional interference effect to be attenuated for related pairs (indeed, we might even observe the opposite pat- tern). Moreover, we would expect increased modulation The suggestion that unitization corresponds to se- mantic relatedness effects is especially intriguing given the emotional enhancement effect of within-object memory binding (Mather & Sutherland, 2011). More specifically, it had been argued that when items are unitized, rather than relying on inter-item binding, their relations are supported by links that are qualitatively similar to within-object links (e.g., Mayes et al., 2007; Tibon et al., 2018). Therefore, unitized associations might be subjected to the enhancing effects of emotions on item memory. In other words, if emotions benefit within-object binding, then unitization of emotional stimuli might alleviate the negative effects of emotion on associative memory. 1 \| INTRODUCTION Then, at a subsequent test phase, they are asked to discriminate be- tween intact (studied pairs), rearranged (studied items with new combination), and new pairs (of unstudied items). Thus, successful performance require memory for the specific pairings of the stimuli that were encoded. Pierce and Kensinger (2011) used this task to investigate the effect of emotion on associative memory. In their ver- sion of the task, studied pairs included either emotionally arousing or neutral words-pairs. Whereas for intact pairs accuracy did not differ, emotionally arousing rearranged word-pairs (and negative ones in particular) were less likely to be correctly identified as rearranged relative to neutral word-pairs. This interaction between emotional effects and retrieval conditions points to the multiplicity of processes involved in associative recognition, and their varying sensitivity to the influence of emotional content. The prevalent dual-process model of recognition memory suggests that recognition involves two separa- ble processes: familiarity and recollection. Familiarity is a general feeling of encountering something or someone which was previously seen, without memory of additional details from that initial encounter, whereas recollection involves the conscious retrieval of additional contextual details (for review see Yonelinas, 2002). Amongst other Furthermore, the current study sought to examine how pre-existing semantic relations between episodi- cally associated stimuli interact with emotional effects on associative memory. Semantic relatedness might affect memory in two different ways. First, congruent HAN et al. 3 of 16 stimuli combinations, such as related (vs. unrelated) pictures, support high Levels of Processing (LOP; Craik & Lockhart, 1972), yielding rich and elaborated encod- ing. In turn, during retrieval, this elaborated encoding supports high levels of recollection (summarized by Craik, 2002). Second, stimuli that share pre-existing relations can be subjected to unitization—an encoding process by which several discrete items are perceived and encoded as one single unit (reviewed by Mecklinger & Jäger, 2009; Yonelinas et al., 2010)—and to be sub- sequently retrieved via familiarity (e.g., Li et al., 2019; Tibon, Ben-Zvi, & Levy, 2014; Zheng, Li, Xiao, Broster, Jiang, & Xi, 2015). This latter suggestion is rooted in the idea that instead of creating inter-item links between the items, unitization results in a joint representation of the encoded items (that is, a single item, rather than several linked items). 1 \| INTRODUCTION Importantly, unitization is not only enabled via explicit instructions to process pairs of memoranda as a single unit, but can also be driven by stimulus- or associative-properties of the encoded items (e.g., Tibon, Gronau, et al., 2014). This notion had been demonstrated in several ERP studies, in which the putative electrophysiological correlate of familiarity— the early old/new effect—was observed for episodi- cally formed associations between semantically related stimuli, but not for episodic associations between se- mantically unrelated stimuli (Li et al., 2019; Rhodes & Donaldson, 2007; Tibon, Ben-Zvi, & Levy, 2014; Tibon, Gronau, et al., 2014; Zheng, Li, Xiao, Broster, Jiang, & Xi, 2015). depend on the relevancy of emotions to the encoding task. This point is further elaborated below, but in short, previous studies suggested that emotional content ef- fects on processing and behavior often emerge when emotions are relevant to the task that is performed (e.g., Engen et al., 2017; Huang et al., 2008; Pessoa, 2009; Stein et al., 2009; Wei et al., 2015), though it is unclear whether this also applies to memory. Accordingly, in the present study we conducted two experiments, in which emotions were either relevant (Experiment 1) or irrel- evant (Experiment 2) to the encoding task. In both ex- periments, an associative memory paradigm was used in order to examine the effects of valence (negative, neu- tral) and relatedness (related, unrelated) of memoranda on associative recognition. 2.1.1 \| Participants To determine the required sample size, we first ex- tracted the behavioral effect size obtained by Pierce & Kensinger (2011; N = 32, Cohen's f = 0.42), who employed a similar experimental design to the one used in the cur- rent study. Next, we extracted the associative memory ERP effects (for the early and late ERP components) from multiple studies that used an associative memory para- digm (Bader & Mecklinger, 2017; Kamp et al., 2016; Li et al., 2017; Li et al., 2019; Rhodes & Donaldson, 2008; Tibon, Gronau, et al., 2014; Zhao et al., 2020; Zheng, Li, Xiao, Broster, Jiang, & Xi, 2015). The effect sizes for the associative memory component reported in these studies were all medium-large, ranging from Cohen's f = 0.3 to 10.7 (N ranging between 17 and 46). Nevertheless, to avoid an overinflated estimation of effect size, we set f = 0.25 as a lower, more conservative value. Based on this effect size, we estimated that power > .8 (α = .01) would require at least 39 participants (actual power = .81), and therefore recruited 47 participants for the study. Forty-seven healthy, right-handed native Chinese speakers (30 females; mean age 22.4 ± 2 years) from Capital Normal University participated in the experiment and were paid ¥30/h. All participants had normal or corrected- to-normal vision, were prescreened for history of neuro- logical or psychiatric disorders, learning disorders, head injury or psychotropic drug use. Informed consent, ap- proved by the Capital Normal University Institutional Review Board, was collected from each participant. Data from five participants were discarded, including one par- ticipant with very poor task performance (associative Pr <0), and four participants with insufficient number of artifact-free ERP trials in one or more experimental condi- tions (N trial < 16). Our final sample therefore included 42 participants (27 females; mean age 22.4 ± 2 years). We subsequently constructed 200 rearranged pairs by combining together pictures belonging to different pairs, but keeping their type and location unchanged, such that there were 50 rearranged pairs for each type. For example, two related neutral pairs A-B (e.g., rabbit-carrot) and C-D (e.g., goat-cabbage) could be recombined to form another related neutral pair A-D (rabbit-cabbage). B and C would also be combined with other items (belonging to related neutral pairs) to form rearranged pairs. 2.1.1 \| Participants The same sam- ple (N = 10) of participants in prior relatedness ratings were recruited again and provided ratings for relatedness. The results confirmed our initial assignment of pairs, and Photoshop 8.0, and were presented at the center of the picture on a gray background (RGB: 150; see Figure 1 for examples). of the late ERP—the marker of recollection—for related compared to unrelated pairs. of the late ERP—the marker of recollection—for related compared to unrelated pairs. An independent sample (N = 12) provided ratings of the pictures on the dimensions of valence, arousal and familiarity, using a scale ranging from 1 (very negative/ calm/unfamiliar) to 9 (very positive/exciting/familiar). Pictures with familiarity ratings below 4 were removed from the pool. 1272 pictures, including 646 negative pictures (with valence scores below 4) and 625 neutral pictures (with valence scores between 4 and 7), were chosen and combined to form negative/negative pic- ture pairs or neutral/neutral picture pairs, resulting in 166 semantically-unrelated negative, 150 semantically- unrelated neutral, 157 semantically-related negative, and 162 semantically-related neutral picture pairs. Another independent sample (N = 10) provided ratings of the pairs on the dimension of relatedness. They were asked to judge how likely it is for the two objects to appear to- gether (Tibon, Gronau, et al., 2014), by responding on a scale ranging from 1 (very unlikely) to 9 (very likely). Only pairs for which the pre-assigned relatedness status was verified (i.e., unrelated stimuli with relatedness score <5, and related stimuli with relatedness score ≥5) by the ma- jority of the raters (at least 6/10 raters) were included in the study. Based on the ratings, 600 pairs were selected, including 150 semantically-related negative pairs, 150 semantically-related neutral pairs, 150 semantically- unrelated negative pairs, and 150 semantically-unrelated neutral pairs. Semantically-related pairs either belonged to the same category (e.g., “desk-sofa”), or were function- ally related (e.g., “rabbit-carrot”). The relatedness scores of related pairs [Mean (SD) = 6.61 (1.25)] were significantly higher than that of unrelated pairs [Mean (SD) = 1.61 (.41); t (299) = 64.20, p < .001]. Negative pictures were significantly more negative and arousing than neutral pictures [valence: Meanneg (SD) = 3.05 (0.66), Meanneu (SD) = 5.06 (0.31), t (599) = −59.22, p < .001; arousal: Meanneg (SD) = 5.43 (1.23), Meanneu (SD) = 2.41 (0.63), t (599) = 45.77, p < .001], but equal to neutral pictures on familiarity (p > .05). 2 \| EXPERIMENT 1 Indeed, previous studies showed that when several items were integrated together (e.g., by creating a mental image of the words comprising a pair interacting with each other. For example, for the word pair “flies-booger”: imagining flies stuck in booger), associative emotional interference can disap- pear or even transform into mnemonic benefits (e.g., Guillet & Arndt, 2009; Murray & Kensinger, 2012). Finally, the current study examined whether emo- tional and relatedness effects on associative memory HAN et al. 4 of 16 Stimuli Our initial stimuli dataset included 1469 object pictures of animals, food, equipment, tools, appliances, etc., from the Hemera Photo-Objects Collection (Hemera Photo Objects, Gatineau, Quebec, Canada), the International Affective Pictures System (Lang et al., 2008) and free in- ternet sources. Colored objects were matched for size, lu- minance and contrast by using adjustment curve in Adobe \| 5 of 16 HAN et al. FIGURE 1 Example stimuli and experimental paradigm. (a) Example stimuli for each condition. Our factorial design included four types of picture pairs, varying in their semantic relatedness (related, unrelated) and valence (negative, neutral). (b) Schematic illustration of the experimental paradigm. At study, participants viewed picture-pairs and indicated which item is more negative (Experiment 1) or more familiar (Experiment 2). At test, they discriminated between intact, rearranged, and new pairs. HAN et al. FIGURE 1 Example stimuli and experimental paradigm. (a) Example stimuli for each condition. Our factorial design included four types of picture pairs, varying in their semantic relatedness (related, unrelated) and valence (negative, neutral). (b) Schematic illustration of the experimental paradigm. At study, participants viewed picture-pairs and indicated which item is more negative (Experiment 1) or more familiar (Experiment 2). At test, they discriminated between intact, rearranged, and new pairs. 2.1.3 \| Procedure showed that the relatedness scores of related pairs [Mean (SD) = 6.67 (1.23)] were significantly higher than that of unrelated pairs [Mean (SD) = 1.65 (.57); t (99) = 64.90, p < .001]. Importantly, there was no difference in related- ness between the rearranged pairs and the original pairs [Unrelated pairs: t (99) = 1.07, p = .29; Related pairs: t (99) = 0.76, p = .35]. Participants were seated at a distance of 70 cm from a Dell monitor in an electrically shielded room. Picture pairs, with a visual area of 10° × 5°, were displayed (using Presentation by Neurobehavioral Systems, Inc) horizon- tally at the center of the monitor against a black back- ground. A standard study-test paradigm was adopted, with the study phase followed by the test phase after a 10 min delay. Four self-paced breaks were provided dur- ing the study phase and during the test phase. Stimuli were presented pseudo-randomly to ensure that no more than three consecutive trials were from the same condition. A total of 400 picture pairs were encoded at the study phase (100 related negative pairs, 100 related neutral pairs, 100 unrelated negative pairs, 100 unrelated neutral pairs), with the remaining 200 pairs serving as new pairs during the test phase. At test, 200 intact pairs (the same pairs shown at study), 200 rearranged pairs (pictures belonging to different study pairs that were recombined together), and 200 new pairs were presented, with each condition containing 50 related negative, 50 related neutral, 50 un- related negative, and 50 unrelated neutral pairs. Test pairs were counterbalanced across subjects, with every picture presented equally often as part of intact, rearranged, or new pairing. At study, each trial began with a gray fixation cross for 1000–1500 ms, followed by the presentation of a pic- ture pair for 2000 ms, during which the participants were asked to memorize the pairs and perform a valence judg- ment task, namely, to judge which one of the two objects is more negative (Figure 1b). They were asked to press the ‘left arrow’ key on the keyboard if they thought that the HAN et al. 6 of 16 Mean numbers of related analyzed trials were 39 (in- tact), 26 (rearranged), and 38 (new) for negative pairs, and 37 (intact), 26 (rearranged), and 43 (new) for neutral pairs. Behavioral analyses y Behavioral measure of interest was associative Pr: a dis- crimination measure of old/new effects for associative memory, defined by subtracting false alarm rates for rearranged pairs from hit rates for intact pairs (Jäger et al., 2006; Snodgrass & Corwin, 1988). This measure was used to dissociate potential response bias (e.g., for related pairs; Ahmad & Hockley, 2014; Liu & Guo, 2019; Tibon, Gronau, et al., 2014; see Supporting Information 1 for an ancillary analysis of response bias) from a true memory advantage. Pr scores were analyzed using a repeated measure ANOVA with relatedness (related or unrelated) and valence (negative or neutral) as repeated factors, and with Pr score as the dependent measure. 2.1.3 \| Procedure Mean numbers of unrelated analyzed trials were 25 (in- tact), 30 (rearranged), and 37 (new) for negative pairs, and 27 (intact), 30 (rearranged), and 41 (new) for neutral pairs. left object was more negative, to press the ‘right arrow’ key if they thought that the right one was more negative, and to press the ‘down arrow’ key if they thought that the two objects had similar valence. Once the study phase was completed, a 10 min break was provided. During this period, participants performed a distractor task of 3- digit backward counting for 5 min and then rested for five additional minutes. 2.1.4 \| EEG recording and preprocessing EEG was recorded using a 64-channel Neuroscan system and the electrode locations adhered to the extended in- ternational 10–20 system. The sampling rate was 500 Hz with a 0.05–100 Hz bandpass filter. Electrooculogram (EOG) was recorded using two electrodes placed out- side the outer canthi of each eye and one infraorbital to the left eye. The left mastoid was used as the reference site online, and EEG signals were re-referenced offline to the average of the left and right mastoid recordings. Impedance was kept below 5 kΩ. EEG/EOG signals were filtered with a bandpass of 0.05–40 Hz. EEG data from the test phase were segmented into 1100 ms epochs, cor- rected to the 100 ms pre-stimulus baseline. Epochs with voltage exceeding ±75 μV were excluded. Independent component analysis (ICA) conducted with the runica algorithm available through the EEGLAB toolbox for MATLAB (v.2019.0, Delorme & Makeig, 2004), was used to isolate and remove EOG blink artifacts. A minimum of 16 trials for each condition was required to ensure an acceptable signal-to-noise ratio, and four participants were excluded for failing to meet the minimal number of trials. Given our interest in associative memory, and to dis- entangle true memory effects from response bias, we fur- ther analyzed accuracy rates (% correct) for intact pairs and for rearranged pairs using a repeated ANOVA with relatedness (related or unrelated) and valence (negative or neutral) as repeated factors, and accuracy rate as the dependent measure. A full 3-way ANOVA which includes all factors (relatedness, valence, and response type) and levels (intact, rearranged, new) within the same model, is included in the Supporting Information 2. 2.1.5 At test, each trial began with a jittered fixation cross presented for 1000–1500 ms, followed by the presenta- tion of a picture pair for 2000 ms. Participants were asked to indicate whether the pair is “intact”, “rearranged”, or “new” as accurately and as quickly as possible. Responses were provided via keyboard keys, counterbalanced across participants. Half of the participants made responses of “intact” and “rearranged” by pressing the key “F” and “G” with left hand, and of “new” by pressing the key “J” with right hand. The other half of participants responded “in- tact” and “rearranged” by pressing the key “H” and “J” with right hand, and “new” by pressing the key “F” with left hand. Data were extracted for correct trials only (e.g., Donaldson & Rugg, 1998; Paller et al., 2003). Repeated measures analyses of variance (ANOVAs) were conducted for in- ferential statistics, with Greenhouse–Geisser correction for non-sphericity when required. Follow-up analyses were performed using repeated measures ANOVAs or t-tests as appropriate. To control for Type I error rates, p-values were corrected for false discovery rate (FDR) with the Benjamini–Hochberg procedure (Benjamini & Hochberg, 1995) at p < .05. Because the current study focuses on mnemonic effects, only main effects and in- teractions that included the factor of response type are reported. A study practice block of 12 trials was provided at the beginning of the experiment, prior to the study phase. An additional test practice block of 18 trials was provided prior to the test phase. During these practice sessions, the experimenter ascertained that the participants under- stood the task. 2.1.5 \| Statistical analyses: General approach 2.1.5 \| Statistical analyses: General approach ERP analyses Both intact and rearranged pairs are comprised of stud- ied items. However, while intact pairs further contain studied associative information, rearranged pairs contain HAN et al. 7 of 16 novel associative information which was not presented at study. Therefore, the intact/rearranged effects, i.e., dif- ferences between ERPs associated with correct “intact” judgments vs. correct “rearranged” judgments, is indica- tive of associative recognition. (e.g., Li et al., 2017; Rhodes & Donaldson, 2008; Zheng, Li, Xiao, Broster, Jiang, & Xi, 2015). Accordingly, we focused on the comparison between intact and rearranged pairs to index associative memory. For completion, we also include the comparison between rearranged and new pairs as an index of item memory in the Supporting Information 3. associative Pr (relatedness × valence) revealed a signifi- cant main effect of valence, F (1, 41) = 24.28, p < .001, 휂2 p = 0.37 (greater Pr scores for neutral vs. negative pairs), and of relatedness, F (1, 41) = 120.01, p < .001, 휂2 p = 0.75 (greater Pr scores for related vs. unrelated pairs). The analysis further revealed a significant 2-way interaction between relatedness and valence, F (1, 41) = 6.61, p = .014, 휂2 p = 0.14, with lower associative Pr for negative pairs (vs. neutral) in the unrelated condition, t (41) = 5.38, p < .001, d = 0.83, but not in the related condition, t (41) = 1.65, p = .107, d = 0.25. associative Pr (relatedness × valence) revealed a signifi- cant main effect of valence, F (1, 41) = 24.28, p < .001, 휂2 p = 0.37 (greater Pr scores for neutral vs. negative pairs), and of relatedness, F (1, 41) = 120.01, p < .001, 휂2 p = 0.75 (greater Pr scores for related vs. unrelated pairs). The analysis further revealed a significant 2-way interaction between relatedness and valence, F (1, 41) = 6.61, p = .014, 휂2 p = 0.14, with lower associative Pr for negative pairs (vs. neutral) in the unrelated condition, t (41) = 5.38, p < .001, d = 0.83, but not in the related condition, t (41) = 1.65, p = .107, d = 0.25. ERP analyses The analysis of accuracy rates for intact pairs (“hits”) revealed a main effect of relatedness, F (1, 41) = 361.91, p < .001, 휂2 p = 0.90, and a 2-way interaction between the two factors, F (1, 41) = 17.35, p < .001, 휂2 p = 0.30, resulting from lower accuracy rates for negative pairs (vs. neutral) in the unrelated condition, t (41) = 2.78, p = .008, d = 0.43, but greater accuracy rates for negative pairs (vs. neutral) in the related condition, t (41) = 2.31, p = .026, d = 0.36. The analysis of accuracy rates for rearranged pairs (“correct rejections”) only revealed a main effect of relatedness, F (1, 41) = 22.70, p < .001, 휂2 p = 0.36, with greater accuracy rates for unrelated vs. related pairs. For the frontal and parietal memory effects, time segments and regions of interest were defined based on previous ERP studies (Bader et al., 2010; Han et al., 2018; Li et al., 2017, 2019; Rugg & Curran, 2007; Speer & Curran, 2007; Wolk et al., 2006; Zheng et al., 2016). Accordingly, two time win- dows, 300–550 ms and 550–800 ms, were used to capture the frontal and parietal memory effects, respectively. Mean amplitudes for statistical analyses in these windows were obtained from frontal (collapsed across F3, Fz, and F4), cen- tral (collapsed across C3, Cz, and C4) and parietal (collapsed across P3, Pz, and P4) scalp locations (Han et al., 2018; Hou et al., 2013; Molinaro et al., 2011). Repeated measures ANOVA was conducted separately for each time window and included four within-subjects factors: relatedness (related or unrelated), valence (nega- tive or neutral), response type (intact or rearranged), and location (frontal, central, or parietal). Taken together, the behavioral results depict the pre- dicted emotional associative interference effect, indicated by reduced Pr scores and accuracy rates for negative vs. neutral pairs. Furthermore, this effect was attenuated by semantic relatedness, with greater emotional interference observed for unrelated vs. related pairs. TABLE 1 Distribution of participants' responses and Pr scores in Experiment 1 Discussion 2.3 conditions are shown in Figure 2. In the early time win- dow (300–550 ms), the ANOVA for the associative mem- ory effect revealed a 2-way interaction between valence and response type, F (1, 41) = 9.29, p = .004, 휂2 p = 0.19. Decomposition of the interaction using paired t-tests at each level of valence, revealed a significant associative memory effect (more positive-going waveforms for intact vs. rearranged) for negative pairs, t (41) = 3.13, p = .003, d = 0.48, but not for neutral pairs (p = .29). Thus, in the early time-window, the associative memory effect emerged for negative pairs, regardless their relatedness, and had a widespread distribution. In the late time win- dow (550–800 ms), the ANOVA only revealed a main ef- fect of response type, F (1, 41) = 38.84, p < .001, 휂2 p = 0.49, suggesting that the late associative memory effect was similarly observed in all conditions, and in all locations. Exploratory analysis of a later associative memory ef- fect (800–1000 ms), which resembled the pattern ob- served in the 550–800 ms time window, is included in the Supporting Information 4. In Experiment 1 we observed an associative emotional interference effect, which was attenuated by semantic re- latedness. Turning to the EEG data, semantic relatedness did not modulate the early associative memory effect, as- sociated with familiarity, nor the late associative memory effect, associated with recollection. That is, the difference waveform for intact vs. rearranged pairs was similar for related and unrelated pairings. Interestingly, however, the associative memory ERP effect was modulated by the va- lence of the stimuli: the early ERP component showed a frontal modulation for negative pairs (with greater frontal negativity for rearranged vs. intact pairings), suggesting unitizability of negative stimuli and the contribution of familiarity to their associative recognition. Nevertheless, even though the modulation of the early ERP effect was similar for related and unrelated pairs, the behavioral at- tenuation of the associative emotional interference effect was only apparent for pairs that were semantically related. FIGURE 2 ERP results for Experiment 1. (a) Grand average ERP waveforms for intact responses (black), rearranged responses (red), and new responses (blue) in the four experimental conditions (related\unrelated × negative\neutral) at three scalp locations (F3, Fz, F4 collapsed as the frontal site; C3, Cz, C4 as the central site; P3, Pz, P4 as the parietal site). 2.2.1 \| Behavioral results Means and SDs for the various behavioral measures of Experiment 1 are shown in Table 1. The ANOVA for Waveforms and topographical distribution of the as- sociative memory effect in the various experimental TABLE 1 Distribution of participants' responses and Pr scores in Experiment 1 Test pair type Response Related Unrelated Negative Neutral Negative Neutral Intact Intact 0.81 (0.08) 0.77 (0.11) 0.51 (0.12) 0.55 (0.14) Rearranged 0.13 (0.06) 0.13 (0.07) 0.40 (0.11) 0.34 (0.11) New 0.06 (0.05) 0.09 (0.07) 0.09 (0.07) 0.10 (0.07) Rearranged Intact 0.37 (0.13) 0.30 (0.12) 0.28 (0.11) 0.21 (0.13) Rearranged 0.53 (0.13) 0.55 (0.14) 0.60 (0.13) 0.64 (0.15) New 0.09 (0.07) 0.15 (0.08) 0.11 (0.07) 0.15 (0.09) New Intact 0.05 (0.05) 0.04 (0.05) 0.03 (0.03) 0.02 (0.03) Rearranged 0.15 (0.07) 0.06 (0.06) 0.19 (0.11) 0.10 (0.07) New 0.79 (0.12) 0.89 (0.10) 0.77 (0.13) 0.87 (0.10) Assoc Pr 0.44 (0.15) 0.47 (0.16) 0.22 (0.15) 0.34 (0.17) Note: standard deviations are shown in parenthesis. HAN et al. 8 of 16 3.1.2 \| Procedure, recording, and analyses Stimuli were the same as in Experiment 1. The procedure was identical to that of Experiment 1, except that partici- pants were asked to perform a familiarity judgment task at study, namely, to judge which one of the two objects pre- sented in each trial was more familiar. They were asked to press the ‘left arrow’ key on the keyboard if they thought that the left object was more familiar, to press the ‘right arrow’ key if they thought that the right one was more fa- miliar, and to press the ‘down arrow’ key if they thought that the two objects do not differ in their familiarity. EEG recording and preprocessing were the same as in Experiment 1. Mean numbers of related analyzed trials were 38 (intact), 26 (rearranged), and 39 (new) for neg- ative pairs, and 40 (intact), 29 (rearranged), and 43 (new) for neutral pairs. Mean numbers of unrelated analyzed tri- als were 23 (intact), 29 (rearranged), and 36 (new) for neg- ative pairs, and 28 (intact), 31 (rearranged), and 42 (new) for neutral pairs. Statistical analyses were the same as in Experiment 1. 3.1.1 \| Participants Forty-seven healthy, right-handed native Chinese speak- ers (32 females; mean age 22.1 ± 1.9 years) from Capital Normal University, with the same characteristics as those who participated in Experiment 1, participated in Experiment 2. Data from seven participants were dis- carded, due to insufficient number of artifact-free ERP tri- als in one or more experimental conditions (N trials < 16). Our final sample therefore included 40 participants (27 females; mean age 21.9 ± 2.0 years). Indeed, in the current experiment, emotional content was explicitly probed (i.e., during study participants com- pleted valence judgments for the stimuli), which might have promoted emotional processing and hindered process- ing of semantic relations. Notably, a growing body of evi- dence suggest that modulation of behavior or processing by emotional content is only triggered when emotions are rel- evant to the task (e.g., Engen et al., 2017; Huang et al., 2008; Pessoa, 2009; Stein et al., 2009; Wei et al., 2015). For exam- ple, Stein et al. (2009) conducted the attentional blink task to estimate whether task relevance impacts prioritization. In this experiment, participants judged either the emotion (relevant condition) or gender (irrelevant condition) of two target facial stimuli depicting different expressions (fearful or neutral). Fearful faces (vs. neutral) induced a stronger attentional blink, but only in the relevant condition. This demonstrates that the processing advantage of emotional stimuli depends on the relevance of emotion to the task. Similarly, in the context of the current task, situating emo- tion as task-relevant might have promoted binding along this dimension. In other words, instead of creating unit- ized links for semantically related (vs. unrelated) items, such links were created for negative (vs. neutral) ones. Experiment 2 was designed to address this possibility. Discussion Shaded areas indicate time-windows used for the analyses of the early memory effects (300–550 ms, light gray) and the late memory effects (550–800 ms, dark gray). (b) Topographical maps of the associative memory effects (intact minus rearranged) in each time window. FIGURE 2 ERP results for Experiment 1. (a) Grand average ERP waveforms for intact responses (black), rearranged responses (red), and new responses (blue) in the four experimental conditions (related\unrelated × negative\neutral) at three scalp locations (F3, Fz, F4 collapsed as the frontal site; C3, Cz, C4 as the central site; P3, Pz, P4 as the parietal site). Shaded areas indicate time-windows used for the analyses of the early memory effects (300–550 ms, light gray) and the late memory effects (550–800 ms, dark gray). (b) Topographical maps of the associative memory effects (intact minus rearranged) in each time window. HAN et al. 9 of 16 We return to this seemingly discrepancy between the be- havioral and the ERP results in our General Discussion. interference effect for semantically related vs. unrelated pairs. We further rationalized that, with emotion being in- cidental to the task, both unitization and LOP would be promoted for semantically related (vs. unrelated) pairs. We therefore predicted that both the early and the late as- sociative memory ERP effects would be greater for related than unrelated pairs. interference effect for semantically related vs. unrelated pairs. We further rationalized that, with emotion being in- cidental to the task, both unitization and LOP would be promoted for semantically related (vs. unrelated) pairs. We therefore predicted that both the early and the late as- sociative memory ERP effects would be greater for related than unrelated pairs. Although somewhat unpredicted, the modulation of the early ERP component for negative (but not neutral) pairs, adheres to the suggestion that valence serves as an organizing principle, i.e., allowing grouping across shared properties. Previous studies (Talmi et al., 2007; Talmi & Moscovitch, 2004) proposed that the common finding of enhanced memory for emotional items can be due to a shared emotional context. In a series of experiments, they showed that the memory advantage for emotional items is eliminated when these are compared with categorized neutral items, suggesting that valence serves as an orga- nizing principle for the items. This organization, along the dimension of emotional valence, possibly overshadowed the effect of semantic relatedness. 3 \| EXPERIMENT 2 In Experiment 2, we investigated the effect of semantic re- latedness on associative recognition of emotional stimuli, when emotions are incidental to the task. To this end, Experiment 2 employed the same behavioral and EEG measures as Experiment 1, but with a different study task. Namely, rather than valence judgment, in Experiment 2 participants were asked to perform a familiarity judgment. 3.2.1 \| Behavioral results Means and SDs for the various behavioral measures of Experiment 2 are shown in Table 2. The ANOVA for We predicted an overall behavioral pattern similar to Experiment 1, that is, a smaller associative emotional 10 of 16 \| Test pair type Response Related Unrelated Negative Neutral Negative Neutral Intact Intact 0.79 (0.08) 0.83 (0.10) 0.48 (0.12) 0.59 (0.14) Rearranged 0.16 (0.06) 0.11 (0.05) 0.43 (0.10) 0.34 (0.10) New 0.06 (0.05) 0.06 (0.07) 0.09 (0.07) 0.07 (0.07) Rearranged Intact 0.36 (0.12) 0.29 (0.11) 0.26 (0.09) 0.27 (0.12) Rearranged 0.54 (0.12) 0.60 (0.13) 0.62 (0.12) 0.64 (0.14) New 0.10 (0.05) 0.11 (0.09) 0.12 (0.07) 0.09 (0.06) New Intact 0.05 (0.06) 0.02 (0.04) 0.04 (0.04) 0.01 (0.02) Rearranged 0.15 (0.08) 0.07 (0.05) 0.21 (0.12) 0.11 (0.08) New 0.80 (0.12) 0.90 (0.08) 0.74 (0.14) 0.89 (0.10) Assoc Pr 0.43 (0.13) 0.54 (0.15) 0.22 (0.12) 0.31 (0.18) Note: standard deviations are shown in parenthesis. HAN et al. TABLE 2 Distribution of participants' responses and Pr scores in Experiment 2 Note: standard deviations are shown in parenthesis. associative Pr revealed main effects of valence, F (1, 39) = 40.31, p < .001, 휂2 p = 0.51, and relatedness, F (1, 39) = 220.81, p < .001, 휂2 p = 0.85, with greater Pr scores for neutral pairs (vs. negative), and for related pairs (vs. unrelated). and response type, F (1, 39) = 5.09, p = .030, 휂2 p = 0.12. Decomposition of the interaction showed a significant associative memory effect for related pairs, t (39) = 3.08, p = .004, d = 0.49, but not for unrelated pairs (p = .81). Thus, in the early time-window, the associative memory effect emerged for related pairs, regardless their valence, and had a widespread distribution. The analysis of accuracy rates for intact pairs revealed main effects of relatedness, F (1, 39) = 370.64, p < .001, 휂2 p = 0.91, and valence, F (1, 39) = 22.64, p < .001, 휂2 p = 0.37, as well as a 2-way interaction between the two factors, F (1, 39) = 12.67, p = .001, 휂2 p = 0.25. Decomposition of this interaction showed that even though the difference in accuracy rates between neutral and negative pairs emerged for both related and unrelated pairs, it was greater in the latter, t related(39) = 2.35, p = .024, d = 0.37; t unrelated(39) = 5.26, p < .001, d = 0.83. 3.2.1 \| Behavioral results For rearranged pairs, the analysis of accuracy rates revealed a main effect of relatedness, F (1, 39) = 12.88, p = .001, 휂2 p = 0.25, and valence, F (1, 39) = 6.59, p = .014, 휂2 p = 0.15, but no signif- icant interaction. In the late time window (550–800 ms), the analysis re- vealed a main effect of response type, F (1, 39) = 38.49, p < .001, 휂2 p = 0.50, a 2-way interaction between relatedness and response type, F (1, 39) = 10.23, p = .003, 휂2 p = 0.21, a 2-way interaction between valence and response type, F (1, 39) = 6.92, p = .012, 휂2 p = 0.15, and a 3-way interaction between relatedness, valence, and response type, F (1, 39) = 8.37, p = .006, 휂2 p = 0.18. To decompose the 3-way interaction, we conducted follow-up ANOVAs with relat- edness and response type as within-subject factors, sep- arately for each valence. For negative pairs, this analysis revealed a main effect of response type, F (1, 39) = 11.46, p = .002, 휂2 p = 0.23, and a significant 2-way interaction be- tween relatedness and response type, F (1, 39) = 17.12, p < .001, 휂2 p = 0.31, resulting from an associative memory effect for related pairs, t (39) = 4.74, p < .001, d = 0.75, but not for unrelated pairs (p = .82). A similar follow-up ANOVA for neutral pairs revealed a main effect for re- sponse type, F (1, 39) = 42.93, p < .001, 휂2 p = 0.52, but no interaction between the factors. As for Experiment 1, an exploratory analysis of a later associative memory effect (800–1000 ms) is included in the Supporting Information 4. The behavioral results of Experiment 2 show a simi- lar pattern of attenuated emotional interference effect for related vs. unrelated pairs, which we observed in Experiment 1. However, unlike Experiment 1, in which this pattern was observed both for hit rates (correct “in- tact” responses) and for the unbiased associative Pr scores, in Experiment 2 it was only observed for the former. 3.2.2 \| ERP results Waveforms and topographical distribution of the associa- tive memory effect are shown in Figure 3. In the early time window (300–550 ms), the analysis revealed a main effect of response type, with more positive-going waveforms for intact pairs (vs. rearranged), F (1, 39) = 4.67, p = .037, 휂2 p = 0.11, and a 2-way interaction between relatedness 3.3 \| Discussion In Experiment 2, participants were more likely to classify negative pairs as intact if they were semantically related, HAN et al. \| FIGURE 3 ERP results for Experiment 2. (a) Grand average ERP waveforms for intact responses (black), rearranged responses (red), and new responses (blue) in the four conditions at three scalp locations. Shaded areas indicate time-windows used for the analysis of the early and late memory effects. (b) Topographical maps of the associative memory effects in each time window. FIGURE 3 ERP results for Experiment 2. (a) Grand average ERP waveforms for intact responses (black), rearranged responses (red), and new responses (blue) in the four conditions at three scalp locations. Shaded areas indicate time-windows used for the analysis of the early and late memory effects. (b) Topographical maps of the associative memory effects in each time window. \| GENERAL DISCUSSION interference effect. Interestingly, in Experiment 2, nega- tive related pairs were more likely to be classified as “in- tact” compared to negative unrelated pairs (as indicated by their accuracy scores). This tendency, however, was not reflected in the unbiased associative Pr scores. Therefore, in the case of Experiment 2, the alleviated number of “in- tact” responses does not represent a memory effect, but rather indicates a response bias. Taken together, the re- sults of the two experiments agree with previous research, showing that emotions can enhance processing, but only when they are relevant to the task (as in Experiment 1; e.g., Engen et al., 2017; Huang et al., 2008; Pessoa, 2009; Stein et al., 2009; Wei et al., 2015). In the current case, during encoding, processing of semantic relations was enhanced for negative pairs, leading to better binding which reduced the associative emotional interference. Nevertheless, this reduction only occurred when emotions were task- relevant (i.e., in Experiment 1). Unlike the selective modulation of the early associa- tive memory effects, the modulation of the late effect was apparent more generally across the various experimental conditions (albeit, as noted above, not for unrelated neg- ative pairs in Experiment 2). Although this late effect was rather broadly distributed and lacked the pronounced pari- etal maxima often associated with the recollection-related late positive component (Rugg & Curran, 2007; Wilding & Ranganath, 2011), retrieval-related modulations with an- terior/central topographic distribution are commonly re- ported in associative recognition ERP studies (e.g., Bader et al., 2010; Han et al., 2018; Kriukova et al., 2013; Mollison & Curran, 2012; Rhodes & Donaldson, 2007, 2008; Tibon, Ben-Zvi, & Levy, 2014; Zheng, Li, Xiao, Broster, & Jiang, 2015) and are interpreted as reflecting recollective processes. Our findings suggest that recollective processes were readily available for intact pairs, regardless their va- lence or semantic relations. Turning to the ERP data, our results revealed that the modulation of the early associative memory effect was task dependent. Specifically, in Experiment 1, where valence was relevant to encoding, the early ERP effect showed greater frontal negativity for intact vs. rearranged negative pairs, regardless their semantic relations. In contrast, in Experiment 2, where familiarity (and not va- lence) was probed during encoding, this modulation only occurred for related (but not for unrelated) pairs, regard- less their valence. \| GENERAL DISCUSSION These results provide evidence for the suggestion that emotions triggered by stimulus' valence can serve as an organizing principle that binds the items together via shared context (Riberto et al., 2019; Talmi & Moscovitch, 2004). Therefore, pairs of emotional stimuli might be more easily unitized compared to non-emotional ones. The results of Experiment 1 support this idea. Namely, in this experiment, the early associative mem- ory effect—the putative electrophysiological correlate of familiarity—showed a modulation for negative pairs, indi- cating that familiarity was readily available for these pairs. One aspect of the associative memory effects that war- rants further attention is the correspondence between the behavioral results and the ERP data. Specifically, in Experiment 1, associative emotional interference was only attenuated when semantically related pairs were retrieved, even though the modulation of the early ERP effect was similar for related and unrelated pairs. We speculate that the production of early mnemonic signals (as indicated in the ERPs) would only affect behavior if these signals are considered diagnostic or trusted. For semantically related pairs, the emotional context adjoins the semantic one (e.g., for gun: bullets pair: negative feeling due to the shooting gun), producing a trustworthy mnemonic signal that attenuates associa- tive emotional interference. In contrast, for stimuli that lack semantic relations, the early signal is cognitively attributed to more general sources (e.g., that the stim- uli were experienced together during the experimental session, rather than within specific pairing). Therefore, these signals are not designated as trustworthy, and do not produce the same behavioral change. This idea coincide with a recent proposal by Bastin et al. (2019) which posits that an attribution system modulates the use of memory traces as a function of expectancies, task context, and goals, leading to subjective experiences and explicit judgments. In the current case, associative fa- miliarity signals might have been attributed to an alter- native source (e.g., such as processing fluency or global familiarity) and were discarded in the face of explicit judgments. Interestingly, this valence-based modulation of the early effect was not observed in Experiment 2, in which valence was not probed during encoding, suggesting that valence might only serve as an organizing principle when it is task relevant (or attended). When valence is not rel- evant to the task, familiarity signals can be elicited when the stimuli comprising the pair are semantically related, as apparent in Experiment 2. \| GENERAL DISCUSSION 4 but this similarly occurred for both intact and rearranged pairs. We come back to this in the General Discussion below. The present study used an associative recognition para- digm, across two experiments, to investigate how semantic relations, valence, and their interaction, affect our ability to retrieve associative information. Both experiments con- formed to the same procedure, aside from the instructions given during the study phase. Namely, in Experiment 1 participants were asked to compare the valence of object pairs, whereas in Experiment 2 they were asked to com- pare their familiarity. As for the ERP data, the results agree with previ- ous studies (e.g., Ahmad & Hockley, 2014; Kriukova et al., 2013; Li et al., 2019; Rhodes & Donaldson, 2008; Tibon, Gronau, et al., 2014), demonstrating modulation of the early associative memory effect by semantic relat- edness. Namely, the early difference between intact and rearranged pairs was only significant when stimuli were semantically related (regardless their valence). The late associative memory effect was more generally distributed, and was only absent for unrelated negative pairs. While this absence might seem puzzling at first sight, a closer look at the behavioral results suggests that this is the only experimental condition (across both experiments) for which the probability to correctly classify intact items (47%) was highly similar to the probability to classify them as rearranged pairs (43%). This suggests that in this con- dition, for which emotional associative interference is not attenuated by semantic relations, recollective processes are impeded. Yet, it remains unclear why the same pattern was not observed in Experiment 1. In both experiments, an associative emotional inter- ference effect had emerged, with reduced associative memory for negative pairs. This finding agrees with pre- vious studies, showing that emotion can impair associa- tive memory (Madan et al., 2012; Mao et al., 2015; Mather & Knight, 2008; Pierce & Kensinger, 2011; Rimmele et al., 2011). Importantly, our results further suggest that this associative emotional interference effect can be re- duced under certain circumstances. In particular, when valence is attended (as in the case of Experiment 1, where the task requires valence judgment) semantic associa- tive relations can attenuate this associative emotional HAN et al. 12 of 16 Taken together, the modulation of the early associative effect observed in the current study, suggests that both se- mantic relationships and emotional context can support unitization which can subsequently promote familiarity- based retrieval. \| GENERAL DISCUSSION Indeed, previous studies have shown that semantic relations can promote unitiza- tion, which further enhances familiarity-based associa- tive recognition (e.g., Ahmad & Hockley, 2014; Kriukova et al., 2013; Li et al., 2019; Rhodes & Donaldson, 2008; Tibon, Gronau, et al., 2014). Arguably, when valence is ir- relevant, semantic relatedness “pops-out” as an organizing principle, and enables unitization along this dimension. HAN et al. 13 of 16 In the current study, the distinction between the two processes supporting recognition memory—familiarity and recollection—relies mainly on the temporal distri- bution, and to some extent also on the spatial distri- bution, of the early and late associative memory ERP effects. Like many other studies (e.g., Bader et al., 2010; Guillaume & Etienne, 2015; Jäger et al., 2006; Kamp et al., 2016; Rhodes & Donaldson, 2008; Tibon, Gronau, et al., 2014; Zheng, Li, Xiao, Broster, Jiang, & Xi, 2015, to list just a few), we associate the early frontal neg- ativity with familiarity, and the late positivity with recollection. While this type of reverse inference has its limitations (Poldrack, 2006), our interpretation builds on decades of intensive research that strongly associates these ERP components with the partic- ular memory processes reported here (reviewed by Mecklinger, 2000; Rugg & Curran, 2007; Wilding & Ranganath, 2011). We do acknowledge that in the cur- rent study, the early ERP component might reflect pro- cessing fluency instead of (or possibly together with) familiarity (see Paller et al., 2012; Paller et al., 2007; Mecklinger et al., 2012 for discussion). In any event, however, even if the links made in our study between electrophysiological measures and specific recognition processes are not entirely conclusive (though strongly suggestive), our data clearly point to a neural distinc- tion, whereby the contribution of the early ERP effect to associative recognition is limited to certain experi- mental conditions, but the contribution of the late ERP effect is more widely available across different types of associated information. recollection. This modulation further depends on the way information was encoded: when valence is relevant to encoding, it acts as an organizing principle that trig- gers early retrieval processes. But when valence is not relevant, other relations (in our case, semantic) serve to organize information. We propose that the neural mod- ulation only relates to behavioral change when the sig- nals are being interpreted as trustworthy, particularly, when pre-existing semantic relations between the vari- ous pieces of information are present. AUTHOR CONTRIBUTIONS Meng Han: Conceptualization; data curation; formal analysis; investigation; methodology; project adminis- tration; visualization; writing – original draft. Bingcan Li: Data curation; formal analysis. Chunyan Guo: Conceptualization; funding acquisition; investigation; methodology; project administration; resources; soft- ware; supervision; writing – original draft. Roni Tibon: Conceptualization; formal analysis; investigation; meth- odology; project administration; validation; writing – orig- inal draft. ACKNOWLEDGEMENTS The present study was supported by the National Natural Science Foundation of China (31671127), Support by Capacity Building for Sci-Tech Innovation – Fundamental Scientific Research Funds (No. 025185305000/200). RT was supported by a British Academy Postdoctoral Fellowship (grant SUAI/028 RG94188, fp170046). We thank Deborah Talmi and Zara Bergström for insightful discussion, and Xiaohuan Li, Yun Chen, and Yubin Peng for assisting in collecting data. One caveat of the current study, is that negative stim- uli were also highly arousing (compared to neutral, see Stimuli section above). Therefore, one potential difficulty in interpretation of the present findings is that valence ef- fects cannot be distinguished from arousal effects, even though prior studies suggest that these might rely on distinct neural process (e.g., Kensinger & Corkin, 2004). Furthermore, the current study only included negative and neutral stimuli, precluding any conclusions regard- ing general emotional effects, or distinction between different kinds of valence. In addition, the study did not include any additional emotional measures, such as mood assessment or assessment of anxiety/stress symptoms, to be used as covariates in statistical analyses. Future studies are thus required in order to generalize our conclusions further. \| GENERAL DISCUSSION This suggests that in real-life situations, where emotional information is often highly relevant and semantically meaningful, ac- tivation of early mnemonic signals can be tightly linked to memory performance. OPEN PRACTICES STATEMENT Materials for the experiments reported here are available on OSF (https://osf.io/7kpq6/). In summary, the current study shows that when items share a context during their encoding, be that semantic or emotional, their associative retrieval can provoke an early neural modulation, arguably indica- tive of familiarity-based retrieval, which accompanies CONFLICT OF INTEREST The authors declare no conflict of interest. DATA AVAILABILITY STATEMENT Data will be available upon request via the MRC Cognition and Brain Sciences Unit data repository. M REFERENCES Ahmad, F. N., & Hockley, W. E. (2014). The role of familiarity in asso- ciative recognition of unitized compound word pairs. Quarterly Journal of Experimental Psychology, 67(12), 2301–2324. https:// doi.org/10.1080/17470218.2014.923007 Guillaume, F., & Etienne, Y. (2015). Target-context unitization effect on the familiarity-related FN400: A face recognition exclusion task. International Journal of Psychophysiology, 95(3), 345–354. https://doi.org/10.1016/j.ijpsycho.2015.01.004 Bader, R., & Mecklinger, A. (2017). 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https://openalex.org/W4253738771	https://www.researchsquare.com/article/rs-7627/latest.pdf	English	null	Urinary continence outcomes of four years of follow-up and predictors of early and late urinary continence in patients undergoing robot-assisted radical prostatectomy	Research Square (Research Square)	2,020	cc-by	7,381	Urinary continence outcomes of four years of follow-up and predictors of early and late urinary continence in patients undergoing robot-assisted radical prostatectomy Page 1/20 Urinary continence outcomes of four years of follow-up and predictors of early and late urinary continence in patients undergoing robot-assisted radical prostatectomy Xing Li Chinese PLA General Hospital Huan Zhang Chinese PLA General Hospital Zhuo Jia Chinese PLA General Hospital Yunpeng Wang Chinese PLA General Hospital Yong Song Chinese PLA General Hospital Limin Liao China Rehabilitation Research Center Xu Zhang (  xzhang@foxmail.com ) Department of urology, Chinese PLA General Hospital/Chinese PLA Medical Academy，28, fuxing road,eijing Research article Keywords: prostate cancer, prostatectomy, robotics, urinary incontinence, continence Posted Date: March 16th, 2020 DOI: https://doi.org/10.21203/rs.2.16991/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Urology on March 18th, 2020. See published version at https://doi.org/10.1186/s12894-020-00601-w. Xing Li Chinese PLA General Hospital Huan Zhang Chinese PLA General Hospital Zhuo Jia Chinese PLA General Hospital Yunpeng Wang Chinese PLA General Hospital Yong Song Chinese PLA General Hospital Limin Liao China Rehabilitation Research Center Xu Zhang (  xzhang@foxmail.com ) Department of urology, Chinese PLA Gen road,eijing Xing Li Chinese PLA General Hospital Huan Zhang Chinese PLA General Hospital Zhuo Jia Chinese PLA General Hospital Yunpeng Wang Chinese PLA General Hospital Yong Song Chinese PLA General Hospital Limin Liao China Rehabilitation Research Center Xu Zhang (  xzhang@foxmail.com ) Department of urology, Chinese PLA G road,eijing Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Urology on March 18th, 2020. See the published version at https://doi.org/10.1186/s12894-020-00601-w. Page 1/20 Abstract Background: The robot-assisted radical prostatectomy (RARP) has been widely applied in recent years; however, only a few studies are reported about long-term urinary continence after surgery. The present study aimed to examine the outcomes of continence rates (CRs) and determine the risk and protective factors of urinary continence in patients with prostate cancer (PCa) undergoing RARP. Methods: This retrospective study included 650 patients treated with RARP with perioperative data and at least one year of follow-up from January 2009 to November 2017. Also, the preoperative, intraoperative, and postoperative parameters of the patients were analyzed. Continence was defined as no pad use. Early and late continence was defined as the return of urinary continence within 3 months and beyond 12 months post-surgery, respectively. CRs were examined from 1–48 months postoperatively. Logistic regression analysis evaluated the association between the predictive factors and urinary continence in the early and late stages. Results: No significant difference was detected in the CR from 12–48 months postoperatively (P=0.766). Logistic regression analysis proved that pelvic lymph node dissection (PLND) was a significant risk factor of urinary continence at 1 month. Nerve-sparing (NS) was a significant protective factor of urinary continence at 1, 3, and 6 months. Advanced age was an independent risk factor of urinary continence at 6, 12, and 24 months. Other variables were not statistically significant predictors of urinary continence. Conclusions: The current results demonstrated that CR gradually improved with time within 1 year and stabilized 1 year after the surgery. PLND, NS, and age were significant determinants of continence in the early and late stages, respectively. These parameters could be used for preoperative identification of patients at high risk and counseling about postoperative expectations for urinary continence. Background Despite advances in surgical technique and methodology, postprostatectomy urinary incontinence (UI) remains a significant adverse event that leads to decreased quality of life [1,2]. Several factors are involved in the recovery of urinary continence after radical prostatectomy (RP) [3,4]. Since patients have learned about this disease from the media, it is necessary to predict the recovery of urinary continence early in order to minimize the patients’ concerns and embarrassment [5]. The technical progress has facilitated the increasing use of robot-assisted radical prostatectomy (RARP) worldwide [6]. Because the procedure can induce different degrees of damage to the bladder and urethra, UI is inevitable despite the assistance of robotic systems [7,8]. This in turn, places physical and psychosocial burden on patients. Reportedly, urinary continence improves only slightly in the 12 months after the surgery [9]. Hence, previous reports mainly addressed continence rates (CRs) and potential predictors within 1 year post- RARP. However, continence may continue to develop at 2 years post-surgery [7,10,11]. Limited data are Page 2/20 Page 2/20 Page 2/20 available for longer follow-up with urinary continence after 24–48 months. In addition, a series of studies reported outcomes at 1 year after surgery, and most of them included a small number of patients (<200). Moreover, the follow-up period was discontinuous or incomplete. For example, Yanagiuchi et al. examined and identified the outcomes at 1 and 3 months post-RARP, while Olgin et al. and Haga et al. analyzed and evaluated the outcomes at 3 and 12 months after the surgery [8,12,13]. In addition, Honda et al. reported continuous outcomes from 1–6 months postoperatively [14]. To the best of our knowledge, continuous follow-up data from 1–48 months on CRs and predictive factors for urinary continence after RARP with a high number of patients have not been collected. Therefore, in this retrospective study, we examined the outcomes of CRs at 1, 3, 6, 12, 24, 36, and 48 months after the surgery and determined the risk and protective factors of urinary continence. Methods Patients diagnosed with clinically localized prostate cancer and received treatment from September 2009 to November 2017 at our institution were studied in a retrospective manner. Only patients who completed the outpatient visits or telephonic interviews for at least one year were enrolled. Patients with incomplete data and those unavailable for follow-up of continence were excluded from the study. Patients who underwent preoperative transurethral resection, enucleation of the prostate, radioactive seed implantation, orchidectomy, bladder neck, urethral, or pelvic surgery, and retropubic radical prostatectomy (RRP) or laparoscopic radical prostatectomy (LRP) were also excluded. Finally, a total of 650 eligible patients were analyzed. The schematic is illustrated in Fig. 1. Data were collected from cases performed by three surgeons. RARP was performed by a transperitoneal approach. Nerve-sparing (NS) procedures were attempted in all potent patients at a clinical stage of T1 or T2, prostate-specific antigen (PSA) ≤10 ng/mL, and Gleason score ≤7. To preserve the urethral length, it was performed proximally close to the prostate while dissecting the urethra. Pelvic lymph node dissection (PLND) was performed selectively to collect samples in intermediate- and high-risk patients [15]. Urethrovesical anastomosis was carried out using 2-0 monofilament sutures with a 5/8 needle. Typically, a catheter was placed and removed at 3 weeks after the surgery. Urethrovesical anastomosis was carried out using 2-0 monofilament sutures with a 5/8 needle. Typically, a catheter was placed and removed at 3 weeks after the surgery. Clinical data on the demographic characteristics and surgery-related variables were retrieved from the patient’s medical records. Together with the follow-up records, the data were assimilated into a database in this retrospective study. The factors assessed included age at the operation, body mass index (BMI), comorbidities such as hypertension (HP), diabetes mellitus (DM), coronary heart disease (CHD), and cerebrovascular diseases (CD), serum PSA level, prostate volume (PV), biopsy-determined Gleason score, clinical stage, operation time (OT), NS and PLND during surgery, and the duration of indwelling catheter (DIC) after the surgery. UI was defined as any leakage of urine after the surgery. Early and late continence was defined as the return of urinary continence within 3 months and beyond 12 months after surgery, respectively [16]. Statistical analysis Page 3/20 Page 3/20 SPSS version 19.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Data are reported as mean ± standard deviation or percentage. The chi-square test was used to compare the CRs at 12, 24, 36, and 48 months. The independent sample t-test or chi-square test was used to compare the predictive factors for urinary continence after RARP. All the above factors that might affect urinary continence were included in the univariate and multivariate logistic regression analysis to identify the risk and protective factors. P<0.05 was considered statistically significant. Results CRs at 1, 3, 6, 12, 24, 36, and 48 months post-surgery were 40.62%, 60.92%, 71.38%, 78.77%, 79.96%, 79.51%, and 76.50%, respectively (Table 1), which improved gradually within one year. The comparison of CRs at 12 and 48 months did not reveal any significant differences in the continence outcomes during the 4-year follow-up (P=0.766). Consequently, CR was found to be stabilized 1 year after the operation. Furthermore, the demographic data and baseline clinical characteristics of the two groups (continence and incontinence groups) at 1 and 48 months were compared (Tables 2–4). Fewer patients had NS, and a large number of patients had PLND in the incontinence group than those in the continence group during the surgery at a 1-month follow-up (P<0.05) (Table 2). Similarly, more patients in the continence group had intraoperative NS than those in the incontinence group, as observed at the 3-month follow-up (P<0.05) (Table 2). Data from the 6- and 12-month follow-ups are presented in Table 3. At a 6-month follow-up, the continence group more commonly had a NS procedure performed (P<0.05). Urinary continence at 6 and 12 months after RARP was associated with age at the time of surgery (P<0.05). Data from the 24- and 48-month follow-ups are presented in Table 4. Patients in the incontinence group were older than those in the continence group at 24-month follow-up (P<0.05). No significant variables were observed at the 48-month follow-up. Univariate and multivariate associations between urinary continence and predictive factors are shown in Tables 5, 6, and 7. In the univariate logistic analysis, PLND was associated with urinary continence at 1- month post-surgery (P=0.009). In the multivariate logistic analysis, PLND was a significant independent risk factor of early urinary continence at 1 month (odds ratio (OR): 1.535, 95% confidence interval (CI): 1.079–2.184, P=0.017; Table 5). Univariate logistic analysis showed that NS was associated with urinary continence at 1 month (OR: 0.372, 95% CI: 0.248–0.557, P<0.001), 3 months (OR: 0.549, 95% CI: 0.356–0.846, P=0.007), and 6 months (OR: 0.561, 95% CI: 0.346–0.909, P=0.019) (Tables 5 and 6). In multivariate logistic analysis, NS was a significant independent protective factor of urinary continence at 1 month (OR: 0.360, 95% CI: 0.231–0.561, P<0.001), 3 months (OR: 0.546, 95% CI: 0.342–0.872, P=0.011), and 6 months (OR: 0.545, 95% CI: 0.324–0.915, P=0.022) (Tables 5 and 6). Discussion Reportedly, the urinary continence is stable up to 12 months after RP [2]. Hence, previous studies have addressed CRs within 1 year post-RARP. Only little data are available at more than 24 months follow-up for urinary continence. In the current study, the CRs were 78.77%, 79.96%, 79.51%, and 76.50% at 1, 2, 3, and 4 years after the surgery, respectively (Table 1). No significant differences were observed in the continence outcomes during the 4-year follow-up. Our results certified that one year after RARP was the stable continence period [17]. Few studies have evaluated the CRs after 12 months. Shao et al. reported that CR was 89.4% at 24 months after RARP, while Xylinas et al. reported that the 24-month urinary continence rate was 88% based on the no-pad definition [7,18]. Murphy et al. reported a 36-month urinary continence rate of 94.7% using the no-pad or safety pad definition [19]. Mandel et al. reported that CRs were 89.5% and 90.9% at 24 and 36 months after the surgery, respectively [20]. The CR of the current study was 76.50% at 48 months after RARP, which has been the longest follow-up on the topic to date. A large number of studies have evaluated the predictors of urinary continence within 1 year after surgery. These studies either included a relatively small number of patients or had a discontinuous follow-up. To the best of our knowledge, this is the first study to evaluate the predictors of continence from 1–48 months after RARP in a large sample. In the current cohort, the CRs were 40.62%, 60.92%, 71.38%, and 78.77% at 1, 3, 6 and 12 months after RARP, respectively (Table 1). These results were in agreement with those from the recent study by Honda et al. that revealed CRs at 1, 3, and 6 months as 40.7%, 63.0%, and 73.1%, respectively [14]. The definition of postoperative urinary continence varied among several studies. Hitherto, there is no consensus on UI post-RP [21]. Herein, we selected the most stringent definition of incontinence: any leakage of urine. Reportedly, the continence rate one year after RARP is 69–97% [1,22]. The overall continence rate in this study was 78.77% at 12 months, without the usage of any pad. Although it is not excellent, it is within the average range. To identify the predictive factors, urinary continence was divided into two categories: early continence (<3 months) and late continence (>12 months) [16]. Results Page 4/20 Univariate logistic analysis showed that younger age was associated with urinary continence at 6 months (OR: 1.028, 95% CI: 1.005–1.052, P=0.019), 12 months (OR: 1.034, 95% CI: 1.007–1.061, P=0.012), and 24 months (OR: 1.970, 95% CI: 1.943–1.998, P=0.017) (Tables 6 and 7). In multivariate logistic analysis, advanced age was a significant independent risk factor of urinary continence at 6 months (OR: 1.026, 95% CI: 1.001–1.051, P=0.038), 12 months (OR: 1.030, 95% CI: 1.002–1.058, P=0.035), and 24 months (OR: 1.968, 95% CI: 1.939–1.997, P=0.038) (Tables 6 and 7). Other variables mentioned above were not statistically significant predictors. Also, no significant predictors of late urinary continence were detected at 48 months (Table 7). advanced age was a significant independent risk factor of urinary continence at 6 months (OR: 1.026, 95% CI: 1.001–1.051, P=0.038), 12 months (OR: 1.030, 95% CI: 1.002–1.058, P=0.035), and 24 months (OR: 1.968, 95% CI: 1.939–1.997, P=0.038) (Tables 6 and 7). Other variables mentioned above were not statistically significant predictors. Also, no significant predictors of late urinary continence were detected at 48 months (Table 7). Discussion Page 5/20 In general, PLND was selectively performed for sampling purposes in intermediate- and high-risk patients [15]. One month after the surgery, PLND in the continence group occurred in 121 patients (45.83%), while it occurred in 217 patients in the incontinence group (56.22%) (P<0.05). Three months post-surgery, no significant differences were detected in both groups. The logistic analysis showed that PLND was a significant independent risk factor of early urinary continence at 1 month. Patients who had undergone Page 5/20 PLND during surgery had a high risk of UI. Lymphadenectomy may give rise to transient damage to nerve vessel bundles (NVBs), which affected the recovery of urinary continence. However, with the recovery of body function, this impact declined gradually. The current logistic analysis showed that NS was a significant independent protective factor of urinary continence at 1,3, and 6 months (Tables 5 and 6). These findings were in line with those from the study by Reeves et al., which found that avoiding damage to the nerves around the prostate improves urinary continence in the first 6 months after the surgery [23]. Michl et al. investigated long-term CRs (12 months) after RP and found a significant difference between the NS and non-NS groups [24]. The studies by Kadono et al. and Steineck et al. also indicated that NS is associated with urinary continence in the long- term [17,25]. The bias in the studies used for the analysis might have influenced the results. However, all the studies showed that NS during the surgery produced satisfactory postoperative continence outcomes. Multiple studies have demonstrated that age is an independent risk factor for the return of continence at 1–12 months after RARP. Lavigueur-Blouin et al. evaluated the early continence after RARP [26]. It showed that advanced age was an independent predictor at 1 month. Kim et al. demonstrated that younger men could have an early recurrence of continence 3 months after RARP [27]. Greco et al. compared the continence outcomes of RARP in older men to those of the younger men [28]. The study showed that CRs at 1, 3, and 12 months were similar in the two groups; however, the older group had a significantly lower continence rate at 6 months. The results of these studies might partially be in agreement with those of our study. Shikanov et al. Discussion demonstrated that age is a predictor of continence return at 12 months after RARP, which is partially in accordance with the current results [29]. Our results show that advanced age is a significant risk factor of continence at 6, 12, and 24 months after the surgery (Tables 6 and 7). Men of advanced age had a high risk of UI. Older men have poor endothelial dysfunction, which affects the vascular supply of the NVBs. In addition, it is difficult to perform pelvic floor exercises (PLE) due to an age-related decrease in the mass of the skeletal muscle and neuronal plasticity [30]. These conditions might affect functional outcomes. Interestingly, the optimal time to remove the indwelling catheter has not yet been determined. Conventionally, the urinary catheter was removed between 10 and 21 days postoperatively [31]. Typically, the catheter is removed 3 weeks after the operation in our center in order to ensure the healing of anastomosis. However, most patients returned home after the operation, and the indwelling catheter was removed at the local clinic. This leads to the inconsistency of catheter removal and the duration of indwelling catheter might be longer than expected. Funding This study received financial support from China Postdoctoral Science Foundation funded project (2018M643880) and National Natural Science Foundation of China (81570688). Conclusions The current study has shown that CR improves gradually with time within 1 year post-surgery and stabilizes after one more year. PLND, NS, and age are the significant determinants of continence in the early and late stages, respectively. Thus, these parameters could be used to identify patients at high risk for UI preoperatively and counsel them on postoperative expectations for urinary continence. Limitations Page 6/20 Nevertheless, the present study had some limitations. First, this was a retrospective study from a single institution, and surgeries were not performed by a single surgeon. Second, we did not analyze all variables due to the undocumented surgical steps of the procedure, variations in surgical experience, and differences in the pathological reports. Third, although a stringent definition of continence was applied, the conditions were reported by the patients rather than based on a quality questionnaire. Fourth, the potential bias in selecting the patients for the procedure might influence the results. In addition, data were missing as many patients were lost to follow-up. Despite these drawbacks, this study utilized a large sample size, and the survey of postoperative urinary continence was time-continuous. potential bias in selecting the patients for the procedure might influence the results. In addition, data were missing as many patients were lost to follow-up. Despite these drawbacks, this study utilized a large sample size, and the survey of postoperative urinary continence was time-continuous. Acknowledgements Not applicable. Abbreviations BMI: Body mass index; CRs: Continence rates; CHD: Coronary heart disease; CD: Cerebrovascular diseases; CI: Confidence interval; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; LRP: Laparoscopic radical prostatectomy; NS: Nerve-sparing; NVBs: Nerve vessel bundles; OT: Operation time; OR: Odds ratio; RARP: Robot-assisted radical prostatectomy; RRP: Retropubic radical prostatectomy; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection; PLE: Pelvic floor exercises; RP: Radical prostatectomy; UI: Urinary incontinence Authors’ contributions XL: Protocol/project development, Data Collection, Data management, Statistical analysis, Manuscript writing HZ: Data Collection, Data analysis ZJ: Data Collection, Data analysis YW: Data Collection, Data analysis YS: Data Collection, Data analysis LL: Critical revision of the manuscript, Manuscript editing XZ: Critical revision of the manuscript, Manuscript editing. All authors read and approved the final version of the manuscript. Competing interests The authors declare that they have no competing interest. Consent for publication Not applicable. Availability of data and materials All the data supporting our findings is contained in the manuscript. The datasets used and/or analysed in the current study is available from the corresponding author on reasonable request. Page 7/20 Author details 1Department of Urology, Medical College of People’s Liberation Army, Chinese People’s Liberation Army General Hospital, Fuxing Road. No: 28 Fengtai District, Beijing, China. 2 Department of Urology, Rehabilitation School of Capital Medical University, China Rehabilitation Research Center, Jiaomen North Road. No: 10 Fengtai District, Beijing, China. Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all individual participants included in the study. This study was approved by the Chinese PLA General Hospital Local Ethics Committee. References 1. Ficarra V, Novara G, Rosen RC, Artibani W, Carroll PR, Costello A, Menon M, Montorsi F, Patel VR, Stolzenburg JU, Van der Poel H, Wilson TG, Zattoni F, Mottrie A. Systematic review and meta-analysis of studies reporting urinary continence recovery after robot-assisted radical prostatectomy. Eur Urol. 2012;62(3):405-17. 2. Litwin MS, Pasta DJ, Yu J, Stoddard ML, Flanders SC. 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Michl U, Tennstedt P, Feldmeier L, Mandel P, Oh SJ, Ahyai S, Budäus L, Chun FKH, Haese A, Heinzer H, Salomon G, Schlomm T, Steuber T, Huland H, Graefen M, Tilki D. Nerve-sparing surgery technique, not the preservation of the neurovascular bundles, leads to improved long-term continence rates after radical p Eur Urol. 2016;69(4):584-9. 25. Steineck G, Bjartell A, Hugosson J, Axén E, Carlsson S, Stranne J, Wallerstedt A, Persson J, Wilderäng U, Thorsteinsdottir T, Gustafsson O, Lagerkvist M, Jiborn T, Haglind E, Wiklund P; LAPPRO steering committee. Degree of preservation of the neurovascular bundles during radical prostatectomy and urinary continence 1 year after surgery. Eur Urol. 2015;67(3):559-68. 26. Lavigueur-Blouin H, Noriega AC Valdivieso R, Hueber PA, Bienz M, Alhathal N, Latour M, Trinh QD, El- Hakim A, Zorn KC. Predictors of early continence following robot-assisted radical prostatectomy. Can Urol Assoc J. 2015;9(1-2):e93-7. 27. Kim JJ, Ha YS, Kim JH, Jeon SS, Lee DH, Kim WJ, Kim IY. Independent predictors of recovery of continence 3 months after robot-assisted laparoscopic radical prostatectomy. J Endourol. 2012;26(10):1290-5. 28. Greco KA, Meeks JJ, Wu S, Nadler RB. Robot-assisted radical prostatectomy in men aged > or =70 years. BJU Int. 2009;104(10):1492-5. 29. Shikanov S, Desai V, Razmaria A, Zagaja GP, Shalhav AL. Robotic radical prostatectomy for elderly patients: probability of achieving continence and potency 1 year after surgery. J Urol. Page 10/20 Page 10/20 2010;183(5):1803-7. Tables Table 1 Continence data from 1-48 months after RARP No. Tables continence (%) 1 month (N = 650) 3 months (N = 650) 264 (40.62%) 396 (60.92%) 6 months (N = 650) 464 (71.38%) 12 months (N = 650) 512 (78.77%) 24 months (N = 469) 375 (79.96%) 36 months (N = 327) 260 (79.51%) 48 months (N = 217) 166 (76.50% ) Abbreviation RARP Robot assisted radical prostatectomy Table 1 Continence data from 1-48 months after RARP Table 1 Continence data from 1-48 months after RARP Abbreviation: RARP: Robot-assisted radical prostatectomy Abbreviation: RARP: Robot-assisted radical prostatectomy Table 2 Comparison of parameters between continence and incontinence patients at 1 and 3 months after operation Page 11/20 Variable 1 Month 3 Months Continence (N = 264) Incontinence (N = 386) P value Continence (N = 396) Incontinence (N = 254) P value Age (years) 65.60 ± 7.04 65.63 ± 7.94 0.959 65.20 ± 7.32 66.27 ± 7.94 0.080 BMI (kg/m2) 24.85 ± 2.75 25.14 ± 2.98 0.209 25.01 ± 2.82 25.02 ± 3.01 0.964 Comorbidities, no (%) HP DM 77 (29.17%) 38 (14.39%) 141 (36.53%) 77 (16.06%) 0.051 0.563 122 (30.81%) 64 (16.16%) 96 (37.80%) 36 (14.17%) 0.066 0.493 CHD CD 20 (7.58%) 9 (3.41%) 43 (11.14%) 11 (2.85%) 0.131 0.685 38 (9.60%) 10 (2.53%) 25 (9.84%) 10 (3.94%) 0.917 0.309 PSA (ng/mL) 27.65 ± 32.92 32.11 ± 36.08 0.109 28.65 ± 32.17 32.86 ± 38.64 0.133 PV (mL) 37.73 ± 20.20 35.80 ± 18.58 0.211 37.15 ± 19.34 35.70 ± 19.15 0.348 Gleason score, no (%) 0.475 0.654 ≤6 79 (29.92%) 114 (29.53%) 117 (29.55%) 76 (29.92%) 7 95 (35.98%) 160 (41.45%) 151 (38.13%) 104 (40.94%) ≥8 78 (29.55%) 97 (25.13%) 113 (28.54%) 62 (24.41%) Unknown 12 (4.55%) 15 (3.89%) 15 (3.78%) 12 (4.73%) Clinical T stage, no (%) 0.962 0.550 T1a,b 1 (0.37%) 1 (0.26%) 1 (0.25%) 1 (0.39%) T1c 46 (17.42%) 68 (17.62%) 69 (17.42%) 45 (17.72%) T2a,b 102 (38.64%) 139 (36.01%) 157 (39.65%) 84 (33.07%) T2c 103 (39.02%) 155 (40.16%) 149 (37.63%) 109 (42.91%) T3a 1 (0.37%) 2 (0.51%) 1 (0.25%) 2 (0.79%) T3b 11 (4.17%) 21 (5.44%) 19 (4.80%) 13 (5.12%) OT (mins) 151.61 ± 42.16 153.81 ± 48.78 0.551 151.02 ± 41.76 155.86 ± 52.30 0.192 NS, no (%) 73 (27.65%) 48 (12.44%) 0.000 87 (21.97%) 34 (13.39%) 0.045 PLND, no (%) 121 (45.83%) 217 (56.22%) 0.009 204 (51.52%) 134 (52.76%) 0.757 DIC (days) 21.22 ± 10.83 21.02 ± 10.09 0.810 21.28 ± 11.46 20.84 ± 8.46 0.601 Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection Page 12/20 Page 12/20 Table 3 Comparison of parameters between continence and incontinence patients at 6 and 12 months after operation 12 months after operation Variable 6 Months 12 Months Continence (N = 464) Incontinence (N = 186) P value Continence (N = 512) Incontinence (N = 138) P value Age (years) 65.17 ± 7.33 66.73 ± 8.08 0.018 65.23 ± 7.36 67.06 ± 8.22 0.012 BMI (kg/m2) 25.05 ± 2.78 24.93 ± 3.14 0.631 25.07 ± 2.84 24.82 ± 3.06 0.362 Comorbidities, no (%) HP DM 150 (32.33%) 73 (15.73%) 68 (36.56%) 27 (14.52%) 0.302 0.698 172 (33.59%) 80 (15.63%) 46 (33.33%) 20 (14.49%) 0.954 0.744 CHD CD 41 (8.84%) 12 (2.59%) 22 (11.83%) 8 (4.30%) 0.244 0.253 46 (8.98%) 13 (2.54%) 17 (12.32%) 7 (5.07%) 0.240 0.126 PSA (ng/mL) 29.15 ± 33.12 33.16 ± 38.85 0.185 29.43 ± 32.66 32.51 ± 42.07 0.223 PV (mL) 37.14 ± 19.11 35.20 ± 19.63 0.245 36.95 ± 18.75 35.24 ± 21.10 0.354 Gleason score, no (%) 0.251 0.257 ≤6 137 (29.53%) 56 (30.11%) 153 (29.88%) 40 (28.99%) 7 178 (38.36%) 77 (41.40%) 197 (38.48%) 58 (42.03%) ≥8 133 (28.66%) 42 (22.58%) 144 (28.13%) 31 (22.46%) Unknown 16 (3.45%) 11 (5.91%) 18 (3.51%) 9 (6.52%) Clinical T stage, no (%) 0.659 0.918 T1a,b 2 (0.43%) 0 2 (0.38%) 0 T1c 78 (16.81%) 36 (19.35%) 88 (17.19%) 26 (18.84%) T2a,b 178 (38.36%) 63 (33.87%) 193 (37.70%) 48 (34.78%) T2c 179 (38.58%) 79 (42.47%) 201 (39.26%) 57 (41.30%) T3a 2 (0.43%) 1 (0.55%) 2 (0.38%) 1 (0.73%) T3b 25 (5.39%) 7 (3.76%) 26 (5.08%) 6 (4.35%) OT (mins) 152.42 ± 44.08 154.15 ± 51.17 0.666 151.66 ± 43.98 157.57 ± 53.49 0.182 NS, no (%) 97 (20.91%) 24 (12.90%) 0.018 103 (20.12%) 18 (13.04%) 0.058 PLND, no (%) 241 (51.94%) 97 (52.15%) 0.961 267 (52.15%) 71 (51.45%) 0.884 DIC (days) 21.34 ± 11.20 20.51 ± 8.00 0.356 21.37 ± 10.96 20.13 ± 7.83 0.215 Page 13/20 Page 13/20 Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection Table 4 Comparison of parameters between continence and incontinence patients at 24 and 48 months after operation Page 14/20 Variable 24 Months 48 Months Continence (N = 375) Incontinence (N = 94) P value Continence (N = 166) Incontinence (N = 51) P value Age (years) 63.93 ± 8.48 65.82 ± 7.62 0.016 65.70 ± 7.78 66.16 ± 7.97 0.719 BMI (kg/m2) 25.16 ± 2.91 24.71 ± 3.05 0.186 25.21 ± 2.89 25.30 ± 2.61 0.832 Comorbidities, no (%) HP DM 138 (36.80%) 60 (16.00%) 27 (28.72%) 11 (11.70%) 0.143 0.299 53 (31.93%) 25 (15.06%) 20 (39.22%) 13 (25.49%) 0.335 0.087 CHD CD 40 (10.67%) 11 (2.93%) 6 (6.38%) 4 (4.26%) 0.212 0.515 21 (12.65%) 5 (3.01%) 6 (11.76%) 2 (3.92%) 0.867 0.748 PSA (ng/mL) 30.00 ± 34.03 33.77 ± 35.59 0.343 30.57 ± 40.59 35.60 ± 38.12 0.433 PV (mL) 35.97 ± 19.66 34.72 ± 15.95 0.567 34.50 ± 18.85 40.11 ± 20.82 0.071 Gleason score, no (%) 0.336 0.501 ≤6 109 (29.07%) 29 (30.85%) 49 (29.52%) 20 (39.22%) 7 151 (40.27%) 42 (44.68%) 63 (37.95%) 19 (37.25%) ≥8 103 (27.47%) 18 (19.15%) 47 (28.31%) 11 (21.57%) Unknown 12 (3.19%) 5 (5.32%) 7 (4.22%) 1 (1.96%) Clinical T stage, no (%) 0.806 0.356 T1a,b 1 (0.27%) 0 0 0 T1c 68 (18.13%) 15 (15.96%) 32 (19.28%) 8 (15.69%) T2a,b 133 (35.47%) 39 (41.49%) 54 (32.53%) 11 (21.57%) T2c 152 (40.53%) 34 (36.17%) 70 (42.17%) 29 (56.86%) T3a 2 (0.53%) 0 2 (1.20%) 0 T3b 19 (5.07%) 6 (6.38%) 8 (4.82%) 3 (5.88%) OT (mins) 155.19 ± 48.98 146.79 ± 42.32 0.128 154.39 ± 49.88 152.14 ± 53.29 0.782 NS, no (%) 65 (17.33%) 12 (12.77%) 0.285 20 (12.05%) 8 (15.69%) 0.498 PLND, no (%) 204 (54.40%) 52 (55.32%) 0.873 91 (54.82%) 33 (64.71%) 0.212 DIC (days) 21.27 ± 11.21 19.94 ± 5.08 0.260 21.14 ± 12.23 20.20 ± 7.93 0.603 Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection Page 15/20 and 3 months Predictors 1 Month 3 Months Univariable Multivariable Univariable Multivariable OR, 95% CI P value OR, 95% CI P value OR, 95% CI P value OR, 95% CI P value Age 1.001, 0.980- 1.021 0.959 1.001, 0.979- 1.023 0.940 1.019, 0.998- 1.041 0.080 1.018, 0.996- 1.041 0.113 BMI 1.036, 0.981- 1.094 0.209 1.023, 0.963- 1.086 0.460 1.001, 0.948- 1.057 0.964 0.998, 0.941- 1.059 0.947 HP 1.398, 0.998- 1.957 0.051 1.376, 0.947- 1.999 0.094 1.365, 0.980- 1.901 0.066 1.404, 0.978- 2.015 0.066 DM 1.138, 0.734- 1.764 0.563 0.999, 0.626- 1.595 0.998 0.857, 0.550- 1.334 0.493 0.748, 0.470- 1.190 0.220 CHD 1.529, 0.878- 2.665 0.134 1.476, 0.817- 2.688 0.197 1.028, 0.605- 1.750 0.917 0.885, 0.508- 1.542 0.665 CD 0.831, 0.340- 2.034 0.685 0.739, 0.289- 1.889 0.528 1.582, 0.649- 3.856 0.313 1.456, 0.582- 3.640 0.422 PSA 1.004, 0.999- 1.009 0.114 1.000, 0.995- 1.005 0.985 1.003, 0.999- 1.008 0.138 1.002, 0.997- 1.006 0.501 PV 0.995, 0.987- 1.003 0.212 0.994, 0.986- 1.002 0.159 0.996, 0.988- 1.004 0.348 0.995, 0.987- 1.004 0.262 Gleason score 0.929, 0.774- 1.116 0.433 0.801, 0.654- 0.981 0.332 0.965, 0.802- 1.160 0.702 0.926, 0.758- 1.131 0.449 Clinical stage 1.060, 0.899- 1.250 0.487 1.013, 0.850- 1.208 0.883 1.077, 0.913- 1.270 0.379 1.071, 0.902- 1.273 0.434 OT 1.001, 0.998- 1.004 0.551 1.000, 0.996- 1.003 0.931 1.002, 0.999- 1.006 0.193 1.002, 0.998- 1.005 0.281 NS 0.372, 0.248- 0.557 0.000 0.360, 0.231- 0.561 0.000 0.549, 0.356- 0.846 0.007 0.546, 0.342- 0.872 0.011 PLND 1.517, 1.018- 2.079 0.009 1.535, 1.079- 2.184 0.017 1.051, 0.767- 1.441 0.757 0.978, 0.690- 1.386 0.901 DIC 0.998, 0.983- 1.013 0.809 0.999, 0.983- 1.014 0.869 0.996, 0.980- 1.012 0.602 0.997, 0.981- 1.013 0.713 Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; Page 16/20 Page 16/20 NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection Table 6 Univariable and multivariable regression analysis for predictors of continence 6 and 12 months Predictors 6 Months 12 Months Univariable Multivariable Univariable Multivariable OR, 95% CI P value OR, 95% CI P value OR, 95% CI P value OR, 95% CI P value Age 1.028, 1.005- 1.052 0.019 1.026, 1.001- 1.051 0.038 1.034, 1.007- 1.061 0.012 1.030, 1.002- 1.058 0.035 BMI 0.986, 0.929- 1.046 0.630 0.993, 0.932- 1.058 0.827 0.970, 0.908- 1.036 0.362 0.983, 0.916- 1.054 0.626 HP 1.206, 0.845- 1.723 0.302 1.179, 0.800- 1.737 0.404 0.988, 0.663- 1.472 0.954 0.937, 0.606- 1.448 0.768 DM 0.910, 0.564- 1.468 0.698 0.834, 0.506- 1.375 0.477 0.915, 0.538- 1.556 0.744 0.873, 0.501- 1.521 0.632 CHD 1.384, 0.800- 2.395 0.246 1.190, 0.672- 2.109 0.551 1.423, 0.788- 2.571 0.242 1.275, 0.686- 2.371 0.443 CD 1.693, 0.681- 4.211 0.258 1.583, 0.617- 4.064 0.339 2.051, 0.802- 5.244 0.134 2.144, 0.806- 5.072 0.126 PSA 1.003, 0.998- 1.008 0.190 1.001, 0.996- 1.006 0.614 1.003, 0.998- 1.008 0.227 1.002, 0.996- 1.007 0.517 PV 0.995, 0.985- 1.004 0.245 0.995, 0.985- 1.004 0.288 0.995, 0.985- 1.005 0.354 0.995, 0.985- 1.006 0.393 Gleason score 0.976, 0.800- 1.192 0.815 0.946, 0.764- 1.173 0.614 1.017, 0.816- 1.267 0.879 0.998, 0.788- 1.264 0.989 Clinical stage 0.973, 0.814- 1.164 0.767 0.972, 0.807- 1.171 0.765 0.996, 0.818- 1.214 0.970 0.991, 0.807- 1.218 0.932 OT 1.001, 0.997- 1.004 0.665 1.001, 0.997- 1.005 0.720 1.003, 0.999- 1.007 0.182 1.003, 0.999- 1.007 0.177 NS 0.561, 0.346- 0.909 0.019 0.545, 0.324- 0.915 0.022 0.596, 0.347- 1.023 0.060 0.609, 0.342- 1.086 0.093 PLND 1.008, 0.717- 1.418 0.961 0.993, 0.683- 1.444 0.971 0.972, 0.668- 1.416 0.884 0.914, 0.606- 1.380 0.670 DIC 0.991, 0.973- 1.010 0.359 0.993, 0.975- 1.012 0.474 0.986, 0.964- 1.008 0.215 0.988, 0.966- 1.011 0.304 Table 6 Univariable and multivariable regression analysis for predictors of continence 6 and 12 months d multivariable regression analysis for predictors of continence 6 Table 6 Univariable and multivariable regression analysis for pre and 12 months Page 17/20 Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection Table 7 Univariable and multivariable regression analysis for predictors of continence 24 and 48 months Page 18/20 Predictors 24 Months 48 Months Univariable Multivariable Univariable Multivariable OR, 95% CI P value OR, 95% CI P value OR, 95% CI P value OR, 95% CI P value Age 1.970, 1.943-1.998 0.017 1.968, 1.939-1.997 0.038 1.008, 0.967- 1.049 0.717 1.009, 0.964- 1.057 0.691 BMI 0.949, 0.877-1.026 0.186 0.956, 0.877-1.042 0.310 1.012, 0.906- 1.131 0.831 0.999, 0.880- 1.136 0.992 HP 0.692, 0.422-1.134 0.144 0.810, 0.474-1.384 0.441 1.376, 0.718- 2.635 0.336 1.484, 0.708- 3.109 0.296 DM 0.696, 0.350-1.383 0.301 0.779, 0.382-1.589 0.492 1.929, 0.902- 4.125 0.090 1.803, 0.803- 4.051 0.153 CHD 0.571, 0.235-1.390 0.217 0.642, 0.255-1.617 0.347 0.921, 0.350- 2.421 0.867 0.847, 0.293- 2.448 0.759 CD 1.471, 0.458-4.726 0.517 1.816, 0.535-6.160 0.339 1.314, 0.247- 6.987 0.749 1.003, 0.158- 6.389 0.997 PSA 1.003, 0.997-1.009 0.346 1.003, 0.996-1.009 0.419 1.003, 0.996- 1.010 0.437 1.002, 0.995- 1.010 0.530 PV 0.996, 0.984-1.009 0.567 0.996, 0.983-1.009 0.580 1.014, 0.999- 1.029 0.077 1.014, 0.998- 1.031 0.093 Gleason score 0.919, 0.699-1.207 0.542 0.892, 0.659-1.208 0.461 0.744, 0.509- 1.089 0.128 0.691, 0.452- 1.058 0.089 Clinical stage 1.013, 0.802-1.280 0.912 1.054, 0.824-1.348 0.677 1.222, 0.886- 1.684 0.221 1.189, 0.835- 1.692 0.337 OT 0.996, 0.991-1.001 0.128 0.997, 0.991-1.002 0.210 0.999, 0.993- 1.005 0.781 0.998, 0.991- 1.005 0.492 NS 0.698, 0.360-1.353 0.287 0.719, 0.352-1.472 0.367 1.358, 0.559- 3.299 0.499 1.163, 0.427- 3.167 0.767 PLND 1.038, 0.659-1.635 0.873 1.145, 0.690-1.902 0.600 1.511, 0.788- 2.896 0.214 1.720, 0.828- 3.573 0.146 DIC 0.983, 0.955-1.012 0.257 0.980, 0.949-1.013 0.228 0.991, 0.958- 1.026 0.605 0.989, 0.950- 1.030 0.604 Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection Abbreviations: BMI: Body mass index; CHD: Coronary heart disease; CD: Cerebrovascular diseases; DM: Diabetes mellitus; DIC: Duration of indwelling catheter; HP: Hypertension; NS: Nerve-sparing; OT: Operation time; PSA: Prostate-specific antigen; PV: Prostate volume; PLND: Pelvic lymph node dissection Figure 1 Study flow diagram Figures Page 19/20 Page 19/20 gure 1 udy flow diagram upplementary Files is is a list of supplementary files associated with this preprint. Click to download. Supplementaryfile.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementaryfile.docx Page 20/20 Page 20/20
https://openalex.org/W4386405969	https://zenodo.org/record/8313172/files/265-282%20last%20paper%20Mohammad%20Noor.pdf	English	null	A brief review of Bornean banded langur Presbytis chrysomelas (Müller, 1838) of Sarawak	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	7,283	Volume 7(4): 265-282 (2023) (http://www.wildlife-biodiversity.com/) Research Article A brief review of Bornean banded langur Presbytis chrysomelas (Müller, 1838) of Sarawak Mohammad Noor-Faezah1,2, Tukiman Nur-Aizatul1,2, Tingga Roberta Chaya Tawie2,3, Mohamad Fhaizal Bukhori2, Jayasilan Mohd-Azlan4, Azroie Denel5, Badrul Munir Md- Zain3, Muhammad Abu Bakar Abdul-Latiff 6, Abd Rahman Mohd-Ridwan2* 1Animal Resource Science and Management, Faculty of Resource Science and Technology Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia y y 2Centre for Pre-University Studies, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 2Centre for Pre-University Studies, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 3School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 3School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 3School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 4Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 4Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 5Sarawak Forestry Corporation, Kota Sentosa, Sarawak, Malaysia 5Sarawak Forestry Corporation, Kota Sentosa, Sarawak, Malaysia 6 5Sarawak Forestry Corporation, Kota Sentosa, Sarawak, Malaysia 6 5Sarawak Forestry Corporation, Kota Sentosa, Sarawak, Malaysia 6 Environmental Management and Conservation Research Unit (eNCORe), Faculty of Applied Sciences 5Sarawak Forestry Corporation, Kota Sentosa, Sarawak, Malaysia 6 Environmental Management and Conservation Research Unit (eNCORe), Faculty of Applied Sciences and Technology (FAST), Universiti Tun Hussein Onn Malaysia (Pagoh Campus), 84000, Muar, Johor, Malaysia *Email: armridwan@unimas.my Received: 26 May 2023 / Revised: 25 August 2023 / Accepted: 29 August 2023/ Published online: 31 August 2023. How to cite: Noor-Faezah, et al. (2023). A brief review of Bornean banded langur Presbytis chrysomelas (Müller, 1838) of Sarawak, Journal of Wildlife and Biodiversity, 7(4), 265-282. DOI: https://doi.org/10.5281/zenodo.8313172 Abstract The critically endangered Bornean banded langur (Presbytis chrysomelas) is exclusively found in Borneo. This species is classified among the world's 25 most endangered primate species. The geographical locations and distribution patterns of P. chrysomelas remain inadequately understood, and their taxonomic classification remains unclear. Although it was once abundant in Sarawak, the distribution of P. chrysomelas has become one of the most restricted among all Presbytis species. Presently, documented sightings of P. chrysomelas span across five specific locations in Sarawak: Samunsam Wildlife Sanctuary, Tanjung Datu National Park, Gunung Pueh National Park, Similajau National Park, and Maludam National Park. Thus, the purpose of this review is to present previous studies conducted on P. chrysomelas in Sarawak, elucidating its taxonomy, characteristics, distributions, important sites, research gaps, threats, and recommendations for further studies. Immediate conservation efforts and attention from the primatologist, relevant authorities and stakeholders are needed to prevent the localized extinction of P. chrysomelas. Keywords: Bornean banded langur, Presbytis chrysomelas, Sarawak 267 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) 267 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) 267 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Taxonomy of Presbytis chrysomelas The Bornean banded langur has always been the subject of taxonomic confusion. Formerly, it was first described as Semnopithecus femoralis (Miller, 1934), S. chrysomelas (Kantha & Suzuki, 2009), P. melalophos (Kantha & Suzuki, 2009), P. melalophos chrysomelas (Oates et al. 1994), and then as a distinct species of P. femoralis chrysomelas (Brandon-Jones et al., 2004). Currently, this species is referred to as P. chrysomelas based on updated DNA data (Meyer et al., 2011; Vun et al., 2011) and morphological characteristics (Groves, 2001). Two subspecies comprised under P. chrysomelas were found distinct based on the colour morphs variation: the black morph (P. c. chrysomelas) and the red morph (P. c. cruciger) (Phillipps & Phillipps, 2016). Introduction Borneo is well renowned for being one of the world’s primate hotspots (Meijaard & Nijman, 2003; Wolf & Ripple, 2022). Borneo inhabits 17 distinct species of non-human primates, spanning across six families and eight genera, including genus Presbytis. The genus Presbytis of the subfamily Colobinae is the most widely distributed group among the non-human primates in Borneo and widely studied in Malaysia; however, most investigations focused at species level on Presbytis frontata, Presbytis femoralis, Presbytis hosei, and Presbytis rubicunda (Abdul-Latiff et al., 2019; Duckworth et al., 2011; Smith, 2015; Meyer et al., 2011; Miller, 1934; Najmuddin et al., 2020; Najmuddin et al., 2021; Mohd-Hatta, 2013). Other studies focused more on resolving the taxonomic ambiguity of the genus (Aifat et al., 2016; Md-Zain, 2001; Md-Zain et al., 2008; Meyer et al., 2011; Meyer, 2012; Vun et al., 2011). Nevertheless, limited attention has been directed towards the study of Presbytis chrysomelas. Presbytis chrysomelas or Bornean banded langur is an understudied rare primate and endemic to Borneo. Currently, information on the sites and distribution of P. chrysomelas is still scarce, and its taxonomy is still disputed (Brandon-Jones et al., 2004; Meyer et al., 2011). Similar to other Presbytis, P. chrysomelas is also synonymously known locally as penatat or penyatat by Bidayuh, lutung by Malays, bijit by Iban, and berangad by Kenyah (Mohd-Hatta, 2013). It has the most restricted distribution of all Presbytis in Borneo. And globally, it has been categorized as a Critically Endangered species on the IUCN Red List (Nijman et al., 2020a) as it has a very high risk of extinction in the wild due to the continuously reduced populations and requires immediate conservation and proper study approaches. Approximately, the population has decreased by up to 80% during the last 30 years and is expected to do so at the same rate over the next 30 years (Nijman et al., 2020a). The distribution region is quite narrow in comparison with its historical range as it was once widely spread. Hence, the objective of this current review is to compile the notable existing records concerning P. chrysomelas in Sarawak with the intention of enhancing scientific documentation. This comprehensive review covers aspects including taxonomy, characteristics, distributions, important sites, threats, recommendations for future research and the crucial need for prompt conservation initiatives. 266 Characteristics of Presbytis chrysomelas The fur colouration of P. chrysomelas is quite variable between subspecies although it is normally jet black with a portion of brown hairs on the dorsum (Mohd-Hatta, 2013). For P. c. chrysomelas, the underside is fawn (pale whitish) up to the chin, wrists, and cheek, as well as down to the belly, inner leg, and ventral part of the tail (Rifqi et al., 2019) (Figure 1a). The chest hairs are dark in colour and curly. Small forehead whorls are observable, commonly in pairs, close to the brow, with a crest that is tall, upright, and narrow in between. Nevertheless, Duckworth et al. (2011) recognized the unusual morphological characteristics of P. c. chrysomelas in Similajau National Park. The facial skin is greyish to pinkish, with dark cheeks and lighter underparts. Additionally, the hair colour variation for subspecies P. c. cruciger is apparent, with red-orange, starting on the head, shoulders, sides of the belly, and toward the thigh and calf (Rifqi et al., 2019) (Figure 1b). The hands, arms, feet, and the rear line of its body, which extends to its tail, are covered in black. The juveniles of both subspecies are either grey/reddish/pale white in colour, with a black cross that runs down over their backs and arms and a pale belly (Phillipps & Phillipps, 2016). 267 268 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Figure 1. The fur colouration between subspecies of P. chrysomelas. a) Black morph P. c. chrysomelas b) Red morph P. c. cruciger (source: http://prcfindonesia.org/lutung-sentarum- hewan-langka-muncul-di-nanga-lauk/). Figure 1. The fur colouration between subspecies of P. chrysomelas. a) Black morph P. c. chrysomelas b) Red morph P. c. cruciger (source: http://prcfindonesia.org/lutung-sentarum- hewan-langka-muncul-di-nanga-lauk/). 269 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) 269 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Figure 2. Map of Bornean Island, illustrating the historical distributions and modern distributions of Presbytis chrysomelas (Phillipps & Phillipps, 2016) Figure 2. Map of Bornean Island, illustrating the historical distributions and modern distributions of Presbytis chrysomelas (Phillipps & Phillipps, 2016) Historical distributions Presbytis chrysomelas is a diurnal and arboreal species that primarily inhabits lowland environments, specifically tropical rainforests, mangrove forests, and swamp forests. This species exhibits the most limited distribution among all members of the Presbytis genus, heavily relying on its specific habitat. Typically, P. chrysomelas is found in groups of around five to six individuals, consisting of one male, two to three females, and their offspring (Bennett, 1992; Payne et al., 1985). One distinguishing feature is the distinct calls of P. chrysomelas, which can be differentiated from those of P. robinsoni, P. femoralis, and P. siamensis through their unique sound "ke-ke-ke-ke-ke" (Phillipps & Phillipps, 2016). Presbytis chrysomelas was once the most prevalent and widely dispersed species in Borneo (Phillipps & Phillipps, 2016) (Figure 2). The historical distribution of the Bornean banded langur as per the previous name P. melalophos recorded in the area extends to the Northwestern of Kalimantan down to Western Sarawak. In Sarawak, it was frequently seen northward extending to Central Sarawak where it meets P. melalophos cruciger. Nonetheless, this distribution was omitted by Payne et al. (1985) of the records of P. c. chrysomelas in Similajau National Park located northeast of Sarawak. 268 Modern distributions Once considered common back in the early twentieth century, the current area of occupancy of P. chrysomelas is less than 5% of its historical range with an approximate population between 200 and 500 individuals (Smith, 2015; Nijman et al., 2020a). Five locations in Sarawak have reported recent sightings of P. chrysomelas based on direct observation and camera trapping, namely, Maludam National Park, Similajau National Park, Tanjung Datu National Park, Samunsam Wildlife Sanctuary, and Pueh National Park. Since many records are historical, the modern distribution requires further research to be done to depict the accuracy of the existence. 269 Important sites With the recent sightings as shown in Table 1, these protected areas are considered important for the conservation of P. chrysomelas in Sarawak. The biggest known population was recorded in the Samunsam Wildlife Sanctuary. A total of 17 groups with 8 to 13 individuals of P. c. chrysomelas has been encountered via line transect survey between November 2004 and December 2005 (Ampeng and Md-Zain, 2012). Meanwhile, the Tanjung Datu National Park is one of Sarawak’s smallest national parks in Sarawak and is situated at the southwestern point of Borneo. In 2018, eight distinct photos of the P. c. chrysomelas were captured during the course of 28 months of camera trapping (Mohd- Azlan et al., 2018). All of the sightings were detected in the beach forest and none in the mixed- dipterocarp forest. The Similajau National Park is a 90 km2 forest consisting of mixed-dipterocarp forest that alternates with kerangas woodland. The Bornean banded langur is considered the most common colobine species found in the park. The first recorded sighting of the Bornean banded langur based on direct observation was back in 1986 in which numerous groups were observed (Duckworth & Kelsh, 1988). The Gunung Pueh National Park located at the northwestern tip of Borneo. Given its location near the Samunsam and Tanjung Datu National Park, it is realistic to anticipate sightings of Bornean banded langur there. A solitary record of the P. c. chrysomelas based on motion- sensor camera trappings of 3,109 trap nights was documented at the lowland forest of Gunung Pueh (Mohd-Azlan, 2022). Finally, the Maludam peat swamp forest was declared a national park in 2000 by the state government of Sarawak due to the documented presence of the proboscis monkey (Nasalis larvatus). Out of the five locations listed, only Maludam reported by Nijman et al. (2020b) has a viable population size of P. c. cruciger, but no exact amount has been recorded. The other three exhibit dispersed populations of P. c. chrysomelas, despite being protected in the wildlife refuges and national parks. 270 271 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Table 1. Recorded sightings of Presbytis chrysomelas in Sarawak based on the published papers of previous years. # Location Records Reference 1. Samunsam Wildlife Sanctuary 17 groups of 8 to 13 individuals were observed via line transect. (December 2004 to December 2005) (Ampeng & Md-Zain, 2012) 2. Important sites Tanjung Datu National Park 8 individuals based on camera trap images independent detections. (July 2013 to October 2015) (Mohd-Azlan et al., 2018) 3. Similajau National Park Several groups at two sites in 1986, at least five groups at three sites in 1995, and four times in 2005 with other colobines were observed. (Duckworth et al., 2011) (Duckworth & Kelsh 1988) (Duckworth 1997) 4. Maludam National Park No exact recorded sighting but the population was reported to be viable. (Nijman et al., 2020b) 5. Gunung Pueh National Park Solitary image from camera trap recorded in lowland forests of lower than 1,100 m asl. (June 2016 to March 2017) (Mohd-Azlan, 2022) 6. The northern part of Borneo (No clear geographical barrier) 26 localities data in the habitat of tropical wet evergreen forest. (1991 to 2001) (Meijaard & Nijman, 2003) Comparison with other endangered primates in Sarawak based on research attention 271 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Comparison with other endangered primates in Sarawak based on research attention In Sarawak, two endemic species of primates have been classed as Critically Endangered by the IUCN Red List (IUCN, 2022) and listed as Totally Protected under the Wild Life Protection Ordinance (1998): orangutan and Bornean banded langur. Nonetheless, high conservation attention has been given to the orangutan and the endangered proboscis monkey, in comparison with the Bornean banded langur, even though all species are in a dire need of conservation action. The Bornean orangutan, the charismatic ape species, has drawn significant conservation attention ever since 1958, as a Totally Protected species under the Wild Life Protection Ordinance. The state government of Sarawak had formulated action plans with the relevant Malaysian government stakeholders and agencies to save orangutans from extinction. The 271 272 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) 272 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) latest is the pledge of the Sarawak government on the policy of zero-loss orangutans and their habitat by 2015 (Ji, 2015). Studies on orangutans also have been done holistically by many researchers. In addition, the proboscis monkey has received extensive conservation and study action in Sarawak as well (Table 2). The recent one is the collaboration between the Sarawak Forestry Corporation and Universiti Malaysia Sarawak for Sarawak Proboscis Monkey Action Plan 2021–2025 (Khan et al., 2021). Moreover, several areas in Sarawak with viable populations of proboscis monkeys had been gazetted as Totally Protected areas, namely, Samunsam Wildlife Sanctuary, Bako National Park, and Kuching Wetland National Park (Kombi & Abdullah, 2016). latest is the pledge of the Sarawak government on the policy of zero-loss orangutans and their habitat by 2015 (Ji, 2015). Studies on orangutans also have been done holistically by many researchers. In addition, the proboscis monkey has received extensive conservation and study action in Sarawak as well (Table 2). The recent one is the collaboration between the Sarawak Forestry Corporation and Universiti Malaysia Sarawak for Sarawak Proboscis Monkey Action Plan 2021–2025 (Khan et al., 2021). Moreover, several areas in Sarawak with viable populations of proboscis monkeys had been gazetted as Totally Protected areas, namely, Samunsam Wildlife Sanctuary, Bako National Park, and Kuching Wetland National Park (Kombi & Abdullah, 2016). On the other hand, the ecology of the P. chrysomelas, particularly based on population estimates, is yet unknown. Four studies on the behavior and ecology of P. chrysomelas have been conducted, four on genetic studies and two on the threats and conservation. The studies on population, distribution, and abundance are limited on the assessment from the IUCN Red List. The study gap between the Bornean banded langur, Bornean orangutan, and proboscis monkey in Sarawak is depicted in Table 2, while the comparison of the number of studies by species over a decade is illustrated in Figure 3. 272 273 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) 273 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Table 2. Comparison of study conducted on Presbytis chrysomelas, Pongo pygmaeus and Nasalis larvatus in Sarawak. # Species IUCN Red List Behaviour and ecology Population, habitat, distribution, and abundance Genetic Parasite and health Threats and conservation 1. Pongo pygmaeus Critically endangered Ampeng et al. Conservation threats The primary threats to the P. chrysomelas in Sarawak include habitat loss and fragmentation and poaching (Ampeng, 2006). As the P. chrysomelas is highly dependent on habitat by being arboreal, the conversion of forests into agricultural land, mainly for oil palm cultivation, as well as residential and commercial development has threatened their sustainability. The monoculture farms of oil palms and the construction of the Pan-Borneo Highway-Sarawak further fractured the forest (Alamgir et al., 2020). The severe forest fragmentation heightens the possibility of extinction due to the continuous loss of habitat connectivity and the increment risk of hunting and predation. However, the impact of declining habitat quality on the long- term persistence of species is still unclear. 272 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) (2016); Husen (2001); Mohd-Hatta (2005); Mohd- Rahmantullah, (2001); Salina et al. (2004); Wesley (2001) Ampeng et al. (2016); Ampeng et al. (2021); Blouch (1997); Gurmaya & Silang (2002); Gumal et al. (2021); Pandong et al. (2018); Pandong et al. (2022); Schaller, (1961). Silang et al. (2006) Kanthaswamy & Smith (2002); Mattle-Greminger et al. (2018); Warren et al., (2001); Zhi et al., (1996) Teo et al. (2019); Thayaparan et al. (2014) Pandong & Anak (2019); Gumal (2022); Gumal & (Tisen, 2010); Jonas et al. (2017); Medway (1976); Pandong et al. (2019); Tisen & Silang (2016); Zander et al. (2014) 2. Presbytis chrysomelas Critically endangered Ampeng (2003); Ampeng (2006); Ampeng & Md-Zain (2007); Ampeng & Md- Zain (2012) Nijman et al. (2020a; 2020b; 2020c) Abdul-Latiff et al. (2019); Aifat et al (2016); Md-Zain (2001); Vun et al. (2011) Ampeng (2006); Smith (2015) 3. Nasalis larvatus Endangered Bennett (1986); Bennett (1988); Bennet & Sebastian, (1988); Budeng, (2011); Kombi & Abdullah (2016); Onuma (2002); Salter et al. (1985); Wan- Azman et al. (2022) Aziz et al. (2015); Azman (2005); Kombi & Abdullah, (2013); Kombi & Abdullah (2016); Laman & Aziz (2019); Tuen & Pandong (2007) Mazlan et al. (2019) Adrus et al. (2019); Hasegawa et al. (2003); Khairi (2012); Julian (2015); Lim et al. (2019); Thayaparan et al. (2013); Thayaparan et al. (2014) Bennett (1986); Kamaruzzaman et al. (2017); Khan et al. (2021) 274 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) 274 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Figure 3. Studies on Bornean orangutan, Bornean banded langur and Proboscis monkey of Sarawak per decade. 0 2 4 6 8 10 12 14 16 1960 1970 1980 1990 2000 2010 2020 Bornean Orangutan Bornean Banded Langur Proboscis Monkey 0 2 4 6 8 10 12 14 16 1960 1970 1980 1990 2000 2010 2020 n Bornean Banded Langur Proboscis Monkey Figure 3. Studies on Bornean orangutan, Bornean banded langur and Proboscis monkey of Sarawak per decade. 275 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) Several samples of P. c. chrysomelas were used in the genetic study to reclarify the taxonomic classification of the genus Presbytis. Vun et al. (2011) discovered that P. chrysomelas is genetically distinct from P. femoralis and P. siamensis, separating chrysomelas at the species level, while the cytochrome b gene revealed that P. chrysomelas is the most primitive among Presbytis. In contrast, the subspecies P. c. cruciger in Kalimantan has received much attention from researchers in recent years. Notable research attention was given to the study of ecology on the P. c. cruciger in Taman Danau Sentarum. However, similar to P. c. chrysomelas in Malaysia, P. c. cruciger in Indonesia is also in need of immediate attention in genetic study. Previous ecological studies on Presbytis chrysomelas The ecological study of P. chrysomelas is limited to the research on ranging behavior done by Ampeng and Md-Zain (2012). They discovered the ranging patterns employed by 17 groups of P. c. chrysomelas in Samunsam Wildlife Sanctuary encompass vertical, cross-horizontal, and straight horizontal movements. They have a short daily movement distance (31.8 to 54.3 m). The abundance and distribution of food resources influence the ranging pattern and ranging distance of P. c. chrysomelas. Research recommendations It is important to realize that there is a notable gap in the study with a focus on the ecological and genetic studies of P. chrysomelas at the species level. Hence, future research should focus on the studies listed in Table 3. Table 3. Recommended potential future study in resolving the research gap for P. chrysomelas in Sarawak. # Aspect Recommendation 1. Population size and distribution The population size and density of P. chrysomelas are still in oblivion. The previous study merely documented the frequency and precise distribution (Ampeng & Md-Zain, 2012; Duckworth et al., 2011; Mohd-Azlan, 2022; Mohd-Azlan et al., 2018; Meijaard & Nijman, 2003; Nijman et al., 2020b). Extensive research is required to determine the population size, distribution, and density of both subspecies in Sarawak. Extensive ground sampling should be conducted by returning to the area within its historical distribution. 2. Daily activity budget The study is limited to the subspecies P. c. cruciger in Indonesia (Musyaffa & Santoso, 2020). No previous research was done on the daily activity budget of P. chrysomelas in Sarawak. Baseline daily behavioral data of P. chrysomelas in Sarawak is needed to develop a strategic conservation action. 3. Feeding ecology Dietary information is still quite limited. Dietary research is necessary to evaluate dietary flexibility and to comprehend the coexistence with other primate species in reducing intraspecific competition through dietary overlap as shown in long-tailed macaque and dusky langur (Ruslin et al., 2019). 4. Population genetics Population genetic diversity concentrating on each subspecies in Sarawak is still rare as much focus is on the genus Presbytis (Aifat et al., 2016; Md-Zain, 2001; Md-Zain et al., 2008; Meyer et al., 2011; Meyer, 2012; Vun et al., 2011). Research on population genetics using a molecular technique is essential to determine the genetic diversity of P. chrysomelas in Sarawak. Indeed, the genetic data of both P. chrysomelas subspecies is critical for resolving its taxonomic classification. 5. Positional behavior Data on positional behavior is needed to specify the locomotors and postures used in response to habitat structure. It is also essential to acquire a thorough understanding of the mechanisms that link ecology, morphology, and behavior (Saunders et al., 2018). 6. Metagenomic Metagenomic studies of P. chrysomelas in Sarawak have not been done before. Conclusion This study contributes to the current knowledge of P. chrysomelas in Sarawak. Despite being listed as Totally Protected by Sarawak's Wildlife Protection Ordinance (WPO) and Critically Endangered on the IUCN Red List, this species remains underappreciated. Substantial research efforts are required to enable the implementation of timely and comprehensive conservation actions aimed at preventing the extinction of this species. Research recommendations Metagenomic study based on fecal samples permits the assessment of gut microbial species (Srivathsan et al., 2016) and the identification of the ecommended potential future study in resolving the research gap for P. chrysomelas in Table 3. Recommended potential future study in resolving the research gap for P. chry Sarawak. 276 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) species of plant diet consumed via the DNA metabarcoding method (Osman et al., 2020). 7. Mitogenomic P. chrysomelas has never been the focus subject of mitogenomic research. Genomic data collected from noninvasive fecal samples is a tool for conservation genomics (Taylor et al., 2021). This information can settle the taxonomic disputes between subspecies of P. chrysomelas, which may be different species due to their diverse fur colourations. 8. Conservation management Effective conservation management focusing on P. chrysomelas species is needed. A continuous monitoring program emphasizing ecological criteria may be used during the conservation planning. This action could be a collaboration between local researchers with local authorities (Sarawak Forestry Corporation and Forest Department Sarawak) and international authorities (Brunei and Indonesia) to conserve this neglected transboundary species. 9. Threats The threat to this species is limited to the study conducted by Ampeng (2006) and Smith (2015). Hence, in-depth research on the threats is crucial in the work of revising the Wild Life Protection Ordinance (WPO) 1998. 276 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) 276 \| Journal of Wildlife and Biodiversity 7(Supplementary issue): 28-45 (2023) References Abdul-Latiff, M. A. B., Baharuddin, H., Abdul-Patah, P., & Md-Zain, B. M. 2019. Is Malaysia’s banded langur, Presbytis femoralis femoralis, actually Presbytis neglectus neglectus? 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https://openalex.org/W4282947158	https://www.proceedings.blucher.com.br/download-pdf/370/37386	Portuguese	null	Blucher Design Proceedings	null	2,022	cc-by	1,126	Actions of the Laboratory of Ergonomics and Interfaces during the Covid-19 pandemic and perspectives for future studies Email: luis.paschoarelli@unesp.br; http://lattes.cnpq.br/8521603444193259. O Mundo enfrenta a pandemia de Covid-19 (2019-nCoV), que é causada pelo Coronavírus da Síndrome Respiratória Aguda Grave 2 (SARS-CoV-2). A condição de pandemia foi declarada em Março de 2020 pela Organização Mundial de Saúde, e no final de Dezembro de 2021, a OMS contava com mais de 279.000.000 casos confirmados e 5.400.000 mortes confirmadas em todo o mundo (Organização Mundial de Saúde 2021). A transmissão do SARS-CoV-2 ocorre principalmente através de gotículas respiratórias que transportam partículas virais que podem ser por fluxo de ar (Tysome e Bhutta 2020; Lee e Hsueh 2020; Lentz e Colt 2020); ou através de aerossóis, especialmente entre doentes e profissionais de saúde (Boswell e Longstaff 2019; Organização Mundial de Saúde 2020), por exemplo, durante a intubação e extubação traqueal, ou procedimentos odontológicos. A utilização de Equipamento de Protecção Individual (EPI) é essencial para controlar a transmissão durante o contacto com doentes infectados (Cook 2020; Livingston, Desai e Berkwits 2020). Os Protetores Faciais são EPIs que complementam a proteção dos olhos, nariz e boca (Lindsley et al. 2014; Pearce 2020). Por conseguinte, é importante que os EPIs, especialmente os Face Shields (FS), tenham características que protejam mais eficazmente os profissionais de saúde (Perencevich, Diekema e Edmondt 2020). A aquisição de Equipamento de Proteção Individual como o Face Shields (FS) foi uma prioridade para os serviços de saúde para os procedimentos de tratamento de doentes em hospitais. A procura mundial mobilizou equipes em diferentes contextos para produzir alternativas que respondessem às necessidades dos profissionais de saúde no controle da transmissão da SARS-CoV-2. Tirando partido desta situação, a equipe do Laboratório de Ergonomia e Interface foi contactada por profissionais médicos e sanitários locais, para desenvolver novas alternativas 1 18⁰ ERGODESIGN & USIHC 2022 (evento on-line) – 7 a 10 de março de 2022 – Campina Grande/PB 1 Campina Grande, 7 a 10 de março Campina Grande, 7 a 10 de março de FS. Uma vez que, de acordo com os profissionais de saúde, os modelos disponíveis encontrados na web não satisfazem plenamente todos os requisitos para essa situação particular. Portanto, o objetivo foi desenvolver um FS baseado nos princípios do Design Centrado no Utilizador (UCD), atendendo à regulamentação brasileira (RDC-356/2020 - Agência Nacional de Vigilância Sanitária. 2020), e que pudesse ser aplicado na frente de batalha da Pandemia da Covid-19 para suprir a urgência da situação. Actions of the Laboratory of Ergonomics and Interfaces during the Covid-19 pandemic and perspectives for future studies O projeto foi conduzido desde o início até ao desenvolvimento do produto final e à distribuição aos profissionais de saúde em 18 dias. Foram desenvolvidos dois diferentes modelos de FS: fabricados manualmente e impressos em 3D. Participaram a equipe da área do Design (representados pelos integrantes do Laboratório de Ergonomia e Interfaces); a equipe da área da Engenharia (representados pela equipe do SENAI-Bauru); e a equipe da área da Saúde (Secretaria Municipal de Saúde - Prefeitura Municipal de Bauru). O feedback da equipe da área de Saúde salientou que o equipamento desenvolvido é seguro de utilizar durante os cuidados a pessoas que podem transmitir o SARS-CoV-2. Esta experiência proporcionou reflexões sobre os novos projetos desenvolvidos no Laboratório de Ergonomia e Interfaces, com destaque para os desafios e limitações em estudos laboratoriais; os cuidados éticos e o desenvolvimento de projetos de pesquisa transdisciplinares. Agradecimentos Este estudo recebeu apoio da Justiça Federal - Seção Judiciária de São Paulo (Processo 5687435/2020-BAUR-01V; e Processo SEI nº 0008659-31.2020.4.03.8001). Referências Agência Nacional de Vigilância Sanitária. 2020b. Resolução - RDC Nº 356 de 23 de março de 2020 [Resolution - RDC Nº 356 of March 23, 2020]. Brasilia, 56-C:1. Acessado em 14 de julho de 2020. Agência Nacional de Vigilância Sanitária. 2020b. Resolução - RDC Nº 356 de 23 de março de 2020 [Resolution - RDC Nº 356 of March 23, 2020]. Brasilia, 56-C:1. 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https://openalex.org/W2615757040	https://europepmc.org/articles/pmc5491777?pdf=render	English	null	Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an α,β-unsaturated thioester	Journal of biological chemistry/The Journal of biological chemistry	2,017	cc-by	10,932	Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an ,-unsaturated thioester Received for publication,April 29, 2017, and in revised form, May 11, 2017 Published, Papers in Press,May 18, 2017, DOI 10.1074/j Taichi Chisuga‡, X Akimasa Miyanaga§, Fumitaka Kudo§, and X Tadashi Eguchi‡§1 From the ‡Department of Chemistry and Materials Science and §Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan Taichi Chisuga‡, X Akimasa Miyanaga§, Fumitaka Kudo§, and X Tadashi Eguchi‡§1 From the ‡Department of Chemistry and Materials Science and §Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan Edited by Wolfgang Peti Thioesterases catalyze hydrolysis of acyl thioesters to release carboxylic acid or macrocyclization to produce the corresponding macrocycle in the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Recently, we reported that the thioesterase CmiS1 from Streptomyces sp. MJ635- 86F5 catalyzes the Michael addition of glycine to an ,-un- saturated fatty acyl thioester followed by thioester hydrolysis in the biosynthesis of the macrolactam antibiotic cremimy- cin. However, the molecular mechanisms of CmiS1-catalyzed reactions are unclear. Here, we report on the functional and structural characterization of the CmiS1 homolog SAV606 from Streptomyces avermitilis MA-4680. In vitro analysis indicated that SAV606 catalyzes the Michael addition of gly- cine to crotonic acid thioester and subsequent hydrolysis yielding (R)-N-carboxymethyl-3-aminobutyric acid. We also determined the crystal structures of SAV606 both in ligand- free form at 2.4 Å resolution and in complex with (R)-N-car- boxymethyl-3-aminobutyric acid at 2.0 Å resolution. We found that SAV606 adopts an / hotdog fold and has an active site at the dimeric interface. Examining the complexed structure, we noted that the substrate-binding loop compris- ing Tyr-53–Asn-61 recognizes the glycine moiety of (R)-N- carboxymethyl-3-aminobutyric acid. Moreover, we found that SAV606 does not contain an acidic residue at the active site, which is distinct from canonical hotdog thioesterases. Site-directed mutagenesis experiments revealed that His-59 plays a crucial role in both the Michael addition and hydro- lysis via a water molecule. These results allow us to propose the reaction mechanism of the SAV606-catalyzed Michael addition and thioester hydrolysis and provide new insight into the multiple functions of a thioesterase family enzyme. Thioesterases (TEs)2 hydrolyze the thioester bond involved in acyl-acyl carrier protein (ACP) and acyl-CoA to release a carboxylic acid. Alternatively, TEs catalyze the macrocycliza- tion to produce a macrolide or macrolactam. TEs play impor- tant roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides and in the metabolism of lipids (1–4). Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an ,-unsaturated thioester Currently, TEs are classified into 25 families in the ThYme database based on their amino acid sequence, substrate speci- ficity, and tertiary structure (5, 6). Most of them have either an /-hydrolase fold or a hotdog fold. The /-hydrolase TEs, which belong to family 2 and families 16–22, possess a con- served catalytic triad (Ser-His-Asp) in the active site (2, 5). The reaction is initiated by the nucleophilic attack of the Ser residue on the carbonyl carbon of the acyl thioester substrate followed by hydrolysis or macrocyclization of the resulting ester inter- mediate. The /-hydrolase TEs release final products or remove aberrant non-extendable units from ACP in polyketide and nonribosomal peptide biosynthesis (2, 3). The hotdog TEs, which belong to families 4–15 and 24, are structurally and evolutionarily related to a family of dehydrata- ses (DHs) (5, 7). They commonly hydrolyze the thioester bond of the acyl-CoA molecule, but some of them act on acyl-ACP. They exist either as a homodimer of a single hotdog fold or as a monomer of a double hotdog fold with an active site located at the interface between the two hotdogs. They normally do not contain the catalytic His residue that is fully conserved in DHs (7). They mostly possess an acidic residue (Glu/Asp) as a nucleophile for generating a covalent anhydride intermediate or general base for activating a water molecule in catalysis in the active site (5, 7). However, some of them (e.g. families 15 and 24) do not contain any acidic residue in the active site. The family 15 TE CalE7, which is involved in the biosynthesis of cali- cheamicin, is proposed to use an Arg residue to activate the thioester carbonyl and stabilize the oxyanion intermediate in the reaction (8). The family 24 TE Rv0098, which catalyzes the hydrolysis of a long-chain fatty acyl-CoA in Mycobacterium tuberculosis, is hypothesized to use two Tyr residues and a Asn residue for activation of the thioester carbonyl and stabilization This work was performed with the approval of the Photon Factory Program Advisory Committee (Proposals 2014G530 and 2016G624) and was sup- ported in part by Ministry of Education, Culture, Sports, Science and Tech- nology Grants-in-Aid for Scientific Research in Innovative Areas 16H06451 (toT. E.).Theauthorsdeclarethattheyhavenoconflictsofinterestwiththe contents of this article. The atomic coordinates and structure factors (codes 5WSX and 5WSY) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1 To whom correspondence should be addressed: Dept. 2 The abbreviations used are: TE, thioesterase; ACP, acyl carrier protein; CMABA, N-carboxymethyl-3-aminobutyric acid; NAC, N-acetylcysteamine; CMABA-NAC, N-carboxymethyl-3-aminobutyryl-N-acetylcysteamine; DH, dehydratase; ESI-MS, electrospray ionization mass spectrometry; HR-FAB- MS, high-resolution fast atom bombardment mass spectrometry; r.m.s.d., root mean square deviation; Boc, t-butoxycarbonyl. cros cros ARTICLE Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an ,-unsaturated thioester of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan. Tel./Fax: 81-3-5734-2631; E-mail: eguchi@chem.titech.ac.jp. Functional analysis of SAV606 -Amino acids are frequently found as important compo- nents of natural products such as macrolactam polyketide anti- biotics (10, 11). These -amino acids are typically derived from proteinogenic -amino acids. However, some of the -amino acids are constructed through the polyketide pathway. In the biosynthesis of the macrolactam antibiotic cremimycin, the starter unit 3-aminononanoic acid is synthesized from non-2- enoyl-ACP and glycine (12). Initially, CmiS1, which belongs to the family 24 TEs, catalyzes the Michael addition of glycine to the -position of the non-2-enoyl-ACP and subsequent hy- drolysis of the thioester to give N-carboxymethyl-3-amino- nonanoic acid (Fig. 1A). N-Carboxymethyl-3-aminonon- anoic acid is then converted to 3-aminononanoic acid by a FAD-dependent oxidase, CmiS2. Homologous genes to cmiS1 and cmiS2 are conserved in the biosynthetic gene clusters for related macrolactam polyketides such as hero- namide, BE-14106, and ML-449, all of which possess a -a- mino medium-chain fatty acid unit at the starter position of their polyketide backbone (13–15), suggesting that the amino groups of these -amino acids are introduced through the same mechanism. To investigate the function of SAV606, we expressed recom- binant SAV606 protein in Escherichia coli. Because CmiS1 cat- alyzes the Michael addition of glycine to non-2-enoyl-ACP and the subsequent hydrolysis of the thioester (12), we speculated that SAV606 catalyzes a similar reaction. We first examined the SAV606 reaction with various lengths of ,-unsaturated fatty acyl N-acetylcysteamine (NAC) in the presence of glycine. We detected the release of NAC with Ellman’s reagent (17) in the reaction with crotonoyl-NAC and glycine (specific activity: 3.3 units/mg, relative activity: 100%), whereas we were not able to observe the release of NAC in the absence of glycine (0.05%). We did not detect the release of NAC in the reaction with butyryl-NAC in the presence of glycine (0.05%). Thus, the substrate of SAV606 must be ,-unsaturated fatty acid thio- esters. These results suggest that SAV606 requires glycine for its reaction before the hydrolysis of the thioester, as is the case for the CmiS1 reaction. Release of NAC was not detected from the reaction with non-2-enoyl-NAC and glycine (0.05%), sug- gesting that SAV606 has a different substrate specificity from CmiS1 and prefers a shorter chain ,-unsaturated fatty acid thioester as substrate. CmiS1 is an exceptionally rare TE because it performs a dual function consisting of the Michael addition of glycine and the hydrolysis of a thioester. 10926 J. Biol. Chem. (2017) 292(26) 10926–10937 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. A thioesterase catalyzing a Michael addition A B S O ACP CmiS1 OH O H2N glycine O OH NH O HO S O ACP NH HO O non-2-enoyl-ACP HS ACP S O OH O H2N glycine O OH NH O HO O NH HO O crotonoyl-NAC SAV606 S SAV606 CmiS1 CMABA-NAC (R)-CMABA H N O HS H N O H N O Figure 1. Reactions of CmiS1 (A) and SAV606 (B). O OH NH O HO HS ACP CmiS1 A CmiS1 B Figure 1. Reactions of CmiS1 (A) and SAV606 (B). of the oxyanion intermediate, although the reaction mecha- nism is still unclear (9). Functional analysis of SAV606 Although CmiS1 belongs to the same family as Rv0098, whose crystal structure is available, the reported function of Rv0098 is only the hydrolysis of a long- chain fatty acyl-CoA (9). Thus, the reported functions of CmiS1 and Rv0098 are quite different. It is unclear how CmiS1 cata- lyzes the Michael addition of glycine to an ,-unsaturated thioester. CmiS1 homologs are distributed not only in macro- lactam-producing strains but also in other bacteria. For exam- ple, the protein SAV606 from Streptomyces avermitilis MA-4680 (16) shows 51% amino acid sequence identity with CmiS1. Here, we describe the functional characterization and structural determination of SAV606. We find that SAV606 cat- alyzes the Michael addition of glycine to crotonic acid thioester and subsequent hydrolysis (Fig. 1B). The structure of SAV606 in complex with the product molecule allows us to propose the mechanism of the Michael addition of glycine to an ,-unsat- urated thioester. We also analyzed the reaction product by HPLC after derivatization with phenyl isothiocyanate. We observed the accumulation of product derivative from the reaction mixture containing both crotonoyl-NAC and glycine (Fig. 2A). We did not observe the accumulation of product derivative when we used crotonic acid as a substrate instead of crotonoyl-NAC, suggesting that the thioester moiety of an ,-unsaturated fatty acyl substrate is required for the reaction. To confirm the chemical structure of the reaction product formed by SAV606 from crotonoyl-NAC and glycine, we car- ried out a large-scale enzymatic reaction. After esterification of the enzyme reaction product with ethanol in acidic conditions, the chemical structure of the resulting ethyl ester was analyzed by NMR and MS. High-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) (positive mode) showed a pseudo-molecular ion peak with m/z 218.1381, which corre- sponds to [MH] of the molecular formula C10H19NO4, indi- cating the expected diethyl ester of N-carboxymethyl-3-ami- J. Biol. Chem. (2017) 292(26) 10926–10937 10927 Table 1 Steady-state kinetic parameters of SAV606 ND, not detected. CMABA-NAC were determined to be 23 min1 and 0.80 mM, ti l Th SAV606 lik l t l th Mi h l ddi Steady-state kinetic parameters of SAV606 ND, not detected. Substrate Km kcat kcat/Km mM min1 min1mM1 Crotonoyl-NAC 0.74 0.14 92 6 120 Glycine 26 5 91 6 3.5 Hex-2-enoyl-NAC 6.6 0.4 17 0.4 2.6 Glycine 120 10 16 1 0.13 Non-2-enoyl-NAC ND ND ND Glycine ND ND ND Substrate specificity of SAV606 To analyze the acyl thioester substrate preference of SAV606, we carried out a kinetic analysis. The kcat and Km val- ues of SAV606 for crotonoyl-NAC were found to be 92 min1 and 0.74 mM (Table 1), respectively, which are comparable with those of CmiS1 for non-2-enoyl-NAC (12). Although SAV606 also shows activity toward hex-2-enoyl-NAC, the Km value was nine times higher than that for crotonoyl-NAC. Next, we ana- lyzed the amino donor preference of SAV606. The Km value of SAV606 for glycine was 26 mM. SAV606 also showed activity toward L-alanine (16% of the activity toward glycine), whereas the enzyme had no activity toward other amino acids such as -alanine, L-valine, and L-serine. CMABA-NAC were determined to be 23 min1 and 0.80 mM, respectively. Thus, SAV606 likely catalyzes the Michael addi- tion of glycine to the -position of the crotonic acid thioester followed by hydrolysis of the thioester to produce (R)-CMABA (Fig. 1B). A thioesterase catalyzing a Michael addition nobutyric acid (CMABA). 1H NMR and 1H,1H COSY showed a contiguous 1H spin system from H2 to H4 of the 3-aminobu- tyrate core structure (Fig. 3). One methylene with an AB-type signal at 3.4 ppm in the 1H NMR spectrum is isolated from the spin system, indicating that the resulting ethyl ester is N-ethoxycarbonylmethyl-3-aminobutyric acid ethyl ester, sug- gesting that the SAV606 reaction product is CMABA. We also synthesized the authentic compound from 3-aminobutyric acid by esterification with ethanol followed by alkylation with ethyl bromoacetate to confirm the structure. To determine the ste- reochemistry at the C3 position of the product CMABA, we derivatized the SAV606 product with phenyl isothiocyanate and then analyzed the resulting 3-phenylthiohydantoin-butyric acid by HPLC with a chiral column. By comparing the retention time of the derivative with those of the R- enantiomer of 3-phe- nylthiohydantoin-butyric acid and its racemic form, the stere- ochemistry at position C3 was determined to be R- (Fig. 2B). Figure 3. 1H NMR spectrum of SAV606 product ethyl ester. A thioesterase catalyzing a Michael addition B enzymatic product authentic racemic CMABA authentic (R)-CMABA 6 10 8 retention time (min) 5 7 9 Au 0.0 0.2 0.4 0.6 A B enzymatic product authentic racemic CMABA authentic (R)-CMABA synthetic CMABA without SAV606 without glycine without crotonoyl-NAC SAV606 reaction 6 10 crotonic acid instead of crotonoyl-NAC 8 retention time (min) retention time (min) 10 12 14 16 18 0.0 0.5 1.0 1.5 Au 5 7 9 Au 0.0 0.2 0.4 0.6 Figure 2. HPLC analysis of phenylthiocyanate derivatives of the product of SAV606 reactions with crotonoyl-NAC and glycine as substrates. PEGASIL ODS SP100 (A) and CHIRAL ART Cellulose-SC (B) columns were used for analysis. HPLC traces (detection at 268 nm) are shown. Au, absorbance units. A synthetic CMABA without SAV606 without glycine without crotonoyl-NAC SAV606 reaction crotonic acid instead of crotonoyl-NAC retention time (min) 10 12 14 16 18 0.0 0.5 1.0 1.5 Au Figure 2. HPLC analysis of phenylthiocyanate derivatives of the product of SAV60 Figure 2. HPLC analysis of phenylthiocyanate derivatives of the product of SAV606 reactions with crotonoyl-NAC and glycine as substrates. PEGASIL ODS SP100 (A) and CHIRAL ART Cellulose-SC (B) columns were used for analysis. HPLC traces (detection at 268 nm) are shown. Au, absorbance units. 1.0 2.0 3.0 4.0 5.0 6.0 7.0 (ppm) 0.0 O O NH O O 2 2 3 4 4 3 1’ 1’ Et Et Et Et Et Et Figure 3. 1H NMR spectrum of SAV606 product ethyl ester. nobutyric acid (CMABA). 1H NMR and 1H,1H COSY showed a contiguous 1H spin system from H2 to H4 of the 3-aminobu- tyrate core structure (Fig. 3). One methylene with an AB-type signal at 3.4 ppm in the 1H NMR spectrum is isolated from the spin system, indicating that the resulting ethyl ester is N-ethoxycarbonylmethyl-3-aminobutyric acid ethyl ester, sug- gesting that the SAV606 reaction product is CMABA. We also synthesized the authentic compound from 3-aminobutyric acid by esterification with ethanol followed by alkylation with ethyl bromoacetate to confirm the structure. To determine the ste- reochemistry at the C3 position of the product CMABA, we derivatized the SAV606 product with phenyl isothiocyanate and then analyzed the resulting 3-phenylthiohydantoin-butyric acid by HPLC with a chiral column. By comparing the retention time of the derivative with those of the R- enantiomer of 3-phe- nylthiohydantoin-butyric acid and its racemic form, the stere- ochemistry at position C3 was determined to be R- (Fig. 2B). A thioesterase catalyzing a Michael addition Substrate-binding pocket SAV606 has a long tunnel that extends about 14 Å from the surface to the bottom of the substrate-binding pocket (Fig. 4B). The location of the substrate-binding pocket of SAV606 is almost identical to those of other characterized hot dog-type TEs such as Rv0098. The substrate-binding pocket of SAV606 is mainly constituted of 10 residues that include Tyr-53, Ile-54, His-59, Asn-61, and Ser-62 from one subunit and Tyr-20, Asn- 70, Met-73, Tyr-74, and Ile-107 from the adjacent subunit (Fig. 4D). In contrast to the canonical hot dog TEs, SAV606 contains no acidic Asp/Glu residue in the active site. The binding of CMABA seemed to induce the movement of some amino acid residues in the substrate-binding pocket. The side chains of His-59, Asn-61, and Tyr-74 rotate to accommodate the CMABA molecule (Fig. 4, C and E). In addition, Tyr-20 and Ile-54 shifts 1 Å to be located closer to the glycine moiety of CMABA. hotdog fold that contains a five-stranded antiparallel -sheet wrapping around one long central helix and two short helices (Fig. 4A). The dimeric structure and / hotdog topology are common in TEs and DHs. The structure contains a few disor- dered loop regions (Asp-56–Gly-58 and Ala-138–Gly-139). The overall structure of SAV606 is similar to that of Rv0098 (PDB code 2PFC; Z-score 22.0, r.m.s.d. of 1.8 Å, sequence identity of 34%) (9) and that of the family 11 TE YbdB from E. coli (PDB code 4K4C; Z-score 10.9, r.m.s.d. of 2.5 Å, sequence identity of 13%) (18). SAV606 is also structurally similar to DHs such as FabZ from Pseudomonas aeruginosa (PDB code 1U1Z; Z-score 13.0, r.m.s.d. of 2.8 Å, sequence identity of 15%) (19) and FabA from P. aeruginosa (PDB code 4B0I; Z-score 10.7, r.m.s.d. of 3.1 Å, sequence identity of 10%) (20), both of which catalyze the dehydration of (R)-3-hydroxyacyl-ACP. The carboxyl group of the glycine moiety is recognized by Tyr-20 (2.7 Å), Tyr-53 (2.7 Å), and Asn-70 (3.0 Å) (Fig. 4D). The amino group of CMABA is sandwiched between Asn-61 (3.3 Å) and Tyr-74 (3.6 Å). Met-73 forms a hydrophobic contact (3.9 Å) with the terminal methyl group of the butyric acid moiety. Ile-54 and Ile-107 show hydrophobic interactions with the methylene group of the glycine moiety (3.7 Å) and the meth- ylene group of the butyric acid moiety (4.1 Å), respectively. A thioesterase catalyzing a Michael addition Table 2 Structural data collection and refinement statistics PDB ID Datasets SAV606 SAV606-CMABA complex 5WSX 5WSY Data collection Beamline PF BL-5A PF BL-5A Wavelength (Å) 1.00000 1.00000 Space group P212121 C2221 Unit-cell parameters a (Å) 49.88 110.49 b (Å) 50.10 118.31 c (Å) 125.01 71.42 Resolution (Å) 50.00-2.40 50.00-2.00 (outer shell) (2.52-2.40) (2.03-2.00) Unique reflections 12,859 (1,828) 32,063 (1,578) Redundancy 6.5 (6.7) 6.5 (5.7) Completeness (%) 99.9 (99.9) 100.0 (99.6) Rmerge (%) 13.7 (57.8) 9.1 (55.6) Mean I/(I) 10.8 (2.3) 21.4 (2.4) Refinement Rwork (%) 20.5 17.8 Rfree (%) 26.1 21.5 r.m.s.d. Bond lengths (Å) 0.013 0.018 Bond angles (degree) 1.597 1.861 No. of non-hydrogen atoms Protein 2,540 2,616 Product 0 22 Solvent 67 136 Average B-factors (Å2) Protein 40.6 34.4 Product 27.4 Solvent 39.6 38.9 Ramachandran plot Favored region (%) 96.0 97.8 Allowed region (%) 4.0 2.2 Outer region (%) 0.0 0.0 Table 2 Structural data collection and refinement statistics Table 2 Table 2 comprising Tyr-53–Asn-61, which is located before the start of the long central helix (helix 2: Ser-62–Glu-83), shows an ordered structure. In addition, the helix 3 region comprising Leu-94–Pro-103 rotates 20° toward the substrate-binding loop (Tyr-53–Asn-61) from the neighboring subunit in the complex structure. Leu-102 moves 6 Å toward the substrate-binding loop from the neighboring subunit and forms hydrophobic contacts with Ile-54 (3.6 Å) and Thr-57 (4.0 Å) (Fig. 4C). Con- sequently, the substrate-binding loop and the helix 3 region cover the internal substrate-binding pocket in the complex structure, suggesting that they might act as the lid of the sub- strate-binding pocket. Analysis of SAV606 reaction with N-carboxymethyl-3-aminobutyryl-NAC We determined the crystal structure of apo SAV606 at 2.4 Å resolution (Table 2). There is one SAV606 dimer per crystallo- graphic asymmetric unit. The dimeric interface buries a surface area of 1052 Å2 per monomer (12% of the total monomer sur- face), indicating that the contact area is large enough for the protein to exist as a dimer. Gel filtration chromatography anal- ysis also showed that SAV606 exists as a homodimer in solution (data not shown). The SAV606 monomer shows a typical / To confirm the reaction pathway, we chemically synthesized racemic N-carboxymethyl-3-aminobutyryl-NAC (CMABA- NAC) as a likely reaction intermediate and examined the reac- tion of SAV606 with it. We detected the release of NAC with Ellman’s reagent, as observed in the reaction with crotonoyl- NAC and glycine. The kcat and Km values of SAV606 for racemic 10928 J. Biol. Chem. (2017) 292(26) 10926–10937 Substrate-binding pocket The side-chain hydroxy group and the backbone amide group of Ser-62 and the side-chain amide group of Asn-70 form hydrogen bonds (2.5 Å, 3.2 Å, and 3.2 Å, respectively) with the carboxyl group of the butyric acid moiety. The CMABA molecule is bent through these tight interactions in the substrate-binding tunnel (Fig. 4B). The two carboxyl groups of CMABA are close to each other (3.3 Å). His-59 is positioned near the carboxyl group of the butyric acid moi- ety, although this residue makes no direct hydrogen bond with the CMABA molecule. His-59 retains a water molecule in the substrate-binding pocket through a hydrogen bond (3.2 Å) (Fig. 4, B and D). This water molecule forms a hydro- gen bond (2.9 Å) with the amino group of the glycine moiety and is located close (3.6 Å) to the -carbon of the butyric acid moiety. Thus, this water molecule seems to be the pro- ton source in the Michael addition step and might also nucleophilically attack the thioester moiety of the reaction intermediate for the hydrolysis. Complex structure of SAV606 with (R)-CMABA To better understand the reaction mechanism of SAV606, we attempted to crystallize SAV606 with glycine, crotonoyl- NAC, or racemic CMABA. We succeeded in the determination of the complex structure with CMABA at 2.0 Å resolution (Table 2 and Fig. 4A). The structure shows clear electron den- sity for (R)-CMABA but not for (S)-CMABA in the substrate- binding pocket, which is located at the dimeric interface (Fig. 4B). The complex structure shows significant ligand-induced conformational changes (r.m.s.d. of 1.30 Å for C atoms of chain A). In the complex structure, the substrate-binding loop 10929 J. Biol. Chem. (2017) 292(26) 10926–10937 10929 Mutational study H59Q mutants were not able to catalyze the hydrolysis of CMABA-NAC (Table 3), suggesting that His-59 mutation also disrupted the hydrolysis of the thioester. To identify the catalytically important residues for the Michael addition and hydrolysis reactions, we mutated the seven polar residues in the active site and analyzed the activity of these variants toward crotonoyl-NAC in the presence of gly- cine by detecting the release of NAC. Y20F, Y53F, N61A, and N70A mutants showed significantly reduced activity compared with the wild type (Table 3). H59A, H59N, and H59Q mutants completely lost activity. These results suggest that these five residues are important for the reaction. In contrast, Ser-62 and Tyr-74 are not critical for catalysis, because S62A and Y74F mutants retained activity (74 and 66% of the wild type, respec- tively). We further investigated the role of residue His-59 because only the His-59 mutants completely lost activity. To determine which reaction step is affected by the mutation of His-59, we carried out HPLC analysis to investigate whether the crotonoyl-NAC substrate was consumed by the H59A, H59N, and H59Q mutants. We were not able to detect the consump- tion of crotonoyl-NAC or the formation of the CMABA-NAC intermediate from the reaction of these mutants (Fig. 5), sug- gesting that the His-59 mutation impaired the first (i.e. the Michael addition) step. In addition, the H59A, H59N, and The seven polar residues in the active site are fully conserved in CmiS1 (Fig. 6A). In addition, these residues are also con- served in HerU, BecU, and MlaU, which are supposed to be responsible for the biosynthesis of three-amino medium-chain fatty acids in the heronamide, BE-14106, and ML-449 biosyn- thetic pathways, respectively (13–15). This suggests that these enzymes share the same mechanism as SAV606 to produce N-carboxymethyl-3-amino fatty acids. Interestingly, Met-73 in SAV606 is replaced by Ala in CmiS1 (Fig. 6A). This substitution might be responsible for the difference in the substrate speci- ficity between SAV606 and CmiS1, as judged from the struc- tural observation that SAV606 Met-73 interacts with the termi- nal methyl group of CMABA. A thioesterase catalyzing a Michael addition B Ile54 Tyr20 Tyr74 Met73 Ile107 Tyr53 Thr57 His59 His59 Ile54 Ile54 Asn70 Ser62 Asn61 His59 Leu102 Leu102 His59 Met73 Tyr74 Ile54 Tyr53 Tyr20 Asn61 A Asn70 apo complex His59 Tyr20 C E D Tyr53 Met73 CMABA CMABA CMABA water CMABA 2.7 2.7 3.2 2.9 Figure 4. SAV606 structures. A, overall structures of apo SAV606 (orange and magenta) and the complex of SAV606 (cyan and green) with CMABA. The (R)-CMABA molecule is shown as yellow sticks. B, surface representation of the substrate-binding pocket in the complex structure. An Fo Fc electron density map contoured at 3.0 was constructed before incorporation of the (R)-CMABA molecule. The water molecule that interacts with His-59 is shown as a red sphere. C, structural comparison of loop regions between the apo and complex structures. The movements of His-59 and Leu-102 are shown as broken arrows. D, the substrate-binding pocket of the complex structure. Ten residues involved in product recognition are shown as sticks. E, structural comparison of the active site between the apo and complex structures. Thr57 His59 His59 Ile54 Ile54 Leu102 Leu102 C CMABA complex A apo A C His59 Met73 Tyr74 Ile54 Tyr53 Tyr20 Asn61 Asn70 E CMABA Ile54 Tyr20 Tyr74 Met73 Ile107 Tyr53 Asn70 Ser62 Asn61 His59 D CMABA 2.7 2.7 3.2 2.9 E B His59 Tyr20 Tyr53 Met73 water CMABA D B His59 Figure 4. SAV606 structures. A, overall structures of apo SAV606 (orange and magenta) and the complex of SAV606 (cyan and green) with CMABA. The (R)-CMABA molecule is shown as yellow sticks. B, surface representation of the substrate-binding pocket in the complex structure. An Fo Fc electron density map contoured at 3.0 was constructed before incorporation of the (R)-CMABA molecule. The water molecule that interacts with His-59 is shown as a red sphere. C, structural comparison of loop regions between the apo and complex structures. The movements of His-59 and Leu-102 are shown as broken arrows. D, the substrate-binding pocket of the complex structure. Ten residues involved in product recognition are shown as sticks. E, structural comparison of the active site between the apo and complex structures. Table 3 tion by forming hydrogen bonds. The ammonium ion of the glycine is likely to be deprotonated by His-59 via the water molecule before the Michael addition. Then, the - position of the butyric acid moiety is protonated by His-59 through the water molecule to produce the CMABA thioester intermediate. His-59 holds the water molecule that is close to both the amino group of the glycine moiety (3.2 Å) and the -carbon of the butyric acid moiety (3.6 Å) of the CMABA in the complex struc- ture, supporting this proton-shuttling mechanism. The side- chain amide group of Asn-70 and the main-chain amide group of Ser-62 likely stabilize the oxyanion intermediate. In the sec- ond step, SAV606 catalyzes the hydrolysis of the CMABA thio- ester intermediate. His-59 might act as a base and orient the water nucleophile for attack at the carbonyl carbon of the inter- mediate. The presence of the introduced glycine moiety of the intermediate seems to be necessary for the hydrolysis step because SAV606 did not catalyze the hydrolysis of crotonoyl- NAC in the absence of glycine or butyryl-NAC in the presence of glycine. The substrate-binding loop (Tyr-53–Asn-61) inter- acts with the glycine moiety but not with the butyryl moiety in the complex structure. This implies the presence of the glycine moiety triggers the closure of the substrate-binding loop, which is crucial for the correct placement of His-59 for hydrolysis. In addition, the carboxyl group of the glycine moiety might assist in the hydrolysis of the thioester by activating the carbonyl group for nucleophilic attack, judging by the short distance (3.3 Å) between the two carboxyl groups of CMABA in the complex structure. Relative activity of SAV606 mutants Relative activity of SAV606 mutants ND t d t t d synthetic CMABA-NAC crotonoyl-NAC authentic wild-type H59A mutant H59N mutant H59Q mutant 6 retention time (min) no enzyme Au 0.0 0.5 1.0 1.5 8 10 12 14 16 18 20 22 24 2.0 Figure 5. HPLC analysis of the reaction of SAV606 His-59 mutants with crotonoyl-NAC and glycine. HPLC traces (detection at 250 nm) are shown. Au, absorbance units. y ND, not detected. Table 3 SAV606 Relative activity with crotonoyl-NAC and glycine Relative activity with CMABA-NAC % % Wild type 100 1 100 7 Y20F 2.6 0.1 5.3 0.1 Y53F 1.3 0.1 1.8 0.6 H59A ND ND H59N ND ND H59Q ND ND N61A 1.3 0.1 ND S62A 74 4 102 5 N70A 5.4 0.4 15 0.2 Y74F 67 6 38 5 Structural comparison between SAV606 and Rv0098 revealed that Rv0098 shares all the amino acid residues impor- tant for the reaction with SAV606. Tyr-33, Tyr-66, His-72, Asn- 74, Asn-83, and Tyr-87 in Rv0098 occupy the same positions as Tyr-20, Tyr-53, His-59, Asn-61, Asn-70, and Tyr-74 in SAV606 (Fig. 6B). Rv0098 has Ala-75 at the position corresponding to Ser-62 in SAV606. We assume that this substitution does not affect the catalysis because the SAV606 S62A mutant retained its activity. Thus, Rv0098 has the essentially same architecture of the active site as SAV606, although the reported function is different. Taken together with the fact that the reported hydro- lytic activity of Rv0098 for long-chain acyl-CoA is 102-104-fold less than those of other TEs (9), Rv0098 might require the Michael addition of glycine or other small amino acid(s) to an ,-unsaturated fatty acid thioester before the hydrolysis of the thioester. Met-73 in SAV606 is substituted by Ala-86 in Rv0098. The small side chain of Ala-86 allows the acyl-binding tunnel to extend over the position of Ala-86 and accommodate a long-chain fatty acyl substrate in the Rv0098 structure (Fig. 6B). This substitution is also observed in CmiS1-type enzymes that utilize a longer acyl substrate than SAV606. retention time (min) Figure 5. HPLC analysis of the reaction of SAV606 His-59 mutants with crotonoyl-NAC and glycine. HPLC traces (detection at 250 nm) are shown. Au, absorbance units. activity in addition to the hydrolytic activity (8, 21–26). In this study we found that SAV606 catalyzes both the Michael addi- tion of glycine to an ,-unsaturated thioester and the hydro- lysis of the thioester, as also observed for CmiS1. We deter- mined the crystal structure of SAV606, revealing the structural basis of the Michael addition of glycine to the acyl substrate by this TE. These findings provide new insights into different cat- alytic functions of TEs. The first Michael addition step of SAV606 is analogous to the reverse reaction of dehydration catalyzed by DHs. Discussion TEs generally catalyze only the hydrolysis of acyl thioesters or macrocyclization (2, 7). However, it was recently reported that some TEs also have protease, peptide ligase, dehydratase, decarboxylase, Claisen cyclase, deacetylase, or transferase 10930 J. Biol. Chem. (2017) 292(26) 10926–10937 10930 Table 3 The water molecule attacks the C3 position of the acyl thioester substrate in the reverse reaction of dehydration, whereas the glycine attacks the C3 position of the crotonic acid thioester in the SAV606 reaction. Because SAV606 shows a moderate struc- tural similarity to DHs such as FabA, we compared the active- site structure of SAV606 with that of FabA. The canonical DH contains a catalytic His-Asp dyad in the active site. In the FabA The complex structure and mutational study allow us to pro- pose the reaction mechanism of SAV606 (Fig. 7). SAV606 first adopts an open conformation so that glycine and crotonic acid thioester would approach the active site. When SAV606 has both glycine and crotonic acid thioester in the active site, the closure of the two hot dogs initiates the Michael addition reac- tion of glycine to the C3 position of the crotonic acid thioester. Tyr-20, Tyr-53, Asn-61, Asn-70, and Tyr-74 orient the glycine molecule in the proper position for the Michael addition reac- J. Biol. Chem. (2017) 292(26) 10926–10937 10931 10931 A thioesterase catalyzing a Michael addition The FabA-ACP complex structure showed that Arg-132, Arg-136, Arg-137, and Lys-161 of FabA are important for recognition of ACP. These FabA basic residues are not conserved in SAV606. The physiological substrate for SAV606 is unclear, although kinetic analysis suggested that crotonic acid thioester is the most likely substrate. SAV606-related metabolites have not been isolated in any culture conditions (29). The gene cluster structure, His-70 from one subunit is placed so as to abstract the proton from the C2 position of the -hydroxy fatty acid thioester intermediate (20, 27). Asp-84 from the other subunit forms a hydrogen bond with the C3 hydroxy group and is pro- posed to protonate the C3 hydroxy group for the -elimination. SAV606 does not have a His-Asp dyad at the corresponding positions and instead has the catalytically important residue His-59 at a different position (Fig. 8). The position of the glycine moiety in the SAV606 structure is also different from that of the C3 hydroxy group of the acyl thioester in the FabA structure. Thus, the position of the molecule (i.e. glycine in the SAV606 reaction and water in the reverse reaction of dehydration) that attacks at the C3 position of the acyl substrate is significantly different between the two structures. We also compared the SAV606 structure with FabA-ACP complex structure (28). The FabA-ACP complex structure showed that Arg-132, Arg-136, Arg-137, and Lys-161 of FabA are important for recognition of ACP. These FabA basic residues are not conserved in SAV606. The physiological substrate for SAV606 is unclear, although kinetic analysis suggested that crotonic acid thioester is the most likely substrate. SAV606-related metabolites have not been isolated in any culture conditions (29). The gene cluster that includes the sav606 gene also contains a nonribosomal peptide synthetase gene, suggesting that this gene cluster is likely responsible for the biosynthesis of a peptide compound. In conclusion, we found that SAV606 from S. avermitilis cat- alyzes the Michael addition of glycine to crotonic acid thioester and the subsequent hydrolysis. We also determined the crystal structure of SAV606 in complex with its product (R)-CMABA. These results develop our understanding of the structure-func- tion relationships of TEs and provide new insight into different catalytic functions of some TEs. A thioesterase catalyzing a Michael addition Tyr20 Tyr33 Tyr53 Tyr66 Asn70 Asn83 Tyr74 Tyr87 His59 His72 Asn61 Asn74 Ser62 Ala75 Ala86 Met73 A B substrate binding loop Figure 6. Structural comparison of SAV606 with related thioesterases. A, amino acid sequence alignment. The secondary structural elements of SAV606 are indicated above the sequence. Residues in the substrate-binding pocket are shown as cyan and green circles. The cyan dotted line above the sequence indicates the substrate-binding loop of SAV606. B, superimposition of the SAV606 (cyan and green) complex structure with the Rv0098 structure (PDB code 2PFC, gray). The ligand molecules of the SAV606 and Rv0098 structures are shown as yellow and dark gray sticks, respectively. A thioesterase catalyzing a Michael addition Tyr20 Tyr33 Tyr53 Tyr66 Asn70 Asn83 His59 His72 Asn61 Asn74 Ser62 Ala75 A B substrate binding loop Tyr20 Tyr33 Tyr53 Tyr66 Asn70 Asn83 Tyr74 Tyr87 His59 His72 Asn61 Asn74 Ser62 Ala75 Ala86 Met73 B Ala86 Met7 Figure 6. Structural comparison of SAV606 with related thioesterases. A, amino acid sequence alignment. The secondary structural elements of SAV606 are indicated above the sequence. Residues in the substrate-binding pocket are shown as cyan and green circles. The cyan dotted line above the sequence indicates the substrate-binding loop of SAV606. B, superimposition of the SAV606 (cyan and green) complex structure with the Rv0098 structure (PDB code 2PFC, gray). The ligand molecules of the SAV606 and Rv0098 structures are shown as yellow and dark gray sticks, respectively. structure, His-70 from one subunit is placed so as to abstract the proton from the C2 position of the -hydroxy fatty acid thioester intermediate (20, 27). Asp-84 from the other subunit forms a hydrogen bond with the C3 hydroxy group and is pro- posed to protonate the C3 hydroxy group for the -elimination. SAV606 does not have a His-Asp dyad at the corresponding positions and instead has the catalytically important residue His-59 at a different position (Fig. 8). The position of the glycine moiety in the SAV606 structure is also different from that of the C3 hydroxy group of the acyl thioester in the FabA structure. Thus, the position of the molecule (i.e. glycine in the SAV606 reaction and water in the reverse reaction of dehydration) that attacks at the C3 position of the acyl substrate is significantly different between the two structures. We also compared the SAV606 structure with FabA-ACP complex structure (28). Preparation of recombinant SAV606 protein The artificial sav606 gene that was optimized for overexpres- sion in E. coli was synthesized by Eurofins Genetics (Tokyo, Japan). This sav606 gene was cloned into the expression vector pCold I (Takara Biochemicals, Ohtsu, Japan) by using the NdeI and XhoI restriction sites to form pCold I-sav606. E. coli BL21 (DE3) cells harboring pCold I-sav606 were grown at 37 °C in Luria-Bertani broth containing ampicillin (50 g/ml). After the optical density at 600 nm reached 0.4, protein expression was 10932 10932 J. Biol. Chem. (2017) 292(26) 10926–10937 OH O OH OH H2N O N NH Tyr53 H2N O Asn70 His59 O N H OH Ser62 NH Asn61 Tyr20 HS H N O S O H N O NH2 O O OH OH H2N O H2N O N NH O H H Tyr20 Tyr53 Asn70 His59 O N H OH Ser62 Asn61 H S O H N O OH OH H2N O O N H OH H2N O HN NH O H H Tyr20 Tyr53 Asn70 His59 Ser62 NH O O Asn61 H S O H N O OH OH H2N O H2N O HN NH Tyr53 Asn70 His59 O N H OH Ser62 NH OH O Asn61 HO Tyr20 S O H N O OH OH H2N O N NH Tyr53 H2N O Asn70 His59 O N H OH Ser62 NH OH O Asn61 O H H Tyr20 S O H N O OH OH H2N O H2N O HN NH Tyr53 Asn70 His59 O N H OH Ser62 NH OH O Asn61 H O H Tyr20 OH O Figure 7. Proposed reaction mechanism of SAV606. A thioesterase catalyzing a Mich A thioesterase catalyzing a Michael addition A thioesterase catalyzing a Michael addition Asn70 Asn70 Tyr20 O Ser62 O Ser62 Asn61 His59 Asn70 Asn70 His59 Figure 7. Proposed reaction mechanism of SAV606. Figure 7. Proposed reaction mechanism of SAV606 His70 Tyr20 His59 Tyr53 Asp84 Asn70 C3 hydroxy group acyl thioester CMABA glycine moiety Figure 8. Superimposition of the SAV606 complex structure (cyan and green) on the P. aeruginosa FabA apo structure (PDB code 4FQ9, gray). The (R)-CMABA molecule of the SAV606 structure is shown as yellow sticks. The complex structure of FabA H70N mutant with 3-hydroxydecanoyl-NAC (PDB code 4B0I) is also superimposed, and the 3-hydroxydecanoyl-NAC mol- ecule is shown as dark gray sticks. Site-directed mutagenesis pCold I-sav606 was used for construction of SAV606 mutants. Site-directed mutagenesis was performed with the following oligonucleotides and their complementary oligonucleotides: Y20F, 5 -GTTTTGGTCCCGTTTAAGG- ACCAC-3 ; Y53F, 5 -GTATCGTCGATAAAGCACGATT- CTGG-3 ; H59A, 5 -GACGATACAGGCGCGCTGAACT- CTGTG-3 ; H59N, 5 -GACGATACAGGCAATCTGAAC- TCTGTG-3 ; H59Q, 5 -GACGATACAGGCCAGCTGAA- CTCTGTG-3 ; N61A, 5 -ACAGGCCATCTGGCGTCTGT- GGAAGTG-3 ; S62A, 5 -CATCTGAACGCGGTGGAAGT- GAAC-3 ; N70A, 5 -AACATCTGCTACGCCCAGATGAT- GTACTA-3 ; Y74F, 5 -AATCAGATGATGTTTTATCTG- GTAGCC-3 . The mutations were confirmed by determining the nucleotide sequences. The plasmids were transformed into E. coli BL21 (DE3) cells, and the mutated enzymes were pre- pared as described above. His70 C3 hydroxy group His70 Tyr20 Tyr53 Asp84 Figure 8. Superimposition of the SAV606 complex structure (cyan and green) on the P. aeruginosa FabA apo structure (PDB code 4FQ9, gray). The (R)-CMABA molecule of the SAV606 structure is shown as yellow sticks. The complex structure of FabA H70N mutant with 3-hydroxydecanoyl-NAC (PDB code 4B0I) is also superimposed, and the 3-hydroxydecanoyl-NAC mol- ecule is shown as dark gray sticks. Preparation of recombinant SAV606 protein Factor Xa protease to remove the His tag and subjected to another round of a His-60 Ni-Superflow affinity chromatogra- phy to collect His-tag free protein. Synthesis of acyl-NAC substrates Crotonic acid (103 mg) was dissolved in CH2Cl2 (5 ml) at 0 °C and stirred for 15 min. A solution of N,N -dicyclohexylcarbo- diimide (274 mg) and 4-(dimethylamino)pyridine (13.7 mg) in CH2Cl2 (5 ml) and NAC (150 mg) were added to the crotonic acid solution simultaneously with two syringes. The mixture was then stirred for 2 days at room temperature. After the slurry was filtered off, the solvent was evaporated. The residue was then purified by silica gel column chromatography (hexane: ethyl acetate 1:2) to provide crotonoyl-NAC (147 mg). Data induced by the addition of isopropyl -D-1-thiogalactopyrano- side (0.2 mM), and the cells were cultured for an additional 20 h at 15 °C. The recombinant SAV606 protein was purified from cell-free extract with a His-60 Ni-Superflow affinity column (Clontech). The protein was then desalted and concentrated using a PD-10 column (GE Healthcare) and an Amicon Ultra 3K centrifugal filter (Merck Millipore, Billerica, MA), respec- tively. For crystallization, the protein was further treated with J. Biol. Chem. (2017) 292(26) 10926–10937 10933 Synthesis of CMABA-NAC 3-Aminobutyric acid (503 mg), water (2.5 ml), NaOH (206 mg), tert-butanol (2.5 ml), and di-tert-butyl dicarbonate (Boc2O, 1.09 g) were successively added to a flask. The reaction mixture was then stirred at room temperature for 16 h. After acidification with KH2PO4 and H3PO4, the aqueous solution was extracted with ethyl acetate. The combined organic layer was concentrated to obtain Boc-3-aminobutanoic acid (919 mg). Next, Boc-3-aminobutanoic acid (568 mg) was dissolved in CH2Cl2 (30 ml) and stirred at 0 °C for 15 min. Solutions of N,N -dicyclohexylcarbodiimide (646 mg) and 4-(dimethyl- amino)pyridine (38.8 mg) in CH2Cl2 (15 ml) and NAC (402 mg) in CH2Cl2 (15 ml) were added to the solution simultaneously with two syringes. The mixture was then stirred for 2 days at room temperature. After the slurry was filtered off, the solvent was evaporated. The crude residue was then purified by silica column chromatography (hexane:ethyl acetate 1:2) to pro- vide Boc-3-aminobutyryl-NAC (676 mg). Synthesis of N-ethoxycarbonylmethyl-3-aminobutyric acid ethyl ester After concentration in vacuo, the oil residue was purified with silica gel chromatography (hexane: ethyl acetate 1:1) to yield N-ethoxycarbonylmethyl-3-ami- nobutyric acid ethyl ester (69.6 mg). Data are: 1H NMR (400 MHz, CDCl3): 4.19 (q, J 7.7 Hz, 2H), 4.15 (q, J 7.2 Hz, 2H), 3.44 (d, J 17.6 Hz, 1H), 3.39 (d, J 17.2 Hz, 1H), 3.12 (sext, J 6.4 Hz, 1H), 2.45 (dd, J 15.2 Hz, 6.4 Hz, 1H), 2.33 (dd, J 15.2 Hz, 6.0 Hz, 1H), 1.28 (t, J 7.0 Hz, 3H), 1.26 (t, J 7.2 Hz, 3H), 1.13 (d, J 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): 172.3, 172.0, 60.8, 60.4, 50.0, 48.5, 41.7, 20.4, 14.2; ESI-MS (positive mode): m/z 218.2 ([MH]). 3-Aminobutyric acid ethyl ester (134 mg) was dissolved in acetonitrile (2.5 ml). K2CO3 (356 mg) was then added to the solution. After stirring the mixture for 15 min at 0 °C, ethyl bromoacetate (121 mg) was added, and the mixture was then stirred for 17 h at room temperature. After evaporation of the solvent, the residue was dissolved in saturated NaHCO3 and extracted with ethyl acetate. After concentration in vacuo, the oil residue was purified with silica gel chromatography (hexane: ethyl acetate 1:1) to yield N-ethoxycarbonylmethyl-3-ami- nobutyric acid ethyl ester (69.6 mg). Data are: 1H NMR (400 MHz, CDCl3): 4.19 (q, J 7.7 Hz, 2H), 4.15 (q, J 7.2 Hz, 2H), 3.44 (d, J 17.6 Hz, 1H), 3.39 (d, J 17.2 Hz, 1H), 3.12 (sext, J 6.4 Hz, 1H), 2.45 (dd, J 15.2 Hz, 6.4 Hz, 1H), 2.33 (dd, J 15.2 Hz, 6.0 Hz, 1H), 1.28 (t, J 7.0 Hz, 3H), 1.26 (t, J 7.2 Hz, 3H), 1.13 (d, J 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): 172.3, 172.0, 60.8, 60.4, 50.0, 48.5, 41.7, 20.4, 14.2; ESI-MS (positive mode): m/z 218.2 ([MH]). A thioesterase catalyzing a Michael addition are: 1H NMR (400 MHz, CDCl3): 6.95 (dq, J 15.6 Hz, 6.8 Hz, 1H), 6.16 (dq, J 15.6 Hz, 1.6 Hz, 1H), 3.46 (q, J 6.0 Hz, 2H), 3.09 (t, J 6.4 Hz, 2H), 1.96 (s, 3H), 1.91 (dd, J 6.8 Hz, 1.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): 190.4, 170.4, 142.0, 129.9, 39.9, 28.3, 23.3, 18.1; electrospray ionization MS (ESI-MS) (pos- itive mode): m/z 188.1 ([MH]). HCl and extracted with ethyl acetate. The organic layer was evaporated to provide crude residue, which was purified by sil- ica gel chromatography (chloroform:methanol 7:1) to yield 3-phenylthiohydantoin-butyric acid (3.5 mg). Data are: 1H NMR (400 MHz, CDCl3): 7.46 (m, 3H), 7.29 (m, 2H), 5.36 (sext, J 6.8 Hz, 1H), 4.31 (d, 19.2 Hz, 1H), 4.17 (d, J 19.2 Hz, 1H), 2.83 (dd, J 15.4 Hz, 6.4 Hz, 1H), 2.78 (dd, J 15.6 Hz, 6.8 Hz, 1H), 1.46 (d, J 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3) 182.5, 173.9, 170.0, 133.0, 129.3, 129.2, 128.4, 48.9, 48.8, 37.8, 29.7, 17.5; ESI-MS (positive mode): m/z 279.1 ([MH]). Non-2-enoyl-NAC was synthesized according to the pub- lished method (12). Butyryl-NAC and hex-2-enoyl-NAC were synthesized using a similar method to the crotonoyl-NAC syn- thesis. From 277 mg of butyric acid, 272 mg of butyryl-NAC was obtained. Data are: 1H NMR (400 MHz, CDCl3): 3.44 (q, J 6.4 Hz, 2H), 3.03 (t, J 6.2 Hz, 2H), 2.56 (t, J 7.4 Hz, 2H), 1.97 (s, 3H), 1.70 (sext, J 7.5 Hz, 2H), 0.96 (t, J 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): 200.3, 170.3, 46.0, 39.9, 28.5, 23.3, 19.3, 13.6; ESI-MS (positive mode): m/z 190.1 ([MH]). From 107 mg of trans-2-hexenoic acid, 64.5 mg of hex-2-enoyl-NAC was obtained. Data are: 1H NMR (400 MHz, CDCl3): 6.94 (dt, J 15.6 Hz, 6.8 Hz, 1H), 6.14 (dt, J 15.6 Hz, 1.2 Hz, 1H), 3.46 (q, J 6.4 Hz, 2H), 3.09 (t, J 6.4 Hz, 2H), 2.20 (dq, J 7.2 Hz, 1.6 Hz, 2H), 1.97 (s, 3H), 1.51 (dq, J 7.6 Hz, 7.2 Hz, 2H), 0.95 (t, J 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): 190.6, 170.4, 146.7, 128.5, 39.9, 34.3, 28.3, 23.3, 21.3, 13.8; ESI-MS (positive mode): m/z 216.1 ([MH]). Synthesis of N-ethoxycarbonylmethyl-3-aminobutyric acid ethyl ester Acetyl chloride (1 ml) was slowly added to ethanol (10 ml) at 0 °C. Then the solution was stirred for 1 h at room temperature. 3-Aminobutyric acid (313 mg) was added to the resulting 10% HCl in ethanol solution. After reflux for 3 h, the solvent was evaporated to provide 3-aminobutyric acid ethyl ester (512 mg). Boc-3-aminobutyryl-NAC (100 mg) was dissolved in CH2Cl2 (1 ml) at 0 °C and stirred for 5 min. Trifluoroacetic acid (TFA, 2 ml) was slowly added to the solution. After stirring for 2.5 h, the mixture was evaporated to provide crude 3-aminobutyryl- NAC. The crude 3-aminobutyryl-NAC was dissolved in aceto- nitrile (3 ml), and K2CO3 (253 mg) was added to the solution. After stirring the mixture for 15 min at 0 °C, tert-butyl bro- moacetate (84.6 mg) was added, and the mixture was then stirred for 16 h at room temperature. After the mixture was filtered, the solvent was evaporated. The residue was purified by silica gel chromatography (chloroform:methanol 25:1) to yield N-(tert-butoxycarbonylmethyl)-3-aminobutyryl-NAC (52.1 mg). N-(tert-Butoxycarbonylmethyl)-3-aminobutyryl- NAC (34.4 mg) was dissolved in TFA (1 ml). After stirring for 2.5 h at room temperature, the solution was evaporated. The crude residue was subjected to HPLC chromatography on a PEGASIL ODS SP100 column (5 m, 250 10 mm; Senshu, Tokyo, Japan) with a linear gradient of 5–15% methanol in water containing 0.1% TFA to yield a purified fraction. The fraction was frozen immediately at 80 °C and lyophilized to yield CMABA-NAC (12.2 mg). Data are: 1H NMR (500 MHz, D2O): 3.80 (d, J 16.8 Hz, 1H), 3.75 (d, J 16.8 Hz, 1H), 3.70 (sext, J 6.5 Hz, 1H), 3.28 (t, J 6.3 Hz, 2H), 3.04 (dd, J 16.6 Hz, 5.8 Hz, 1H), 2.83 (m, 3H), 1.84 (s, 3H), 1.25 (d, J 6.7 Hz, 3H); 13C NMR (125 MHz, D2O): 199.1, 174.4, 169.5, 51.4, 45.6, 45.2, 38.3, 28.4, 21.8, 15.5; ESI-MS (positive mode): m/z 263.2 ([MH]). p p y y g 3-Aminobutyric acid ethyl ester (134 mg) was dissolved in acetonitrile (2.5 ml). K2CO3 (356 mg) was then added to the solution. After stirring the mixture for 15 min at 0 °C, ethyl bromoacetate (121 mg) was added, and the mixture was then stirred for 17 h at room temperature. After evaporation of the solvent, the residue was dissolved in saturated NaHCO3 and extracted with ethyl acetate. Isolation of the ethyl ester of the SAV606 product Isolation of the ethyl ester of the SAV606 product 5 mM crotonoyl-NAC (35 mg) and 200 mM glycine were mixed with 20 M SAV606 in 50 mM Tris-HCl (pH 7.5) buffer in a total volume of 37.5 ml. The reaction was carried out at 28 °C for 20 h. After the reaction, protein was removed from the reac- tion mixture by using an Amicon Ultra centrifugal filter. The resulting low-molecular-weight solution was lyophilized. Because it is difficult to isolate the SAV606 product itself, we treated the product with 10% HCl in ethanol solution in reflux for ethyl esterification. The product derivative was extracted with CHCl3 and then purified by silica gel column chromatog- raphy (hexane:ethyl acetate 1:1) to give 20 mg of N-ethoxy- carbonylmethyl-3-aminobutyric acid ethyl ester. Data are: 1H NMR (400 MHz, CDCl3): 4.18 (q, J 7.2 Hz, 2H), 4.13 (q, J 7.1 Hz, 2H), 3.43 (d, J 17.2 Hz, 1H), 3.38 (d, J 17.2 Hz, 1H), 3.10 (sext, J 6.4 Hz, 1H), 2.44 (dd, J 15.0 Hz, 7.0 Hz, 1H), 2.32 (dd, J 15.0 Hz, 5.8 Hz, 1H), 1.26 (t, J 7.0 Hz, 3H), 1.25 (t, J 7.2 Hz, 3H), 1.11 (d, J 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): 172.4, 172.1, 60.9, 60.5, 50.0, 48.7, 41.8, 20.5, 14.3; HR-FAB-MS (positive mode): m/z calculated for C10H20NO4: 218.1392 ([MH]); found: 218.1381. For analysis of the SAV606 reaction product by HPLC, 2 mM acyl substrate (crotonoyl-NAC, butyryl-NAC, or crotonic acid) and 5 mM glycine were mixed with 10 M SAV606 in 50 mM Tris-HCl (pH 7.5) in a volume of 300 l. After incubation at 28 °C for 3 h, the product was derivatized with phenyl isothio- cyanate as described above. The residue was dissolved in meth- anol. HPLC analysis was carried out using an instrument using a 996 Photodiode Array Detector (Waters, Milford, MA) and an L-6250 Intelligent Pump (Hitachi) equipped with a PEGASIL ODS SP100 column (5 m, 250 4.6 mm; Senshu) with an isocratic solvent system (water:methanol:TFA 65:35:0.1). The flow rate was 1.0 ml/min, and the detection wavelength was 268 nm. Enzyme assay using CMABA-NAC and Ellman’s assay For the reaction of SAV606 with CMABA-NAC, 2 mM CMABA-NAC was mixed with 1.6–10 M SAV606 in 100 mM Tris-HCl (pH 7.5) in a volume of 50 l. After incubation at 28 °C for 5 min, the reaction was quenched by the addition of an equal volume of acetonitrile. 5,5 -Dithiobis(2-nitro- benzoic acid) (5 l of 20 mM solution in acetonitrile) was then added to each solution. The absorbance at 412 nm was observed with a V-630 UV/Vis spectrometer. For determi- nation of kinetic parameters for CMABA-NAC, the concen- tration of CMABA-NAC was varied between 0.1 mM and 4 mM. Steady-state kinetic parameters were determined from the Michaelis-Menten equation. Isolation of the ethyl ester of the SAV606 product To investigate whether crotonoyl-NAC was consumed in the presence of SAV606 His-59 mutants, 200 M crotonoyl- NAC and 100 mM glycine were mixed with 100 M SAV606 wild type or His-59 mutant in 50 mM Tris-HCl (pH 7.5) in a volume of 50 l. After incubation at 28 °C for 10 min, the reaction was quenched by the addition of an equal volume of acetonitrile. HPLC analysis was carried out using an instru- ment using a 996 Photodiode Array Detector and an L-6250 Intelligent Pump equipped with a PEGASIL ODS SP100 col- umn (5 m, 250 4.6 mm) with a gradient of solvents A (0.1% TFA in water) and B (0.1% TFA in methanol): 0–5 min in 5% B, 5–15 min in 10–15% B linear gradient, and 15–25 min in 60% B. The flow rate was 1.0 ml/min and the detection wavelength was 250 nm. Analysis of SAV606 reaction with acyl-NAC and glycine using Ellman’s assay Analysis of SAV606 reaction with acyl-NAC and glycine using Ellman’s assay To investigate the substrate specificity of SAV606 with Ell- man’s assay (17), 2 mM fatty acyl-NAC (crotonoyl-NAC, butyr- yl-NAC, hex-2-enoyl-NAC, or non-2-enoyl-NAC) and 200 mM glycine were mixed with 0.5–15 M SAV606 in 50 mM Tris-HCl (pH 7.5) in a volume of 50 l. After incubation at 28 °C for 0.5–35 min, the reaction was quenched by the addition of an equal volume of acetonitrile. Ellman’s reagent (5 l of 5,5 - dithiobis(2-nitrobenzoic acid), 20 mM solution in acetonitrile) was then added to each solution. The absorbance at 412 nm was observed with a V-630 UV/Vis spectrometer (Jasco, Tokyo, Japan). One unit was defined as the amount of enzyme that releases 1 mol of NAC per minute. For deter- mination of kinetic parameters for fatty acyl-NACs (croton- oyl-NAC or hex-2-enoyl-NAC) by Ellman’s assay, the con- centration of fatty acyl-NAC was varied between 0.25 mM and 15 mM, and the concentration of glycine (200 mM) was fixed. For determination of kinetic parameters for glycine, the concentration of glycine was varied between 1 mM and 200 mM, and the concentration of crotonoyl-NAC (5 mM) was fixed. Steady-state kinetic parameters were determined from the Michaelis-Menten equation. Analysis of the stereochemistry of SAV606 product For determination of the stereochemistry of the SAV606 product, the isolated N-ethoxycarbonylmethyl-3-aminobutyric acid ethyl ester was converted to 3-phenylthiohydantoin-bu- tyric acid as described above. The 3-phenylthiohydantoin-bu- tyric acid obtained was dissolved in methanol. HPLC analysis was carried out using a Hitachi instrument (Chromaster 5110 Pump and UV Detector L-7405; Hitachi, Tokyo, Japan) equipped with a CHIRAL ART Cellulose-SC column (5 m, 250 4.6 mm; YMC, Kyoto, Japan) with an isocratic solvent system (hexane:ethanol:TFA 70:30:0.1). The flow rate was 1.0 ml/min, and the detection wavelength was 268 nm. Crystallization, data collection, and structural determination SAV606 apocrystals were grown using sitting-drop vapor dif- fusion by mixing protein solution (10 mg/ml in 5 mM Tris-HCl (pH 7.5), 1% glycerol, and 50 mM CoA) with an equal volume of reservoir solution (0.1 M imidazole (pH 9.0) and 1.0 M sodium citrate) at 20 °C. SAV606 cocrystals with CMABA were grown using sitting-drop vapor diffusion by mixing protein solution (10 mg/ml in 5 mM Tris-HCl (pH 7.5), 1% glycerol, and 10 mM synthetic racemic CMABA) with an equal volume of reservoir solution (0.1 M imidazole (pH 9.0) and 1.0 M sodium citrate) at 20 °C. Before X-ray data collection, crystals were soaked in res- ervoir solution containing 25% (v/v) ethylene glycol or 25% (v/v) glycerol as a cryoprotectant and flash-frozen in the liquid nitrogen stream. All diffraction data were collected at the 10935 Synthesis of 3-phenylthiohydantoin-butyric acid N-Ethoxycarbonylmethyl-3-aminobutyric acid ethyl ester (69.3 mg) was dissolved in 6 M HCl. After reflux for 4 h, the solvent was evaporated to provide CMABA (45.0 mg), which was used directly for the next step. CMABA (7.7 mg) was dis- solved in 2 ml of coupling solution (acetonitrile:pyridine:trieth- ylamine:water 10:5:2:3). After stirring for 10 min at 0 °C, phe- nyl isothiocyanate (40 l) was added. After the mixture was stirred for 4 h at room temperature, it was acidified with 1 M 10934 J. Biol. Chem. 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https://openalex.org/W4210724110	https://www.frontiersin.org/articles/10.3389/fsurg.2021.786158/pdf	English	null	Predictive Factors for Drain Placement After Laparoscopic Cholecystectomy	Frontiers in surgery	2,022	cc-by	6,211	Predictive Factors for Drain Placement After Laparoscopic Cholecystectomy Giacomo Calini 1†, Pier Paolo Brollo 1†, Rosanna Quattrin 2 and Vittorio Bresadola 1* 1 Department of Medicine, General Surgery Department and Simulation Center, Academic Hospital of Udine, University of Udine, Udine, Italy, 2 Department of Organization of Hospital Services, Academic Hospital of Udine, Udine, Italy Purpose: Currently, surgical drainage during a laparoscopic cholecystectomy (LC) is still placed in selected patients. Evidence of the non-beneﬁcial effect of the surgical drain comes from studies with a heterogeneous population. This preliminary study aims to identify any clinical, demographic, or intraoperative predictive factors for a surgical drain placement during LC as the ﬁrst step to identify population for a prospective randomized study. Method: The study was conducted in a single referral center and academic hospital between 2014 and 2018. Patients who underwent unconverted LC were divided into two groups: Group A (drain) and Group B (no drain). We explored baseline, preoperative, intraoperative characteristics, and postoperative outcomes. Edited by: Sanoop Koshy Zachariah, Kerala Institute of Medical Sciences (KIMS), India Results: Between 409 patients who underwent LC: 90 (22%) patients were in Group A (drain). Age >64 years, male sex, cholecystitis, Charlson comorbidity index (CCI) ≥1, experienced surgeon, intraoperative technical difﬁculties, need for an additional trocar, operative time >60 min, and estimated blood loss >10 ml were predictive factors at univariate analysis. While at multivariate analysis, cholecystitis (odds ratio [OR]: 2.8, 95% CI:1.5–5.1; p < 0.001), CCI ≥1 (OR:1.9, 95% CI:1.0–3.5; p = 0.05), intraoperative technical difﬁculties (OR: 3.6, 95% CI:1.8–6.2; p < 0.001), need of an additional trocar (OR: 2.5, 95% CI: 1.4–4.4; p < 0.005), and estimated blood loss >10 ml (OR: 3.0, 95% CI:1.7–5.3; p < 0.0001) were predictive factors for a surgical drain placement during LC. Reviewed by: Dario D’Antonio, ASL Napoli 2 Nord, Italy Atil Çakmak, Ankara University, Turkey Correspondence: Vittorio Bresadola vittorio.bresadola@uniud.it orcid.org/0000-0002-0098-3540 †These authors share ﬁrst authorship Reviewed by: Dario D’Antonio, ASL Napoli 2 Nord, Italy Atil Çakmak, Ankara University, Turkey Correspondence: Vittorio Bresadola vittorio.bresadola@uniud.it orcid.org/0000-0002-0098-3540 †These authors share ﬁrst authorship Conclusions: This study identiﬁed predictive factors that currently drive the surgeons to a surgical drain placement after LC. Randomized prospective studies are needed to deﬁne the use of drain placement in these selected patients. Specialty section: This article was submitted to Visceral Surgery, a section of the journal Frontiers in Surgery Received: 29 September 2021 Accepted: 29 December 2021 Published: 02 February 2022 Citation: Calini G, Brollo PP, Quattrin R and Bresadola V (2022) Predictive Factors for Drain Placement After Laparoscopic Cholecystectomy. Front. Surg. 8:786158. doi: 10.3389/fsurg.2021.786158 Specialty section: This article was submitted to Visceral Surgery, a section of the journal Frontiers in Surgery Specialty section: This article was submitted to Visceral Surgery, a section of the journal Frontiers in Surgery Received: 29 September 2021 Accepted: 29 December 2021 Published: 02 February 2022 BRIEF RESEARCH REPORT published: 02 February 2022 doi: 10.3389/fsurg.2021.786158 Keywords: preliminary study, predictive factor, laparoscopic cholecystectomy, drain placement, retrospective study Citation: Calini G, Brollo PP, Quattrin R and Bresadola V (2022) Predictive Factors for Drain Placement After Laparoscopic Cholecystectomy. Front. Surg. 8:786158. doi: 10.3389/fsurg.2021.786158 In the past decades, cholecystectomy has become one of the most frequently performed surgical procedures, both in the elective and in the urgency/emergency setting (1). The laparoscopic approach is now the ﬁrst choice and in most cases, it allows to complete the surgical procedure with excellent patient outcomes, adding the laparoscopic advantages in terms of postoperative management (2–4). The usefulness of surgical drain placement after a cholecystectomy has long February 2022 \| Volume 8 \| Article 786158 Frontiers in Surgery \| www.frontiersin.org Drain Placement After Laparoscopic Cholecystectomy Calini et al. been debated. Although numerous randomized clinical trials and meta-analyses have already clearly shown that the use of surgical drain does not improve the postoperative outcomes of patients, surgical drains are still used in selected populations (5–7). Over the years, the evolution of surgical technique and the increasing life expectancy of the general population has led to an expansion of surgical indications to a wider and heterogeneous pool of patients (8–12). The progress of surgical technique, the increasing experience of operators, and the accumulation of scientiﬁc evidence greatly inﬂuenced a more restricted use of surgical drain at the end of a cholecystectomy (13). However, it still remains a device used in conditions that often raise serious concerns about its usefulness or even raise doubts about its potentially harmful eﬀects. These considerations are particularly relevant for patients who are more fragile for clinical and age-related reasons, such as the elderly (4, 14). To date, the evidence of the non-beneﬁcial eﬀect of the surgical drain comes from studies with a heterogeneous population (5, 6, 15, 16). We hypothesize that the intraoperative drain placement might still have a role in selected populations. Hence, deﬁning the predictive factors for surgical drain placement in the current practice is the ﬁrst step to identify the population for a prospective randomized study. In light of the above, the aim of this preliminary study was to identify any predictive factors for the intraoperative drain placement in patients undergoing laparoscopic cholecystectomy (LC). METHODS In our center, LC has been performed according to a standardized technique (19). The patient is positioned in stirrups with the primary operator standing between the legs. A blunt umbilical trocar is positioned with an open technique. After exploration of the peritoneal cavity, other 2–3 trocars are inserted under vision. The dissection of the Calot’s triangle is performed to reach the “critical view of safety” with the aim to identify and dissect the cystic duct and cystic artery. Clipping and division of the structures are then carried out. Intraoperative cholangiography and gallbladder needle decompression are not routinely performed. Retrograde dissection of liver bed is completed and the gallbladder removed by using an endobag. An abdominal surgical drain is not routinely placed; when it does, we place an 18 Fr tubular close drain (19). This study was conducted in an Italian Academic Hospital (AH) from March 2020 to September 2020. It was consisted of a retrospective analysis of hospital discharge data regarding all patients who underwent LC, identiﬁed by the International Classiﬁcation of Diseases, Ninth Revision, Clinical Modiﬁcation (ICD-9-CM) code 51.23, as the primary procedure not associated with any other signiﬁcant surgery during the 2014–2018 period. Data were collected, anonymously and in aggregate form, from the Regional Socio-Health Information System (SISSR) and analyzed by extracting information about the demographic characteristics of patients, urgency and diagnosis upon admission to the hospital, surgery procedure, perioperative course, comorbidities, and complications using ICD-9-CM codes. In the second step, two experienced surgeons reviewed all cases to exclude those with malignant biliary disease and with cholelithiasis complicated by acute pancreatitis or obstruction of the common bile duct. After local IRB approval of the study, hospital clinical computerized records were consulted to implement information not available in the regional database. All analyses were performed using the statistical software SPSS, V.20. The description of the data was done in the form of mean and SD for quantitative data and frequency and proportion for qualitative data. Age was described both as a continuous variable and as a qualitative variable. The population was divided into elderly (>65 years) and non-elderly (<65 years). The ﬁrst analysis of the data was done to test statistically signiﬁcant diﬀerences between the two groups of patients, Group A (with surgical drain) and Group B (without drain placement), with regard to preoperative, intra-operative, and postoperative variables. Citation: codes) and Charlson comorbidity index (CCI) with no age- adjustment (17); (3) surgical procedure: admission urgency (elective and non-elective), ﬁrst surgeon (experienced surgeon with more than 10-year laparoscopic experience and resident), intraoperative technical diﬃculties described in the surgical report (deﬁned as ﬁbrotic adherence, intentional puncture to decompress the gallbladder, gallbladder rupture with bile contamination of the operating ﬁeld, bleeding from the cystic artery, diﬃcult individuation of the anatomical structures, liver steatosis/cirrhosis, and subtotal cholecystectomy), need for an additional trocar, operating time, and estimated blood loss. The ICD-9-CM diagnosis codes used to classify comorbidities related to cholecystectomy are reported in the Supplementary Material. CCI was grouped into 3 classes: 0, 1–2, and ≥3; we used the CCI without age to avoid the collinearity in multivariate analysis. The clinical severity of cholelithiasis was divided into two categories: (1) cholelithiasis only (ICD-9-CM codes: 574.20, 574.50, 574.90, 574.21, and 575.3) and (2) cholecystitis with or without cholelithiasis (ICD-9-CM codes: 574.10, 574.40, 574.70, 575.1, 574.00, 575.0, 575.11, and 575.12). The events considered as predictors of negative clinical outcome occurring in the two groups after LC were: (1) postoperative complications, as described in the postoperative notes, discharge summary, and post-discharge follow-up/imaging (bile leak, intra-abdominal ﬂuid, wound complications, and incisional hernia), and their severity level expressed by the Clavien-Dindo classiﬁcation (CDC) (18); (2) surgical re-intervention within 30 days; and (3) increase in the postoperative hospital stay. Frontiers in Surgery \| www.frontiersin.org RESULTS In total, 409 patients underwent LC as the primary procedure during hospitalization in our AH between 2014 and 2018. An intraoperative surgical drain was placed in 90 patients (Group A) and was not placed in 319 patients (Group B). Preoperative and operative data in both groups are shown in Table 1. In regard to the preoperative data, group A was found to be statistically older (44.4 vs. 26.0%, p < 0.001), with a higher incidence of cholecystitis (52.2 vs. 21.3, p < 0.0001) and with a CCI (with no age) ≥1 (35.6 vs. 19.4, p < 0.01). There were statistically signiﬁcant diﬀerences between the two groups in all operative data: group A presented a longer operative time (112.6’ + 30.0’ vs. 77.8’ + 27.4’, p < 0.0001), more intraoperative technical diﬃculties (77.8 vs. 42.3%, p < 0.0001), need of an additional trocar (50.0 vs. 21.9%, p < 0.0001), and estimated blood loss >10 ml in more patients (68.9 vs. 36.4%, p < 0.0001), as compared with group B. Among the postoperative outcomes of interest in Table 2, group A and group B had a statistically signiﬁcant diﬀerence in the occurrence of postoperative surgical complications (18.9 vs. 8.8%, p < 0.01), overall postoperative complications (28.9 vs. 14.7%, p < 0.01) and mean postoperative hospital length of stay (2.3 + 2.1 vs. 1.2 + 0.7 days, p < 0.0001). Univariate analysis for the predictive factors that drove the decision of surgeon to place the drain after a cholecystectomy is reported in Table 3. Predictive variables were age > 64 years (OR = 2.3; 95% CI: 1.4–3.7; p < 0.0001), male sex (OR = 1.6; 95% CI: 1.0–2.5; p < 0.05), cholecystitis with or without cholelithiasis (OR = 4.0; 95% CI: 2.5–6.6; p < 0.0001), CCI (with no age) value ≥1 (OR = 2.3; 95% CI: 1.4–3.8; p < 0.01), experienced surgeon as ﬁrst operator (OR = 2.2; 95% CI: 1.3–3.5; p < 0.005), the presence of an intraoperative technical diﬃculty (OR = 4.8; 95% CI: 2.7– 8.2; p < 0.0001), the need of an additional trocar (OR: 3.5, 95% CI: 2.1–5.8; p < 0.0001), operative time >60 min (13.6, 95% CI: 4.2–44.1; p < 0.0001) and estimated blood loss >10 ml (OR: 3.9, 95% CI: 2.3–6.4; p < 0.0001). METHODS An independent t-test for quantitative data and chi- square test for qualitative ones were used. The second endpoint was to explore the predictive factors that supported the decision of surgeon to insert a drain after LC. Univariate and multivariate analyses with odds ratios (ORs) and 95% CIs were used. The Patients were allocated into two groups: Group A (patients with surgical drain placement during LC) and group B (patients without drain placement). Both groups of patients were investigated with respect to the following variables: (1) general characteristics: age, sex, body mass index (BMI) (underweight: <18.5; normal weight 18.5–24.9; overweight 25.0– 29.9; and obese ≥30.0); (2) preoperative clinical conditions: severity of cholelithiasis, comorbidity (by using ICD-9-CM February 2022 \| Volume 8 \| Article 786158 Frontiers in Surgery \| www.frontiersin.org 2 Drain Placement After Laparoscopic Cholecystectomy Calini et al. TABLE 1 \| Pre- and intra-operative data for patients undergoing laparoscopic cholecystectomy (LC). Group A (drain) (n = 90) Group B (no drain) (n = 319) p-value DEMOGRAPHIC AND BASELINE CHARACTERISTICS Age 0–64 years 50 (55.6) 236 (74.0) <0.001 >64 years 40 (44.4) 83 (26.0) Mean (SD) 59.8 (14.8) 52.7 (15.2) Sex Male 44 (48.9) 120 (37.6) 0.05 Female 46 (51.1) 199 (62.4) BMI <18.5 – 4 (1.3) 0.07 18.5–24.9 13 (14.4) 95 (33.9) ≥25 68 (75.6) 207 (64.9) Missing 9 (10.0) 13 (4.1) Severity of cholelithiasis Cholelithiasis only 43 (47.8) 251 (78.7) <0.0001 With cholecystitis 47 (52.2) 68 (21.3) Comorbidity ICD-9-CM codesa Present 12 (13.3) 36 (11.3) 0.6 Absent 78 (86.7) 283 (88.7) Charlson comorbidity index (without age) 0 58 (64.4) 257 (80.6) <0.01 1–2 27 (30.0) 54 (16.9) ≥3 5 (5.6) 8 (2.5) Hospital admission Urgent 19 (21.1) 58 (18.2) 0.5 Planned 71 (78.9) 261 (81.8) SURGICAL CHARACTERISTICS First operator resident surgeon 52 (57.8) 237 (74.3) <0.005 experienced surgeon 38 (42.2) 80 (25.1) Missing – 2 (0.6) Intraoperative technical difﬁcultiesb Present 70 (77.8) 135 (42.3) <0.0001 Absent 20 (22.2) 184 (57.7) Need of an additional trocar Yes 45 (50.0) 70 (21.9) <0.0001 No 45 (50.0) 247 (77.4) Missing – 2 (0.6) Operative time (min) Mean (SD) 112.6 (30.0) 77.8 (27.4) <0.0001 Estimated blood loss ≤10 ml 28 (31.1) 203 (63.6) <0.0001 >10 ml 62 (68.9) 116 (36.4) SD, standard deviation; BMI, body mass index. aDescribed in Table 1. RESULTS At multivariate analysis in Table 3, cholecystitis (OR: 2.8, 95% CI: 1.5–5.1; p < 0.001), CCI (with no age) value ≥1 (OR:1.9, 95% CI: 1.0–3.5; p = 0.05), intraoperative technical diﬃculties (OR: 3.6, 95% CI: 1.8–6.2; p < 0.001), need of an additional trocar (OR: 2.5, 95% CI: 1.4–4.4; p < 0.005), and estimated blood loss >10 ml (OR: 3.0, 95% CI: 1.7–5.3; p < 0.0001), were the predictive factors that drove the surgeons to surgical drain placement after LC. METHODS bDeﬁned as ﬁbrotic adherence, intentional puncture to decompress the gallbladder, gallbladder rupture with bile contamination of the operating ﬁeld, bleeding from the cystic artery, difﬁcult individuation of the anatomical structures, liver steatosis/cirrhosis, and subtotal cholecystectomy. TABLE 1 \| Pre- and intra-operative data for patients undergoing laparoscopic cholecystectomy (LC). choice of potential predictive factors for the multivariate model was based on the results of univariate analysis, with p < 0.05 as the criterion for inclusion. The factors that did not maintain an association in the ﬁnal model were considered not to be associated after correction by the confounding factors. Statistical signiﬁcance was set at a p < 0.05. DEMOGRAPHIC AND BASELINE CHARACTERISTICS DEMOGRAPHIC AND BASELINE CHARACTERISTICS Age DISCUSSION This study highlights how the surgical drain is used in a small number of patients undergoing unconverted LC from a purely descriptive point of view. In fact, in the patients under consideration, a surgical drain was placed in only 22% of the cases. This reﬂects the careful selection of the patient to use February 2022 \| Volume 8 \| Article 786158 Frontiers in Surgery \| www.frontiersin.org 3 Drain Placement After Laparoscopic Cholecystectomy Calini et al. TABLE 2 \| Postoperative outcomes. Group A (drain) (n = 90) Group B (no drain) (n = 319) p-value Overall postoperative complications 26 (28.9) 47 (14.7) <0.01 CDC 1 13 (14.4) 32 (10) 2 7 (7.8) 9 (2.8) 3 6 (6.7) 6 (1.9) No complications 64 (71.1) 268 (84.3) Missing – 3 (0.9) Surgical postoperative complications 17 (18.9) 28 (8.8) <0.05 Bile leak 1/17 (5.9) – Indra-abdominal ﬂuid collection 4/17 (23.5) 1/28 (3.6) Wound complication 10/17 (58.8) 24/28 (85.7) 0.1 Incisional hernia 2/17 (11.8) 3/28 (10.7) Re-operation 30-day 3 (3.3) 6 (1.8) 0.1 Postoperative stay (days) mean (+SD) 2.3 (+2.1) 1.2 (+0.7) <0.0001 CDC, Clavien-Dindo classiﬁcation; SD, standard deviation. Guidelines (28, 29), plays a key role. For example, in the case of fragile patients with emergency admission, it is preferable to adopt a conservative treatment with percutaneous drainage and antimicrobial therapy scheduling elective surgery subsequently, reducing the risk to place a drain (30–34). However, the results of the study by Ferrarese et al. (31), while conﬁrming the safety and feasibility of LC in elderly patients both in the elective and emergency regimes, showed a statistically signiﬁcant increase in the placement of surgical drain in patients operated in the emergency regime. In the present study, the ﬁrst operator is associated with the surgical drain placement after LC. In particular, residents place fewer drains than experienced surgeons. However, we believe that this association is more than a diﬀerence in management itself due to the patient selection. Indeed, the experienced surgeon attends during the surgery of residents, partake in the decision-making process, and takes the lead in the most complex intraoperative scenario. Therefore, experienced surgeons are the ﬁrst surgeon for surgeries with a greater risk of intra- and postoperative complications, as demonstrated in the literature (19, 35, 36). DISCUSSION (5) underline how the placement of intraoperative surgical drain has proved useful only in cases of diﬃcult ligation of the cystic duct or hemostasis of the operative ﬁeld and in the case of a suspected biliary leak. Conversely, they did not show a statistically signiﬁcant diﬀerence in the onset of infected intra-abdominal collections between the group of patients with and without surgical drain after LC for acute cholecystitis. They conclude that the surgical drain should not be used prophylactically during LC because it does not reduce the morbidity and does not prevent local complications (such as, surgical site infection); rather it could be a risk factor for their onset (5). Qiu and Li (20) and Picchio et al. in their meta- analysis (6), have reported an increased number of complications, such as fever, wound infections, hemorrhage, wound herniations, increased postoperative pain, and hospital stay in patients with surgical drain placement. In our study, intraoperative drain placement was more frequent in older male subjects with BMI ≥25. This is particularly interesting considering that older age, male sex, and obesity are associated with a more severe acute cholecystitis clinical presentation (7, 21–25). Among the clinical conditions, the choice to place surgical drain correlates with the greater severity of the disease, particularly with the diagnosis of cholecystitis. This evidence is in agreement with what has been reported in the meta-analysis by Picchio et al. (6). Besides, these patients are clinically more complex, as expressed by their CCI (with no age). Both conditions put this group of subjects at a higher risk of perioperative complications, often the reason for the decision to use the drain at the end of surgery (26, 27). The emergency admission to the hospital has not shown to be signiﬁcant in the decision to place surgical drain. Probably, the correct selection of the patient undergoing surgery, in accordance with the 2018 Tokyo this device, according to what is widely reported in the current literature (5, 6, 8, 13). In their study, Park et al. (5) underline how the placement of intraoperative surgical drain has proved useful only in cases of diﬃcult ligation of the cystic duct or hemostasis of the operative ﬁeld and in the case of a suspected biliary leak. DISCUSSION Indeed, a greater complexity of the procedure, identiﬁed with the need to place an additional trocar, the ﬁnding of estimated blood losses >10 ml or a longer operating time, correlates with the choice to place the surgical drain after laparoscopic cholecystectomy in our study. At the univariate analysis, the potential preoperative predictors for a possible drain placement were found to be as follows: age over 64, male sex, diagnosis of cholecystitis, and a CCI (with no age) score ≥1. In their study, Bawahab et al. (15) show how the clinical condition, the preoperative complications, and the diagnosis of cholecystitis have a statistically signiﬁcant correlation with the abdominal drain placement at the end of a LC. On the other hand, no study found an association between the CCI and abdominal drain placement during an LC. Among the intraoperative variables, as we have already mentioned, the need to insert an additional trocar, the presence of estimated blood loss >10 ml, and the operative time exceeding 60 min have been conﬁrmed as predictors for the surgical drain placement. According to our bibliographic research, while increased operative time has already been correlated with the increased frequency of surgical drain use (15), no studies have reported the same evidence regarding the use of the additional trocar and intraoperative estimated blood loss. In particular, the diagnosis of cholecystitis, presence of intraoperative technical diﬃculties, need of an additional trocar, and an estimated blood loss >10 ml are found to be independent predictive factors for surgical drain placement in the multivariate analysis. The overall postoperative complications and those speciﬁcally related to surgery (wound infections, abdominal collections, biliary leaks, and incisional hernias) have shown a statistically signiﬁcant correlation with the intraoperative placement of surgical drain. This correlation is conﬁrmed by several sources in the literature (6, 13). For example, Tzovaras et al. (16) report how drain placement is associated with an increased frequency of postoperative pain and biliary leakage; therefore, its routine use in all elective cholecystectomies is not indicated. This aspect ﬁnds conﬁrmation in Lewis et al. (37), who showed CDC, Clavien-Dindo classiﬁcation; SD, standard deviation. this device, according to what is widely reported in the current literature (5, 6, 8, 13). In their study, Park et al. Frontiers in Surgery \| www.frontiersin.org CDC, Clavien-Dindo classiﬁcation; SD, standard deviation. DISCUSSION Total LC (n = 409) Group A (n = 90) Univariate OR (95%CI) p-value Multivariate OR (95%CI) p-value Age 0–64 years 286 (69.9) 50 (17.5) – >64 years 123 (30.1) 40 (32.5) 2.3 (1.4–3.7) <0.0001 Sex Male 164 (40.1) 44 (26.8) 1.6 (1.0–2.5) 0.05 Female 245 (59.9) 46 (18.8) – BMI <18.5 4 (1.0) – – 18.5–24.9 130 (31.8) 22 (16.9) – ≥25 275 (67.2) 64 (24.7) 1.6 (0.9–2.7) 0.07 Cholelithiasis severity Cholelithiasis only 294 (71.9) 43 (14.6) – With cholecystitis 115 (28.1) 47 (40.9) 4.0 (2.5–6.6) <0.0001 2.8 (1.5–5.1) <0.001 Comorbidity ICD−9–CM codesa Present 48 (11.7) 12 (25.0) 1.2 (0.6–2.4) 0.6 Absent 361 (88.3) 78 (21.6) – Charlson comorbidity index 0 315 (77.0) 58 (18.4) – ≥1 94 (23.0) 32 (34.0) 2.3 (1.4–3.8) <0.01 1.9 (1.0–3.5) 0.05 Hospital admission Urgency 77 (18.8) 19 (24.7) 1.2 (0.7–2.1) 0.5 Planned 332 (81.2) 71 (21.4) – First operator Resident surgeon 289 (70.7) 52 (18.0) – Experienced surgeon 118 (28.9) 38 (32.2) 2.2 (1.3–3.5) <0.005 Missing 2 (0.5) – – Intraoperative technical difﬁcultiesb Present 205 (50.1) 70 (34.1) 4.8 (2.7–8.2) <0.0001 3.6 (1.8–6.2) <0.001 Absent 204 (49.9) 20 (9.8) – Need of an additional trocar Yes 115 (28.1) 45 (39.1) 3.5 (2.1–5.8) <0.0001 2.5 (1.4–4.4) <0.005 No 292 (71.4) 45 (15.4) – Missing 2 (0.5) – – Operative time ≤60 min 105 (25.7) 3 (2.9) – >60 min 304 (74.3) 87 (28.6) 13.6 (4.2–44.1) <0.0001 Estimated blood loss ≤10 ml 231 (56.5) 3 (12.1) – >10 ml 178 (43.5) 62 (34.8) 3.9 (2.3–6.4) <0.0001 3.0 (1.7–5.3) <0.0001 LC, laparoscopic cholecystectomy; SD, standard deviation; BMI, body mass index. aDescribed in Table 1. bDeﬁned as ﬁbrotic adherence, intentional puncture to decompress the gallbladder, gallbladder rupture with bile contamination of the operating ﬁeld, bleeding from the cystic artery, difﬁcult individuation of the anatomical structures, liver steatosis/cirrhosis, and subtotal cholecystectomy. Total LC (n = 409) Group A (n = 90) Univariate OR (95%CI) p-value Multivariate OR (95%CI) p-value Described in Table 1. bDeﬁned as ﬁbrotic adherence, intentional puncture to decompress the gallbladder, gallbladder rupture with bile contamination of the operating ﬁeld, bleeding from the cystic artery, difﬁcult individuation of the anatomical structures, liver steatosis/cirrhosis, and subtotal cholecystectomy. raise an economic issue as both hospitalization and materials drive cost. that no patient without surgical drain required reoperation or subsequent drainage procedure for subhepatic collections formation, as well as no diﬀerence in the frequency of wound infections. DISCUSSION Conversely, they did not show a statistically signiﬁcant diﬀerence in the onset of infected intra-abdominal collections between the group of patients with and without surgical drain after LC for acute cholecystitis. They conclude that the surgical drain should not be used prophylactically during LC because it does not reduce the morbidity and does not prevent local complications (such as, surgical site infection); rather it could be a risk factor for their onset (5). Qiu and Li (20) and Picchio et al. in their meta- analysis (6), have reported an increased number of complications, such as fever, wound infections, hemorrhage, wound herniations, increased postoperative pain, and hospital stay in patients with surgical drain placement. In our study, intraoperative drain placement was more frequent in older male subjects with BMI ≥25. This is particularly interesting considering that older age, male sex, and obesity are associated with a more severe acute cholecystitis clinical presentation (7, 21–25). Among the clinical conditions, the choice to place surgical drain correlates with the greater severity of the disease, particularly with the diagnosis of cholecystitis. This evidence is in agreement with what has been reported in the meta-analysis by Picchio et al. (6). Besides, these patients are clinically more complex, as expressed by their CCI (with no age). Both conditions put this group of subjects at a higher risk of perioperative complications, often the reason for the decision to use the drain at the end of surgery (26, 27). The emergency admission to the hospital has not shown to be signiﬁcant in the decision to place surgical drain. Probably, the correct selection of the patient undergoing surgery, in accordance with the 2018 Tokyo February 2022 \| Volume 8 \| Article 786158 Frontiers in Surgery \| www.frontiersin.org 4 Drain Placement After Laparoscopic Cholecystectomy Calini et al. TABLE 3 \| Univariate and multivariate analysis for drain placement after LC. Total LC (n = 409) Group A (n = 90) Univariate OR (95%CI) p-value Multivariate OR (95%CI) p-value TABLE 3 \| Univariate and multivariate analysis for drain placement after LC. TABLE 3 \| Univariate and multivariate analysis for drain placement after LC. Frontiers in Surgery \| www.frontiersin.org REFERENCES 11. Wakabayashi G, Iwashita Y, Hibi T, Takada T, Strasberg SM, Asbun HJ, et al. Tokyo Guidelines 2018: surgical management of acute cholecystitis: safe steps in laparoscopic cholecystectomy for acute cholecystitis (with videos). J Hepato-Biliary-Pancreat Sci. (2018) 25:73–86. doi: 10.1002/jhbp.517 1. Tsui C, Klein R, Garabrant M. Minimally invasive surgery: national trends in adoption and future directions for hospital strategy. Surg Endosc. (2013) 27:2253–7. doi: 10.1007/s00464-013-2973-9 1. Tsui C, Klein R, Garabrant M. Minimally invasive surgery: national trends in adoption and future directions for hospital strategy. Surg Endosc. (2013) 27:2253–7. doi: 10.1007/s00464-013-2973-9 12. Yokoe M, Hata J, Takada T, Strasberg SM, Asbun HJ, Wakabayashi G, et al. Tokyo Guidelines 2018: diagnostic criteria and severity grading of acute cholecystitis (with videos). J Hepato Biliary Pancreat Sci. (2018) 25:41–54. doi: 10.1002/jhbp.515 2. Okamoto K, Suzuki K, Takada T, Strasberg SM, Asbun HJ, Endo I, et al. Tokyo Guidelines 2018: ﬂowchart for the management of acute cholecystitis. J Hepatobiliary Pancreat Sci. (2018) 25:55–72. doi: 10.1002/jhbp.516 2. Okamoto K, Suzuki K, Takada T, Strasberg SM, Asbun HJ, Endo I, et al. Tokyo Guidelines 2018: ﬂowchart for the management of acute cholecystitis. J Hepatobiliary Pancreat Sci. (2018) 25:55–72. doi: 10.1002/jhbp.516 13. Gurusamy KS, Samraj K, Mullerat P, Davidson BR. Routine abdominal drainage for uncomplicated laparoscopic cholecystectomy. Cochrane Database Syst Rev. (2007) CD006004. doi: 10.1002/14651858.CD006004.pub2 3. Keus F, de Jong JA, Gooszen HG, van Laarhoven CJ. Laparoscopic versus open cholecystectomy for patients with symptomatic cholecystolithiasis. Cochrane Database Syst Rev. (2006) CD006231. doi: 10.1002/ 14651858.CD006231 14. Mayol J, Martinez-Sarmiento J, Tamayo FJ, Alvarez Fernández-Represa J. Complications of laparoscopic cholecystectomy in the ageing patient. Age Ageing. (1997) 26:77–81. doi: 10.1093/ageing/26.2.77 4. Lord AC, Hicks G, Pearce B, Tanno L, Pucher PH. Safety and outcomes of laparoscopic cholecystectomy in the extremely elderly: a systematic review and meta-analysis. Acta Chir Belgica. (2019) 119:349–56. doi: 10.1080/00015458.2019.1658356 15. Bawahab MA, Abd El Maksoud WM, Alsareii SA, Al Amri FS, Ali HF, Nimeri AR, et al. Drainage vs. non-drainage after cholecystectomy for acute cholecystitis: a retrospective study. J Biomed Res. (2014) 28:240– 5. doi: 10.7555/JBR.28.20130095 5. Park JS, Kim JH, Kim JK, Yoon DS. The role of abdominal drainage to prevent of intra-abdominal complications after laparoscopic cholecystectomy for acute cholecystitis: prospective randomized trial. Surg Endosc. (2015) 29:453–7. doi: 10.1007/s00464-014-3685-5 16. Tzovaras G, Liakou P, Fafoulakis F, Baloyiannis I, Zacharoulis D, Hatzitheoﬁlou C. Is there a role for drain use in elective laparoscopic cholecystectomy? DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsurg. 2021.786158/full#supplementary-material The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. DISCUSSION However, in other studies, such correlations are not conﬁrmed with the statistical consistency (5, 20). As a result of our study, patients undergoing LC with surgical drain placement have a longer length of hospital stay than those without the placement of this device, as conﬁrmed in previous studies (5, 6). In addition, if our study did not compare costs, a longer length of hospital stay associated with the use of surgical drain The main limitation of this study is to be non-randomized, unbalanced, and retrospective. In addition, number of patients and multi-surgeon decisions on drain placement could limit results of this study. However, this preliminary study aimed to describe predictive factors for surgical drain placement during an LC into the current practice. Deﬁning the predictive factors is the ﬁrst step in identifying the target population for a future prospective study to evaluate the drain placement. The results Frontiers in Surgery \| www.frontiersin.org February 2022 \| Volume 8 \| Article 786158 5 Drain Placement After Laparoscopic Cholecystectomy Calini et al. AUTHOR CONTRIBUTIONS of this study need to be carefully interpreted, according to the limitations and the aim of the study. GC and PB contributed to the study conception, study design, data acquisition, data interpretation, and manuscript drafting. RQ contributed to the study design, data acquisition, data analysis, and manuscript drafting. VB contributed to the study conception, study design, data interpretation, and manuscript drafting. All authors contributed to the article and approved the submitted version. GC and PB contributed to the study conception, study design, data acquisition, data interpretation, and manuscript drafting. RQ contributed to the study design, data acquisition, data analysis, and manuscript drafting. VB contributed to the study conception, study design, data interpretation, and manuscript drafting. All authors contributed to the article and approved the submitted version. In conclusion, the placement of intraoperative drain after LC is done in selected patients as per the decision of the leading surgeon, with the theoretical purpose of early detection and minimizing complications. At the multivariate analysis, cholecystitis, CCI (with no age) ≥1, intraoperative technical diﬃculties, the need for an additional trocar, and estimated blood loss >10 ml were the predictive factors for surgical drain placement during LC. Otherwise, surgical drain placement is often associated with increased complications, length of stay, and re-intervention. Since studies showing high-quality evidence in patients with these characteristics are lacking, randomized prospective studies are needed to deﬁne the use of surgical drain during an LC in these speciﬁc populations. FUNDING Open access funding was provided by University of Udine. REFERENCES A controlled randomized trial. Am J Surg. (2009) 197:759–63. doi: 10.1016/j.amjsurg.2008.05.011 6. 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Clavien PA, Barkun J, de Oliveira ML, Vauthey JN, Dindo D, Schulick RD, et al. The Clavien-Dindo classiﬁcation of surgical complications: ﬁve-year experience. Ann Surg. (2009) 250:187–96. doi: 10.1097/SLA.0b013e3181b13ca2 8. Coccolini F, Catena F, Pisano M, Gheza F, Fagiuoli S, Di Saverio S, et al. Open versus laparoscopic cholecystectomy in acute cholecystitis. Systematic review and meta-analysis. Int J Surg. (2015) 18:196–204. doi: 10.1016/j.ijsu.2015.04.083 19. Bresadola V, Pravisani R, Pighin M, Seriau L, Cherchi V, Giuseppe S, et al. Clinical strategies to aim an adequate safety proﬁle for patients and eﬀective training for surgical residents: the laparoscopic cholecystectomy model. Ann Med Surg. (2016) 11:58–61. doi: 10.1016/j.amsu.2016.09.006 9. Kuwabara K, Matsuda S, Fushimi K, Ishikawa KB, Horiguchi H, Fujimori K. Relationships of age, cholecystectomy approach and timing with the surgical and functional outcomes of elderly patients with cholecystitis. Int J Surg. 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Frontiers in Surgery \| www.frontiersin.org February 2022 \| Volume 8 \| Article 786158 REFERENCES doi: 10.1002/jhbp.504 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 30. Endo I, Takada T, Hwang T-L, Akazawa K, Mori R, Miura F, et al. Optimal treatment strategy for acute cholecystitis based on predictive factors: Japan- Taiwan multicenter cohort study. J Hepato Biliary Pancreat Sci. (2017) 24:346– 61. doi: 10.1002/jhbp.456 Copyright © 2022 Calini, Brollo, Quattrin and Bresadola. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 31. Ferrarese AG, Solej M, Enrico S, Falcone A, Catalano S, Pozzi G, et al. Elective and emergency laparoscopic cholecystectomy in the elderly: our experience. BMC Surg. (2013) 13(Suppl. 2):S21. doi: 10.1186/1471-2482-13-S2-S21 32. Lau H, Lo CY, Patil NG, Yuen WK. Early versus delayed-interval laparoscopic cholecystectomy for acute cholecystitis: a metaanalysis. Surg Endosc. (2006) 20:82–7. doi: 10.1007/s00464-005-0100-2 February 2022 \| Volume 8 \| Article 786158 Frontiers in Surgery \| www.frontiersin.org 7
https://openalex.org/W3107286258	https://ijods.org/index.php/ds/article/download/8/4	English	null	Classification Breast Cancer Revisited with Machine Learning	International Journal of Data Science	2,020	cc-by-sa	5,032	Classification Breast Cancer Revisited with Machine Learning Hanna Arini Parhusip a,1,, Bambang Susanto a,2, Lilik Linawati a,3,Suryasatriya Trihandaru a,4, Yohanes Sardjono b,5, Adella Septiana Mugirahayu b,6 a Mathematics Department, Universitas Kristen Satya Wacana, Jl.Diponegoro 52-60, Salatiga, 50711, Indonesia b Mathematics Department, Universitas Kristen Satya Wacana, Jl.Diponegoro 52-60, Salatiga, 50711, Indonesia b BATAN , Jl. Babarsari, Tambak Bayan, Caturtunggal, Kec. Depok, Kabupaten Sleman, Daerah Istimewa Yogyakarta 55281, Indonesia 1 hanna.parhusip@uksw.edu; 2bambang.susanto@uksw.edu; 3lilik.linawati@uksw.edu; 4suryasatriya@uksw.edu; 5sardjono.batan@gmail.com; 6662017002@student.uksw.edu corresponding author p , y , p g , g , , b BATAN , Jl. Babarsari, Tambak Bayan, Caturtunggal, Kec. Depok, Kabupaten Sleman, Daerah Istimewa Yogyakarta 55281, Indonesia 1 hanna.parhusip@uksw.edu; 2bambang.susanto@uksw.edu; 3lilik.linawati@uksw.edu; 4suryasatriya@uksw.edu; 5sardjono.batan@gmail.com; 6662017002@student.uksw.edu * corresponding author ARTICLE INFO The article presents the study of several machine learning algorithms that are used to study breast cancer data with 33 features from 569 samples. The purpose of this research is to investigate the best algorithm for classification of breast cancer. The data may have different scales with different large range one to the other features and hence the data are transformed before the data are classified. The used classification methods in machine learning are logistic regression, k-nearest neighbor, Naive bayes classifier, support vector machine, decision tree and random forest algorithm. The original data and the transformed data are classified with size of data test is 0.3. The SVM and Naive Bayes algorithms have no improvement of accuracy with random forest gives the best accuracy among all. Therefore the size of data test is reduced to 0.25 leading to improve all algorithms in transformed data classifications. However, random forest algorithm still gives the best accuracy. Article history Received April 21, 2020 Revised April 30, 2020 Accepted May 10, 2020 Keywords: machine learning accuracy breast cancer This is an open access article under the CC–BY-SA license. ISSN 2722-2039 ISSN 2722-2039 International Journal of Data Science Vol. 1, No. 1, June 2020, pp. 42-50 42 This is an open access article under the CC–BY-SA license. Introduction Data from medical field for instance breast cancer has been several times were discussed by many researchers and the research on this topic is still developed since breast cancer (BC) is one of the most common cancers among women worldwide [7]. This paper employed 7 Machine Learning algorithms using data set breast cancer provided by UCI Machine Learning Repository available in internet where tha data of breast cancer has been classified into 2 classes,i.e. Malignant (M) and Benign (B) breast cancer. The used algorithms are Logistic Regression,Nearest Neighbor, Support Vector Machine (SVM), Kernel SVM, Naive Bayes, Decision Tree, dan Random Forest Classification (RFC). There have been several journals discussed those algorithms and applied to the case of breast cancer [6] [8] [9] [10]. However those journals have not presented all algorithms in one report as shown here. Using these classifications, the paper here proposes accuracy of each algorithm. 2. Machine Learning Method 2.1. Data Identification There are 30 features in the data in 3 cathegories obtained from 569 persons who are 212 persons Benign (B) breast cancer and 357 persons are Malignant Cancer (M) .The features are collected into 3 classes,i.e. Feature mean, Feature standard error (se), Feature Worst taken from internet created by University of Wisconsin, Clinical Sciences Center in 1995 (Web 1). Additionally, 2 types of text data in one column to identify the patients belong to, dataset Malignant (M) and Benign (B) breast cancer. The given data are visualized by using Kde Plot and Violin plot to distiquised 2 chategorised more eaily as shown in Figure 1. Figure 1 illustrates the distribution of all features for both categories of breast cancer. Concave_point mean, concavity mean, area mean, and perimeter mean have indicated different distributions for both breast cancers compared to the other features. This conclusion is also depicted in Figure 2. By drawing in different sides (left and right ), we can distiquish the distributions more clearly. The results showed that the texture_mean, median of Malignant Breast Cancer (M) and Benign Breast cancer is most likely separated which indicated that it is good for classification. However, fractal_dimension_mean has unclear separation leading to bad feature for classification. With these initial study, we employ each algorithm to the given data. Introduction Study on breast cancer has been developed in the last 30 years historically from education and support to partnership in scientific research in USA [1] to improve awareness and social support which are also done in other countries in the world. In Indonesia, the research on breast cancer is also growing since this disease is the second cause mortality for mostly women in Indonesia. The research group in Science and Mathematics Faculty has involved in the breast cancer study which namely learning and investigating the used of Boron Neutron Capture Therapy since 2014. One result was measuring the internal dose of radiation for workers at Boron Neutron Capture Therapy (BNCT) facility based on Cyclotron 30 MeV with BSA The internal dose exposed to the radiation worker is 9.08E-9 µSv [2]. The boron accumulation in EMT-6 tumors of different sizes was evaluated and resulted that in larger tumors (approximately 400 mg) boron accumulated but was significantly higher in smaller tumors (approximately 100 mg) [3]. There have been many other results in the study of Boron Neutron Capture Therapy in the group,such as [4][5] which have no study on classification data. Since later on classification will be needed to improve the reseach of using BNCT facility to treat breat cancer, the article here focus on classification on big data which may appear in the measurement. Classification on big data and the related algorithms are now rapidly growing since development of big data has been supported by existence of “Open Data” provided by various organizations and ISSN 2722-2039 International Journal of Data Science 43 Vol. 1, No. 1, June 2020, pp. 42-50 made available to citizens and businesses [6]. Data from medical field for instance breast cancer has been several times were discussed by many researchers and the research on this topic is still developed since breast cancer (BC) is one of the most common cancers among women worldwide [7]. International Journal of Data Science Vol. 1, No. 1, June 2020, pp. 42-50 43 ISSN 2722-2039 made available to citizens and businesses [6]. Data from medical field for instance breast cancer has been several times were discussed by many researchers and the research on this topic is still developed since breast cancer (BC) is one of the most common cancers among women worldwide [7]. made available to citizens and businesses [6]. 2.2.1. Logistic Regression Kde plot of data distribution Horizontal : concave_point mean Horizontal : symmetrical mean y _p Horizontal : concavity mean Horizontal : compactness mean Horizontal : Smoothness mean Horizontal : area mean Horizontal : perimeter mean Horizontal : texture mean Horizontal : radius mean Horizontal : fractal dimension mean Fig 1. Kde plot of data distribution Fig 2. Distribution of Malignant Breast Cancer (M) and Benign Breast cancer for each feature. Horizontal : compactness mean Horizontal : concavity mean Horizontal : compactness mean Horizontal : area mean Horizontal : texture mean Horizontal : fractal dimension mean Horizontal : concavity mean Horizontal : compactness mean Horizontal : Smoothness mean Horizontal : Smoothness mean Horizontal : area mean Horizontal : perimeter mean Horizontal : perimeter mean Horizontal : texture mean Horizontal : radius mean Horizontal : radius mean Horizontal : fractal dimension mean Fig 1. Kde plot of data distribution Fig 2. Distribution of Malignant Breast Cancer (M) and Benign Breast cancer for each feature. Fig 2. Distribution of Malignant Breast Cancer (M) and Benign Breast cancer for each feature. Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) 2.2.1. Logistic Regression Logistic regression is one of classification algorithms to relate a feature as an input with a discrite output with a certain probability limited to values between 0 and 1. . This logistic regression is similar to Ordinary Least Squares (OLS) regression where target variable has dicotomy scales such as yes and no, good and bad, low and high. The curve is given by natural algorithm of target variable. The logistic regression can be formulated as = exp β + βX. By taking the natural logarithm of both side,one yields ln = β + βX. Since we have several features variables, we may write ln = β + βX. Since we have several features variables, we may write = ⋯ (1) (1) = ⋯ The parameters are determined uses maximum likelihood estimation (MLE). The parameters are determined uses maximum likelihood estimation (MLE). Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) 44 International Journal of Data Science ISSN 2722-2039 Vol. 1, No. 1, June 2020, pp. 42-50 Horizontal : symmetrical mean Horizontal : concave_point mean Horizontal : concavity mean Horizontal : compactness mean Horizontal : Smoothness mean Horizontal : area mean Horizontal : perimeter mean Horizontal : texture mean Horizontal : radius mean Horizontal : fractal dimension mean Fig 1. Kde plot of data distribution ta Science ISSN 2722-2039 pp. 42-50 Horizontal : concave_point mean Horizontal : compactness mean Horizontal : area mean Horizontal : texture mean Horizontal : fractal dimension mean stribution a Science ISSN 2722-2039 p. 42-50 Horizontal : concave_point mean Horizontal : compactness mean Horizontal : area mean Horizontal : texture mean Horizontal : fractal dimension mean t ib ti International Journal of Data Science Vol. 1, No. 1, June 2020, pp. 42-50 44 ISSN 2722-2039 ISSN 2722-2039 Horizontal : symmetrical mean Horizontal : symmetrical mean Horizontal : concave_point mean Horizontal : concavity mean Horizontal : compactness mean Horizontal : Smoothness mean Horizontal : area mean Horizontal : perimeter mean Horizontal : texture mean Horizontal : radius mean Horizontal : fractal dimension mean Fig 1. 2.2.2. Support Vector Machine Support Vector Machine (SVM) is one of methods in supervised learning,i.e the data set have been labeled. This algorithm separates data into different classes by hyperplane in linear case or a curve in a nonlinear case by maximing the margin leading to optimization problem. The optimal separating hyperplane or other more general functions in multidimensional cases are defined into a ISSN 2722-2039 International Journal of Data Science 45 Vol. 1, No. 1, June 2020, pp. 42-50 special coding called a kernel that can be set up depending on the given problem. Additionally, data can also contain outliers. Therefore the algorithm adds some regularization parameters. This algorithm has been tested for breast cancer data in several literatures [11][10] [12] and also for other big data [13]. The paper here shows that the algorithm is used in different sizes of test data with different kernels. International Journal of Data Science Vol. 1, No. 1, June 2020, pp. 42-50 ISSN 2722-2039 45 Vol. 1, No. 1, June 2020, pp. 42-50 special coding called a kernel that can be set up depending on the given problem. Additionally, data can also contain outliers. Therefore the algorithm adds some regularization parameters. This algorithm has been tested for breast cancer data in several literatures [11][10] [12] and also for other big data [13]. The paper here shows that the algorithm is used in different sizes of test data with different kernels. 2.2.4. Naive Bayes Naïve Bayes Classifier (NBC) is a method classification based on Bayesian theorem. Naive stands for relying on the assumtion that all features are unrelated in the class though they can be related. All properties are considered to have possible contribution independently. Bayes theorem relies on the event probability from the prior as the assumption knowledge. If we have hypothesis H and given an event E, Bayes states that the relation probability before the event occur as P(H) and probability from hypothesis after the event occur is called P(H\|E) and formulated as P(H\|E) is a certain proportion of P(H). This proportion is named as likelihood that gives a kind of similarity for a certain probability and the likelihood is given by P(E\|H)/P(E). Thus one yields \|" = # $\|%# % # $ (2) (2) \|" = # $\|%# % # $ where P(H\|E) posterior probability. In the computation, one has to define a certain radius to guarantee that some elements in the given dataset on the same class. In this case, the Euclidean distance is used. As usual in the machine learning, data set are splitted into 2,i.e. training data and test data. Prior probability is defined as the number of element in current class divided by the number of the element in all classes. We define also margin probability P(X) to determine the elements in the circle divided by the total elements. Furthermore, the likelihood is then can be prescribed as P(\|current class) is equal to the number of elements in the current class divided by the toral elements. Thus the posterior probability in the algorithm is :P(current class\|X)= likelihood*prior probability/ margin probability. Though Naive Bayes Classifier has been employed in breast cancer data by other authors [12][9] , this paper revisits the algorithm to learn how the algorithm works compared to other algorithms in Machine Learning. One may find the simple presentation of this method in the level of education [15]. 2.2.3. K-Nearest Neighbor Another classification algorithm is the k-nearest-neighbors (K-NN) algorithm and it can be used also for regression. Several authors have used the K-NN for classification and regression [14] It is developed due to some behaviour of data may have similar things in close proximity based on the used distance definition. The given data are splitted into sample data dan query data. We examine the closeness of the sample data dan query data. By sorting the obtained distances from the smallest to the largest , one has a list of distances. Take the first-K of these distances and collect the obtained labels. The most appeared labels are then collected to identify the classes. Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) 2.2.6. Random Forest Classification Random Forest Classification can be considered as a collection of decision trees which is also a supervised learning algorithm. . By averaging out the impact of several decision trees, random forests tend to improve prediction. Randomness is governed by selecting data samples randomly from a given dataset where the best tree will be obtained by voting. The final prediction is taken from the most votes. Compared to decision trees, Random forest has no problem on overfitting with more time consuming due to complexity of trees. To calculate the importance of each feature Gini index is used by droping a variable [8]. The accuracy of each algorithm is checked using Confusion matrix which is frequently used to predict accuracy of classification algorithm and we use the library in sklearn to do the computation of accuracy matrix. 3. All features are studied to have the correlation between 2 different features and we obtained the matrix of correlations.The positive correlations are shown by several pairs of features,i.e. perimeter mean and radius worst, area mean and radius worst, texture mean and texture worst, area worst and radius worst which are shown in Figure 3. Fig 3. Positive correlation for Perimeter mean vs Radius worst, Area mean vs Radius worst, Texture mean and Texture Worst, Area worst and radius worst. Fig 3. Positive correlation for Perimeter mean vs Radius worst, Area mean vs Radius worst, Texture mean and Texture Worst, Area worst and radius worst. 2.2.5. Decision Tree Algorithm Once the best split is found, we can use it as a node in our decision tree. Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) 2.2.5. Decision Tree Algorithm When the atributes are categorical and numeric then decision tree algorithm has ability to classify with these type of data. The algorithm constructs terminal Nodes, Recursive Splitting, Building a Tree. The terminal nodes contain the maximum tree depth and the minimum node records. The first node is called the root node of the tree and from the root node other nodes are made until to the maximum tree depth is obtained. There must be minimum nodes satisfied in the algorithm. Thus the number of nodes is growing until the terminal nodes. A node may have zero children (a terminal node), one child (one side makes a prediction directly) or two child nodes. We will refer to the child nodes as left and right in the dictionary representation of a given node. By recursive splitting the tree is made starting from 2 groups of data,i.e. two lists of rows given the index of an attribute and a split value for that attribute which then the cost function evaluating the split is called the Gini index. The spliting is succesful if the Gini score is 0, and the worst case in splitting will have Gini score 0.5 and the function is called gini_index() The rows in the first group all belong to class 0 and the rows in the second group belong to class 1, so it’s a perfect split. Given 46 International Journal of Data Science ISSN 2722-2039 Vol. 1, No. 1, June 2020, pp. 42-50 a dataset, we must check every value on each attribute as a candidate split, evaluate the cost of the split and find the best possible split we could make. Once the best split is found, we can use it as a node in our decision tree. International Journal of Data Science ISSN 2722-2039 Vol. 1, No. 1, June 2020, pp. 42-50 46 ISSN 2722-2039 a dataset, we must check every value on each attribute as a candidate split, evaluate the cost of the split and find the best possible split we could make. Once the best split is found, we can use it as a node in our decision tree. a dataset, we must check every value on each attribute as a candidate split, evaluate the cost of the split and find the best possible split we could make. 3.2 Classification Result The algorithms have been provided in the python library callled sklearn. We employ all the algorithms above for the studied data. As usual in machine learning algorithm, one must split data into training data and test data. For the first study, the test data is 0.3 part of the original data. Additionally, if data should also be transformed to avoid big range between different features. International Journal of Data Science Vol. 1, No. 1, June 2020, pp. 42-50 ISSN 2722-2039 47 Using the given algorithm, Table 1 has presented the result of logistic regression for classification for data cancer. The obtained confusion matrix is CM = &103 5 1 62- Using the given algorithm, Table 1 has presented the result of logistic regression for classification for data cancer. The obtained confusion matrix is CM = &103 5 1 62- Table 1. Result on logistic regression precision recall f1-score support B 0.99 0.95 0.97 108 M 0.93 0.98 0.95 63 accuracy 0.96 171 macro avg 0.96 0.97 0.96 171 weighted avg 0.97 0.96 0.97 171 Table 1. Result on logistic regression The algorithm of Logistic Regression has been employed. The table shows that the Benign Breast Cancer (C) contains 108 persons where 5 persons are missclasified, and hence the variable recall in the table has defined the prediction has 0.95 value or 95 % correctness. Moreover the Malignant Breast Cancer (M) has 63 perrson with 1 misclassification. Therefore the recall column gives 0.98 ,that is the number of correct predicted divided by the total data. For all data, the algorithm gives 0.965 accuracy or 96.5% accurary. Futhermore, the algorithm is again used where the data are transformed by min-max normalization. . The obtained confusion matrix for the transformed data is as follows,i.e. CM = &93 15 0 63-. Table 2. The result of logistic regression with min-max normalization Table 2. The result of logistic regression with min-max normalization precision recall f1-score support B 1.00 0.86 0.93 108 M 0.81 1.00 0.89 63 accuracy 0.91 171 macro avg 0.90 0.93 0. 91 171 weighted avg 0.93 0.91 0. 91 171 By normalization Min-Max for the given data, we obtain breast cancer with 108 data of Benign Breast Cancer (C) and 15 persons have been misclassified. The Malignant Breast Cancer (M) contains 63 persons with none misclassification. As a result, we get 91,2 % accuracy. 3.2 Classification Result The k-nearest neighbor algorithm is the second used algorithm. We the same splitting parameters where the test size data is 0.3 , one yields the CM matrix, i.e . CM = &103 5 4 59- . The k-nearest neighbor algorithm is the second used algorithm. We the same splitting parameters where the test size data is 0.3 , one yields the CM matrix, i.e . CM = &103 5 4 59- . The same as in the previous algorithms, the table has shown the Benign Breast Cancer (C) has 108 data with 5 misclassification and Malignant Breast Cancer (M) contains 63 data with 5 misclassification. Finally, one concludes that total accuracy is 94.7%. For each algorithm, we redo the algorithm where the given data are normalized. Other algorithms are not shown separately. The final studies for all algorithms are listed in Table 3. Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) 48 8 International Journal of Data Science ISSN 2722-2039 Vol. 1, No. 1, June 2020, pp. 42-50 Table 3. Comparison accuries given by each algorithm No Algorithm Accuracy using original data Data transformed using Accuracy using transformed data 1 Logistic Regression 96,5% min-max normalization 91,2% 2 Nearest Neighbor 94,7% min-max normalization 91,8% 3 SVM 63,2% min-max normalization 94,2% 4 Kernel SVM 63,2% min-max normalization 94,2% 5 Naive Bayes 92,4% min-max normalization 78,9% 6 Decision Tree 92,4% Model Overfitting 94,2% 7 RFC 96,5% Standard Scale 97,1 % Table 3. Comparison accuries given by each algorithm Table 3 shows that the RFC has given best accuracy to predict breast cancer classification. indicating best accuracy is still obtained. Compared to other authors , some differencies achieved,e.g. logistic regression has 96.5% accuracy in this research where as other author obtained 97.18%. RFC in this reseach has 96,5% and other researcher obtained 95.25% [8]. In the case of KNN, efficiency for different values of K has been shown by other author [16]. Table 3 shows that the RFC has given best accuracy to predict breast cancer classification. indicating best accuracy is still obtained. Compared to other authors , some differencies achieved,e.g. logistic regression has 96.5% accuracy in this research where as other author obtained 97.18%. RFC in this reseach has 96,5% and other researcher obtained 95.25% [8]. In the case of KNN, efficiency for different values of K has been shown by other author [16]. 3.2 Classification Result Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) Table 4. Comparison accuries given by each algorithm for different test size No Algorithm Accuracy using original data Test size =0.3 Accuracy using transformed data Test size =0.3 Accuracy using transforming data Test size =0.25. 1 Logistic Regression 96,5% 91,2% 95.86 % 2 Nearest Neighbor 94,7% 91,8% 95.10 % (minkownski distance) 3 SVM 63,2% 94,2% 97.20% (linear separation) 4 Kernel SVM 63,2% 94,2% 96,50% (rbf separation) 5 Naive Bayes 92,4% 94,2% 91.60% 6 Decision Tree 92,4% 94,2% 95.80% (criterion=entropy) 7 RFC 96,5% 97,1 % 98.60% (criterion =entropy) Table 4. Comparison accuries given by each algorithm for different test size Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) International Journal of Data Science Vol. 1, No. 1, June 2020, pp. 42-50 ISSN 2722-2039 49 Vol. 1, No. 1, June 2020, pp. 42-50 The result of Table 4 is obtained by using test size =0.3,i.e the training data are 70% from the given data. However, transforming data may improve the accuracy as seen in the case of SVM. In the case of KNN, the accuracy is reduced. We expect the accuracy should be better in all algorithms after data are rescaled. We tried to use smaller test size. We show that the test size in transforming data may give different accurary and confusion matrix as listed in Table 4. The result of Table 4 is obtained by using test size =0.3,i.e the training data are 70% from the given data. However, transforming data may improve the accuracy as seen in the case of SVM. In the case of KNN, the accuracy is reduced. We expect the accuracy should be better in all algorithms after data are rescaled. We tried to use smaller test size. We show that the test size in transforming data may give different accurary and confusion matrix as listed in Table 4. Reducing test size has led to improve accurary in all algorithms as we expected that the accuracy should rely on the trasforming data to be classified since we want to have reasonable range of data in all features. 3.2 Classification Result Futhermore, one may study the impact for each feature to the algorithm to the result prediction .The result shows that the importantness of features are concave points_worst, radius_worst, and perimeter_worst as the highest three first scores .One may also illustrate in a histogram as shown in Figure 4. Fig 4. The important levels for features Fig 4. The important levels for features Compared to other author, the result on Naive Bayes Classifier gave 94.762% using 10 features in the study [9] .This may happen since the features used in the research here are 31 features . Other author stated that accurary was 95.5% in other literature [12] where Naive Bayes Classifier was improved with cross validation. Compared to other author, the result on Naive Bayes Classifier gave 94.762% using 10 features in the study [9] .This may happen since the features used in the research here are 31 features . Other author stated that accurary was 95.5% in other literature [12] where Naive Bayes Classifier was improved with cross validation. 4. This paper has shown that some algorithms in Machine Learning are tested to use for analysis observing data of breast cancer obtained from UCI Machine Learning Repository available in internet. The algorithms are logistic regression, support vector machine, ,k-nearest neighbourhood, naive bayesian, decision tree and random forest classification. After data are transformed, the 30% of test data give no improvement on accuracy of classification. By reducing 5% test data from 30%, accuracy has been improved and random forest classification gives the best accuracy. References [1] [1] R. Janet et al., “A Historical Perspective on Breast Cancer Activism in the United States: From Education and Support to Partnership in Scientific Research,” J Womens Heal., vol. 21, no. 3, pp. 355–362, 2012, doi: 10.1089/jwh.2011.2862. [2] M. Muhammad, A. W. Harto, and Y. Sardjono, “Monte Carlo N Particle Extended ( MCNPX ) Radiation Shield Modelling on Boron Neutron Capture Therapy Facility Using D-D Neutron Generator,” vol. 4, no. 2, pp. 58–65, 2019, [Online]. Available: https://ejournal.uksw.edu/ijpna/article/view/3318/1404. [3] A. A. Khan, C. Maitz, C. Quanyu, and F. Hawthorne, “BNCT induced immunomodulatory effects contribute to mammary tumor inhibition,” PLoS One, vol. 14, no. 9, pp. 1–14, 2019, doi: 10.1371/journal.pone.0222022. [4] S. Dyah, P. Bagaswoto, and S. Yohannes, “In Vitro and In Vivo Test of Boron Delivery Agent for BNCT,” Indones. J. Phys. Nucl. Appl., vol. 4, no. 2, 2019, doi: https://doi.org/10.24246/ijpna.v4i2.39- 44. [5] S. G. Pinasti, “Measurement of Yttrium-90 Biodistribution in Selective Internal Radiation Therapy ( SIRT): a Comparison Between PET AND SPECT IMAGING,” vol. 4, no. 2, pp. 45–57, 2019, [Online]. Available: https://ejournal.uksw.edu/ijpna/article/view/2769/1403. [6] I. Issam, J. Stéphane, N. Karl, and M. Carole, “The Big Data Revolution for Breast Cancer Patients,” Eur J Breast Heal., vol. 14, no. 2, pp. 61–62, 2018, doi: 10.5152/ejbh.2018.0101. [7] H. G. Russnes, O. C. Lingjærde, A. L. Børresen-Dale, and C. Caldas, “Breast Cancer Molecular Stratification: From Intrinsic Subtypes to Integrative Clusters,” Am. J. Pathol., vol. 187, no. 10, pp. 2152–2162, 2017, doi: 10.1016/j.ajpath.2017.04.022. [8] S. Jabeen and K. Jilani Abdul, “Predicting Breast Cancer using Logistic Regression and Multi-Class Classifiers,” Int. J. Eng. Technol., vol. 7, no. 4.20, pp. 22–26, 2018. [9] R. Megha and S. Arun Kumar, “Breast Cancer Prediction using Naïve Bayes Classifier,” Int. J. Inf. Technol. 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Acknowledgment The paper here supported by the internal research center from Universitas Kristen Satya Wacana SK no.121/Pen./Rek./3/V/2020 entitled Klasifikasi kanker dengan metode Clustering dan COTHONS (Coding Python for Schools) period 2019/2020. Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning) International Journal of Data Science ISSN 2722-2039 Vol. 1, No. 1, June 2020, pp. 42-50 50 International Journal of Data Science ISSN 2722-2039 Vol. 1, No. 1, June 2020, pp. 42-50 References Sokolova, “Big Data Classification Using the SVM Classifiers with the Modified Particle Swarm Optimization and the SVM Ensembles,” Int. J. Adv. Comput. Sci. Appl., vol. 7, no. 5, pp. 294–312, 2016, doi: 10.14569/ijacsa.2016.070541. [14] B. Sadegh, Imandoust, and B. Mohammad, “Application of K-Nearest Neighbor (KNN) Approach for Predicting Economic Events: Theoretical Background,” Int. J. Eng. Res. Appl., vol. 3, no. 5, pp. 605– 610, 2013, [Online]. Available: https://wwijera.com/papers/Vol3_issue5/DI35605610.pdf. [15] M. . Bárcena, M. A. Garín, A. Martín, A. . Tusell, and E. Unzueta, “A Web Simulator to Assist in the Teaching of Bayes’ Theorem,” J. Stat. Educ., vol. 27, no. 2, 2019, doi: https://doi.org/10.1080/10691898.2019.1608875. [16] Z. Shichao, L. Xuelong, Z. Ming, Z. Xiaofeng, and W. Ruili, “Efficient kNN Classification With Different Numbers of Nearest Neighbors,” IEEE Trans. Neural Networks Learn. Syst., vol. 29, no. 5, pp. 1774–1785, 2018, doi: 10.1109/TNNLS.2017.2673241. Hanna Arini Parhusip et al (Classification Breast Cancer Revisited with Machine Learning)

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